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Electron Spin Resonance Volume 106

A Specialist Periodical Report

Electron Spin Resonance Volume IOB

A Review of Recent Literature t o mid-1986 Senior Reporter M. C . R . Symons, Department of Chemistry, University of L eicester Reporters J. F. Gibson, Imperial College, London G . R . Hanson, Monash University, Australia C . P. Keijzers, University of Nijmegen, The Netherlands R . P. Mason, National Institute of Environmental Health Sciences, North Carolina, USA C. Mottley, Luther College, Iowa, USA J. R. Pilbrow, Monash University, Australia A. Schweiger, ETH-Zentrum, Zurich, Switzerland

The Royal Society of Chemistry Burlington House, London, W I V OBN

ISBN 0-85 186-85 1-7 ISSN 0305-9578

Copyright 0 1987 The Royal Society of Chemistry All Rights Reserved No part of this book may be reproduced or transmitted in any form or by any means graphic, electronic, including photocopying, recording, taping or information storage and retrieval systems without written permission f r o m The Royal Society of Chemistry ~

~

Printed in Great Britain at the Alden Press, Oxford, London and Northampton

Foreword

This is

the

Inorganic and

concerned with 'Contents' are

organic and

Bio-inorganic bio-organic

listed herein

half of Volume 10. Volume 10A was

e.s.r.

spectroscopy

and

its major

for the benefit of those who have not seen this

In particular, I call attention to the Chapters on Spin-Labels

Volume.

and

on

Free Radical Studies in Biology and Medicine whjch may well be of interest to readers of the present Volume.

Also, the review on Loop-gap Resonators is, of

course, particularly relevant. The only Chapter 'missing' is one on 'Theoretical Aspects' by Dr. A . Hudson who was, unfortunately, unable to prepare it in time. This is now scheduled for Volume 11A since theory is equally applicable for either volume. As I mentioned in Volume 10A, there remains the question of these Volumes. price

reasonable.

of

All we can hope for is that those who value this production

will do their best to ensure that their institutes and that we

viability

Every effort is being made for rapid publication and to keep the friends buy

copies so

can continue with what I hope is a really valuable contribution to the

e.s.r. literature.

I am most grateful to all the authors for surmounting a l l sorts of problems in order

to get their contributions to me on time.

writing a Review a year considerable task

or

so before

it

is due -

It is so easy to agree to one

forgets the very

that this actually represents - so I am very appreciative of

the efforts made by my collaborators. Martyn C. R . Symons

Contents

CHAFTER 1

Spin

- Spin Interactions in Weakly Interacting Dimers

By C.P. Keijzers

1 Introduction

1

2

Spin-Hamiltonian

3

Isotropic Exchange

5

3.1

Spin-Hamiltonian

5

3.2

Analytical Expressions for J

6

3.2.1 3.2.2 3.2.3 3.3

10 11

Experimental Determinations of J

11

3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 3.3.9 3.4

7

Localized Method Molecular Orbital Method The Above Approaches

Merging Effect Susceptibility with EPR Singlet - Triplet Mixing ENDOR Undiluted Compounds with Small Exchange Heteronuclear Dimers Ferromagnetic Coupling Cluster Systems Paramagnetic Host Crystals

12 12 13 14 14 15 16 16 18

18

Calculation of J

20

4 The Zero Field Splitting Tensor

4.1

Analytical Expressions f o r

4.1.1 4.1.2 4.2

us

The Dipole - Dipole Interaction The Spin - Orbit Contribution

25

Experimental

28

5 Antisymmetric Exchange

5.1

-+ Analytical Expression of d 5.1.1 5.1.2

5.2

21 21 22

The Dipole - Dipole Interaction The Spin - Orbit Contribution

29 29 29

Experimental

32

References

35

vii

...

Contents

Vlll

CHAFTER 2

Transition-metal Ions By J.F.

Gibson

1 Introduction

39

2

General

40

Theory

42

Analysis of Spectra, Computing

3

Jahn - Teller Effects

47

Mixed Valence Complexes

49

Phase Transitions

51

Paramagnetic Ligands

54

Binuclear and Oligonuclear Complexes

55

s

61

= 1/2

d1 Configuration Tervalent Titanium, Zirconium and Hafnium Quadrivalent Vanadium, Niobium and Tantalum Quinquevalent Chromium and Molybdenum Sexivalent Manganese, Technetium and Rhenium d5 Configuration Zerovalent Vanadium and Univalent Chromium Bivalent Manganese, Tervalent Iron, Ruthenium and Osmium Quinquevalent Platinum d7 Configuration

Univalent Iron and Bivalent Cobalt Tervalent Nickel, Palladium and Platinum d9 Configuration

Bivalent Copper Zerovalent Cobalt and Univalent Nickel 4

61

61 62 64 66 66 66 68 69 69

69 70 72 72 75

S = l

77

2 Configuration

77

Univalent Cobalt and Divalent Nickel

5

45

77

S = 3/2

77

d 3 Configuration

77

Tervalent Chromium and Quadrivalent Rhenium

77

ix

Contents

78

d7 Configuration

Zerovalent Manganese, Univalent Iron and Bivalent Cobalt

6 S

=

78

79

5/2

79

d5 Configuration

80

Bivalent Manganese Tervalent Iron

83 85

7 s = 3 Univalent Manganese

a5 87

References

CHAPTFX 3 Metalloproteins

By G.R.

Hanson and J . R .

Pilbrow

1 Introduction

93

2 Copper Enzymes

94

2.1 Type 1 Copper Enzymes

94

2.2 Type 2 Copper Enzymes

95

2.3 Multi-centred Copper Enzymes

97

99

3 Iron Proteins 3.1

Non Heme Iron Proteins

3.2

Heme Iron Proteins

4 Iron Sulphur Proteins

99 100 105 105

4.1

[ 2Fe-2S](2+’1+)

4.2

[ 3Pe-3/4S](3+’2+’1+’0) Cluster Containing Enzymes

106

4.3

[4Fe-4S](3f’2+’2+71+) Cluster Containing Enzymes

107

5 Nickel 5.1

-

Cluster Containing Enzymes

Iron - Sulphur and Related Iron - Sulphur Enzymes

Hydrogenases

5.2 Other Nickel Containing Enzymes

6 Molybdenum Containing Enzymes

109 109 111

111

6.1 Molybdenum Iron Enzymes

111

6.2 Mononuclear Oxomolybdenum Enzymes

112

7 Vanadium

114

Contents

X

8 Paramagnetic Metal Substituted Enzymes

9

114

8.1

Substituted Zinc Enzymes

114

8.2

Substituted Magnesium Enzymes

116

8.3

Extrinsic Metal Binding Site in Proteins

118

Mitochondrial Electron Transport Chain

119

9.1

Succinate Dehydrogenase

119

9.2

Cytochrome Oxidase

120

9.3

Bacterial Cytochrome Oxidases

122

9.4

Other Mitochondrial Reaction Centres

122

10 Photosynthesis

123

10.1

Photosystem I

124

10.2

Photosystem I1

125

11 Statistical Model f o r E.p.r. Line Broadening

127

1 2 Relaxation

128

References

130

CHAPTER 4 ENDOR Methodology By A . Schweiger

1 Introduction

138

2

Basic Instrumentation and Experimental Techniques

139

2.1

Spectrometers, r.f. Power Matching

139

2.2

ENDOR Resonators

139

2.3

Frequency Option

140

2.4

Modulation Schemes

141

2.5

Sensitivity

142

3 Analysis of ENDOR Spectra 3.1

ENDOR Frequencies 3.1.1 3.1.2

First Order Frequencies Second Order Frequencies

143 143 143 145

3.2

Transition Probabilites

148

3.3

Signs of Hyperfine and Quadrupole Coupling Constants

149

3.4

Evaluation of the Magnetic Parameters

150

xi

Contents

3.4.1 Liquid Phase ENDOR 3.4.2 Single Crystal ENDOR 3.4.3 Powder ENDOR

4 Theoretical Approaches, Mechanisms and Conditions

150 150 153 155

4.1 Theory of Liquid Phase ENDOR

155

4.2

156

Negative ENDOR

4.3 ENDOR-Detected Nuclear Magnetic Resonance

157

4.3.1 Mechanisms 4.3.2 Applications

158 158

5 Advanced ENDOR Techniques

159

5.1 Orientation Selection in Powders, Frozen Solutions and Nematic Glasses

159

5.2 TRIPLE Resonance

161

5.2.1 5.2.2 5.2.3 5.2.4 5.3

Special TRIPLE General TRIPLE Experimental Aspects Applications

ENDOR-Induced ESR 5.3.1 Experimental Aspects 5.3.2 Applications 5.3.3 TRIPLE-Induced ESR

5.4 ENDOR with Circularly Polarized r.f. Fields

165 165 166 168 168

Basic Idea Experimental Aspects Applications CP-TRIPLE

168 169 170 170

5.5 Polarization Modulated ENDOR

170

5.5.1 Basic Idea 5.5.2 Experimental Aspects 5.5.3 Applications

170 171 171

5.4.1 5.4.2 5.4.3 5.4.4

5.6 Orientation-Modulated ENDOR

173

5.7 Nuclear Zeeman Correlated ENDOR

173

5.8 Nuclear Spin Decoupling in ENDOR

174

5.9 Coherence Effects and Multiple Quantum Transitions

175

5.9.1 Coherence Effects 5.9.2 Multiple Quantum Transitions 6

161 162 163 163

175 175

Outlook

176

References

177

Contents

xii CHAPTER 5 Spin Trapping Free Radical Metabolites of Inorganic Chemicals

By R.P. Mason and C. Mottley

1 Introduction

185

1.1

Spin Trapping

185

1.2

Review Articles

186

2 Carbon Dioxide Anion Radical

186

2.1 Methanobacterium formicium

186

2.2

187

Carbon Tetrachloride-derived

3 Sulfur Dioxide-, Bisulfite- or Sulphite-Derived Radicals

188

4

191

Azidyl Radical

5 Hydrogen Atom

192

6

Oxygen-derived Radicals

193

6.1 Superoxide Anion Radicals

193

6.2 Hydroxyl Radical

193

7 Conclusion

195

References

195

CHAPTER 6 Inorganic and Organometallic Radicals

By Martyn C.R. Symons

1 Introduction 1.1

Books and Reviews

1.2 Techniques 2 Trapped and Solvated Electrons 2.1

Theoretical Advances

198 198 199

200 200

2.2 Electrons in Fluids

200

2.3 Electrons in Solids

201

3 Atoms, Atom Clusters and Atom - Ligand Complexes 3.1

3.2

202

Monatomic Centres

202

3.1.1 Atoms 3.1.2 Cations 3.1.3 Anions

202 205 207

Metal Clusters

3.3 Metal Atoms and Ligands

208 209

...

Contents

Xlll

3.3.1 4

Metal Carbonyls

2 10

Diatomic Radicals (AB)

213

4.1

Introduction

213

4.2

Di-Metal Centres

213

4.3

Mono-Metal Centres

213

4.4

Non-Metal Centres

214

Triatomic Radicals (AB ) and Related Species 2

215

6 Tetraatomic Radicals (AB3 ) and Related Species

218

5

7

Pentaatomic Radicals (AB ) and Related Species including Higher Coordinated Spe2ies 7.1

AH 4 Species

219

7.2

A0

2 20

4

Species

8 Other Radicals

222

8.1

Some Boron-containing Radicals

222

8.2

Nitrogen Derivatives

222

9 Radicals in Inorganic Materials

224

9.1

Jntroduction

224

9.2

Paramagnetic Centres in Oxides

224

9.3

Silicon and Germanium

225

9.4

I1

V I Compounds

226

9.5

Minerals and Related Materials

-

10 The Use of Spin-Traps

227 228

10.1 lntroduction

228

10.2

230

Pure Metal Carbonyls

11 Transition Metal Carbonyls and Related Species

2 30

11.1

Introduction

230

11.2

Pure Metal Carbonyls

230

11.3 Metal Carbonyls and Other Ligands

12

219

Radicals in the Gas Phase

232 233

12.1

Introduction

233

12.2

Doublet-State Radicals: Structure and Mechanism

233

Contents

xiv

234

12.3 Triplet-State Species

235

References

240

AUTHOR INDEX

Summarised Contents of Volume 10A

CHAPTER 1 Organic Radicals in Solution B y B.J.

Tabner

1) Introduction; 2) Carbon-centred Radicals; 3) Nitrogen-centred Radicals; 4 ) Oxygen-centred Radicals; 5) Nitroxides; 6 ) Sulphur-centred Radicals; 7 ) Radical Cations; 8) Radical Anions; 9 ) CIDEP : References

.

CHAPTER 2 Organic Radicals in Solids B y T.J. Kemp

1) Introduction and Bibliography; 2) Technical, Analytical and Theoretical Developments; 3 ) Spectroscopic Aspects; 4 ) Mechanistic Studies; 5 ) Molecules of Biological Interest; 6 ) Radicals at Surfaces and Semi-conductors: References. CHAPTER 3 Triplets and Biradicals By A . Hudson

1) Introduction; 2) Triplets and Radical Pairs in Fluid Solution; 3) Ground and Thermally Excited Triplets, Quintets and Nonets; 4 ) Photoexcited Triplets; 5) Photoexcited Biomolecules; 6) Excitons and Energy Migration; References. CHAPTER 4

Applications of ESR in Polymer Chemistry By D.J.T.

H i l l , J.H.

O ' D o n n e l l , and P.J.

Pomery

1) Introduction; 2) Polymer Degradation; 3) Polymerization; 4 ) Polymer Structure; References.

CHAPTER 5 Spin Labels: Biological Systems B y Ching-San Lai

1) Introduction; 2 ) Proteins; 3 ) Nucleic Acids; 4 ) Properties of Phospholipid Bilayers; 5) Lipid - Protein Interaction; 6) Membrane Fluidity of Cells; 7 ) Modification of Membrane Functions by Drugs; 8) Immunology; 9) Miscellaneous; 10) Synthesis; References.

Contents CHAPTER 6 Free Radical Studies in Biology and Medicine By N.J.F.

Dodd

1) Introduction; 2) Tissues; 3) Radiation Effects in Biological Molecules; 4 ) Radical Reactions of Drugs and Toxic Chemicals; 5) Enzymes; 6) Oxygen Radicals; References. CHAPTER 7 Loop-Gap Resonators By James S . Hyde and W . F r o n c i s z

1) Introduction; 2) Technical Background; 3) Multifrequency ESR; 4 ) Phase Noise and Dispersion; 5) Double Resonance; 6) Pulse ESR; 7) Other Loop-Gap Resonators; References. AUTHOR INDEX

Ex

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And the increased versatility comes from the rangr of software packages that integrate the data system with the same choice of magnets. bridges. cavities. power supplies and optional plug-ins that have made the E R / 2 0 0 thc industrv standard.

Get the facts on how to expand your EPR capabilities. For documentation on the new E R / 3 0 0 Series or a discussion with a technical representativr, write or call: Dr. Arthur H. Heiss. IBM Instruments. Inc., Orchard Park, PO. Box 3332. Danbury, CT 06810, ( 2 0 3 ) 796-2454. Outside the U.S., please contact your Brrrkcr-Sprctrospin representative

1 Spin-Spin Interactions in Weakly Interacting Dimers BY C. P. KEIJZERS 1 Introduction

As such, the subject of "Spin-Spin Interactions" has not beenthe subject of discussion in this series. Under different titles, such as "Transition Metal Ions", "Triplet Biradicals" or "Inorganic and Organometallic Radicals", various theoretical and experimental results have been discussed that are related to this subject (see, for instance, reference 1) but an integrated discussion has not been provided. In the past years, several groups have applied themselves specifically to the study of various aspects of the "exchange" phenomenon in order to obtain a better understanding of the physical interactions that are underlying the various terms in the effective spin-Hamiltonian with which the EPR spectra of systems with spinspin interactions are described. An understanding of the magnetic exchange interactions propagated by multi-atom bridges could give insight into, for instance, the pathways of electron transfer in biological electron transport chains. It could also be used as a guideline for the preparation of new and interesting polymetallic complexes or one- and two-dimensional magnetic exchange systems with magnetic properties that can be predicted, both in nature and in magnitude. It is not the intention of this contribution to be an all inclusive review of spin-spin interaction studies, this would be impossible in view of the breadth of the field and the vast literature. Instead, the subject is limited to the spin-Hamiltonian

which is applied for the description of magnetic exchange in weakly interacting dimers or other discrete (transition) metal complexes. A review is given of the fundamental theory that is necessary for the interpretation of the Hamiltonian ( 1 ) and selected papers from the literature are cited in which these interactions are either calculated or experimentally determined. For the experimental work, the attention is focussed mainly to EPR which means that various [For. r e f e r e n c e s see p a g e 35 1

2

Electron Spin Resonance

other techniques which are relevant to the subject (like for instance susceptibility, NMR, Mbsbauer, optical spectroscopy) are not discussed. Also lineshape and linewidth studies in (low dimensional) magnetic systems are not discussed. This extensive, complicated but very interesting subject would warrant a separate contribution in this series. For the time being, we refer to some and especially also to the extensive work of Soos (for instance references 5 and 6 ) . Many revie~s~'~'' , textbookk0'11'12r13r14and conference proceedings15 are available that have a bearing on the subject of this contribution. Especially the last one will be referenced often: it contains contributions covering a wide range of experimental and theoretical topics in this field. 2

Spin-Hamiltonian

The spin-Hamiltonian (1) is the usual Hamiltonian for the ions in zero magnetic description of the interaction of two S = 1 2 field. The tensor can be decomposed into its trace, a symmetric tensor and an antisymmetrical tensor:

(One has to pay attention when "J"-values from different authors are to be compared, because €or the isotropic part also the formulations + + + + -JS1.S2 and +JS1.S2 are used). The effect of J is a separation of the four spin-functions into a singlet and a triplet. Es splits the triplet into a doublet and a singlet (in case of axial symmetry) or -b into three singlets. d, finally, mixes the singlet with all three triplet functions. The essential requirement for the antisymmetric + + + term, d.SlxS2 is the absence of a centre of symmetry between the + + magnetic sites containing S1 and S 2 . If an inversion centre would -+ + exist, S 1 and S2 would interchange under the inversion operation and would change its sign. Since the Hamilton operator must be z1X$2 invariant for any symmetry operation of the system, this means that "d must change sign as well. Hence, 3 = 0. In the principal axes system of Es and in the basis of the eigenfunctions of the Hamiltonian matrix is:

ns,

1: Spin-Spin Interactions

3 ITx


. If the Hamiltonian is defined

Jc

=

h(1) + h(2)

+

hint

(8)

where h(i) are the one-electron Hamiltonians, including the intramonomer interactions hA(l) and hB(2) (of which v A and ( P B are eigenfunctions) and the interactions with the nuclei of the "other" 2 monomer, and hint is 3- , then the energies of J1 S I T are: '12

where

and where it is assumed that the monomers A and B are identical. The resulting singlet-triplet splitting is 25

=

(1 - SiB)-l 12jAB

+ 4hABSAB

-

4hmSiB

- 2kAB S2 AB' (11)

This localized method, but with orthogonalized orbitals, was used 45 by Anderson 2 7 f 2 a r 2 9 and by Hay, Thibeault and Hoffmann . Essentially they followed the same procedure but Anderson applied it €or an infinite lattice whereas Hay et al. calculated J for a dimer. Kahn43 rephrased the Anderson approach for the interaction between two identical single-ion doublet states. The first step is to calculate the two highest singly occupied (sometimes called magnetic) molecular orbitals Qb (bonding) and (antibonding) of the triplet state. In a weakly interacting dimer these MO's are approximately:

The next step is to determine the orthogonalized magnetic orbitals:

9

I : Spin-Spin Interactions 9i and q ;

have metal and ligand character, but they are essentially

centered on the treatment these construction of gonal localized resulting in:

A' =

monomers A and B , respectively (In the Anderson steps were 1. calculation of Bloch functions and 2. Wannier functions). Another way of obtaining ortho-orbitals is Lijwdin orthogonalization (9I = $9) ,

s

(A

+ SAB)/(l + ASAB)

After construction of the singlet and triplet functions (7) (with S A I B l= 0 1 ) the singlet-triplet separation is:

where j is as defined in (lo) but with orthogonal orbitals. Since this integral is positive, the exchange constant J becomes necessarily positive as well and a ferromagnetic coupling is the result. Since normally chemical bonds contain electron pairs of opposite spin, the antiferromagnetic contribution in (11) should prevail. A description with orthogonalized orbitals necessitates, therefore, a configuration interaction treatment in order to obtain an antiferromagnetic term. The reason is that triplet and singlet functions constructed with €unctions (13) or (14) contain charge transfer (ionic) configurations in the neutral dimer state. Explicit admixture of these configurations is then required in order to remove these components. This lowers the singlet state because no ionic triplets are possible. After a second order perturbation treatment , the exchange constant is45:

where

A somewhat different procedure starts with an excited singlet 1 state JZ (9i(l)9;1(2) + 9;(1)9;(2)) which is constructed from the

Electron Spin Resonance

10

two ionic configurations and which is lower in energy than these by an amount jA'B I . The resulting J-value differs in the denominator by 45 the absence of jA1B143'44. The integral l A l B 1 is often ignored but Girerd et a1.44 stressed its importance €or the understanding of the actual meaning of the antiferromagnetic contribution in the orthogonal magnetic orbital approach. In the approach with non-orthogonal magnetic orbitals, in principle a single-configuration representation is adequate for describing correctly the antiferro- or ferromagnetic coupling. This approach was further elaborated by Kahn and coworkers43 I 4 4 who considered also excited charge transfer configurations, apart from the ground configurations which would lead to the expression (11) for J. Again for a symmetrical dimer, J is found to be 2 =

(1

+

( h ~ ~ - h ~ S ~ ~ + l ~ ~ - k ~ ~ S ~ ~ - j ~ (17)

k

~

~

-

k

~

where J ( l ) is the first order value in equation ( 1 1 ) . The various integrals are defined in (10) and ( 1 6 ) .

-

3.2.2 Molecular Orbital Method. Hay et a1.45 started from the MO's $b and $a (equation (12)). They considered the following configurations:

. F -

d T

4 s1

+ s2

A

$a

-4-

b ' s3

When Jlb and $, are of different symmetry, S 3 will be of different symmetry from S1 and S 2 and in any case they assume that S 3 corresponds to an excited state much higher in energy. Therefore, the lowest singlet state, $ s , will be an approximately equal mixture of S1 and S 2 . The calculated exchange constant is:

This expression was simplified byassumingthathaa - hbb is small as compared to jab and by neglecting kaa - kbb and terms of order (A) 2 . The resulting expression is: 1 ab

I : Spin-Spin Interactions

2J = -kab

11

+ l(k 2

aa

+

kbb)

-

2 ( haa-hbb) 2Jab

The authors emphasize the key role of haa - hbb = E a - E~ as a measure of the singlet-triplet energy splitting ( E is ~ the energyof MO i (equation (12)). 3.2.3 The Above Approaches - which lead to the relations (161, (17) and (19) are at the same level of approximation: only the unpaired electrons are explicitly taken into account in the exchange interaction. The doubly occupied orbitals play a role only in so far as they influence the energy and the (de)localization of the unpaired electron €unctions. The validity of this approximation was tested by Wormer and van der A ~ o i r d after ~ ~ an all-electron calculation of the

exchange interaction in the 02-02dimer. Omission of the closed shells leads to a four-electron model that is much cheaper, of course, than the all-electron calculations. The J values for this model follow qualitatively the orientational (one O2 relative to the other) dependence of the all-electron J values; guantitatively they are quite inaccurate, however. Recently, also Charlot et al. 4 6 pointed out that a limited configuration interaction is not able to explain the exchange in a series of azido bridged copper dimers. A full CI calculation where all the core-levels of the azido bridge are included is necessary (and capable!) in order to understand the exchange in these systems. However, the calculations are necessarily limited to model systems. 3 . 3 Experimental Determinations of J. - The most powerful method of determining J is, of course, the measurement of the magnetic susceptibility. Excellent books have been written which discuss the large range of experimental techniques (see for instance reference 14). They are applied mainly to pure, (paralmagnetic compounds. For structures with isolated dimers (inter-dimer exchange negligible) they can yield, n e v e r t h e l e s s , i n f o r m a t i o n about the intra-dimer exchange interaction. However, magnetic susceptibility and magnetization data for one triplet state or for two doublet states do not differ much in the low-temperature range, and complications in interpretation arise from low-lying singlet states, inter-dimer interactions and zero field splittings. The most useful collabora47 tive evidence comes from EPR studies .

With

of concentrated paramagnetic systems, information

Electron Spin Resonance

12

about the exchange interactions can be obtained via the analysis of the lineshape and the linewidth as a function of the orientation of the magnetic field. Especially the dimension of the exchange interaction influences the relaxation €unction. However, as stated in the introduction, we will not go into details and refer to some reviews2’3,4. 3.3.1 Merging effect. - Another method of measuring J with EPR is the so called “merging effect”. If a concentrated paramagnetic crystal contains more than one magnetically inequivalent sites then the spectrum will show one line for each site when the magnetic field is not along a crystallographic symmetry direction, provided that the exchange is small enough as compared to the difference between the resonance frequencies of the sites (hyperfine structure is generally not resolved in EPR spectra of magnetically condensed compounds). In these directions, the linewidths are much larger than along the symmetry directions due to the exchange between the differently oriented sites. One way of obtaining J from these spectra is by fitting them to the generalized Bloch equations. In this manner J values were found for tropylium bis(l,2-dicyanoethylene dithiolato)nickelate (111)48, for [ Cu2 (dien)2C12 ] (C104) (dien = diethylaminetriamine)4g,and for three Cu2C18 dimers which are crystallized in layers which layers are shielded from each other by bulky groups5’. In all systems the exchange constant is temperature dependent. Possible causes for the increase with decreasing temperature were alreay mentioned in previous literature and some are cited in the first two papers where J does increase with decreasing temperature. The decrease of J with decreasing temperature in the third paper is ascribed to a decrease of the overlap of the atomic orbitals in the superexchange pathway because of a decrease of the thermal lattice vibrations. The second way to obtain J from spectra of crystals with more than one site, is from the frequency dependence of the linewidth, in case IJI 2 hv, where v is the microwave frequency. In these cases, the spectrum shows a single line but its width depends on the microwave frequency. This effect was used, for instance, for “(CH 3 4 ] 2Cu(mntI2 (mnt = maleonitriledithiolato)51’52 and for the determination of the exchange coupling between magnetically inequivalent ions in adjacent layers in the quasi-two-dimensional salts of (CnH2n+lNH ) CuClq ( n = 1,2,3)5 3 . 3 2

3.3.2 Susceptibility with EPR.

-

Another way to measure the singlet-

triplet separation is via the temperature dependence of the EPR

1 : Spin-Spin Interactions

13

intensity. If all interactions (Zeeman, hyperfine, zero-field splitting) are small as compared with J, the well known BleaneyBowers54 equation can be applied for dimers of spin-doublets:

NgLui

’M =

2

kT 3+exp(-2J/kT)

+

This procedure was applied by Richardson and K r e i l i ~ kfor ~ ~ the determination of the interaction between bis(hexafluoroacety1acetonato) copper(I1) with a series of pyridylnitronyl nitroxide radicals. However, measuring the area of an EPR signal by double integration causes always (large) errors in the estimate of the exchange interaction. For small J-values, the intensities have to be computed by calculating the energies and transition probabilities with the complete spin-Hamiltonian and taking into account the Boltzmann distribution. This approach was followed, for instance, by Hefni et al. in order to determine the exchange within a fluoride bridged copper (11) dimer (J = -0.375 f 0.125 cm-l)56. If the rnonoiners have higher spin-states, different equations must be used for the magnetic susceptibility (see for instance reference 57). 3.3.3 Singlet-Triplet Mixing. - If J is very small (on the order of 0.001 cm-’) mixing of singlet and triplet functions may be observed. Possible mechanisms are the Zeeman i n t e r a ~ t i o n (provided ~~ that the two monomers have a different g-value/tensor) and the anisotropic metal or ligand hyperfine i n t e r a c t i ~ n r ~ 6 2~ . ~ If~mixing ~ ~ ~ ~ ~ ~ ’ occurs, the EPR spectra can be described in terms of AB spincoupling theory63t64f65.From the position and the intensity of the singlet-triplet transitions, the J-value can be derived. Even if singlet-triplet transitions cannot be observed, information about J58,59 and about the tensors of the individual monomers58 can be obtained from shifts in the fine- and hyperfine structure splittings. These transitions were observed, for instance, in powdered samples of Me -dien-bridged copper dimers (Me5-dien = 1,1,4,7,7-pentamethyl5 diethylenetriamine)66 where a value of 0.1 cm-l was derived for J. A much better resolution was obtained in single crystal measurements of diamagnetic host crystals heavily doped with paramagnetic, and accidentally Jahn-Teller active , copper molecules67 6 8 16’ 70. The detailed X- and Q-band spectra show signals of Jahn-Teller monomers and of various, ferrodistortive and a n t i f e r r o d i s t o r t i v e , d i m e r s . For the antiferrodistortive dimers of hexakis pyridine-N-oxide copper(II), the exchange constant varies from 0.0224 to 0.0329 cm-’ €or

14

Electron Spin Resonance

different counter-ions6" 69' 70. These values were obtained by simulating the X- and Q-band spectra with the full spin-Hamiltonian, including Zeeman-, nuclear hyperfine, nuclear quadrupole, zero-fieldand exchange interactions. The trend can be qualitatively understood from the crystal structures and the magnitude of the Jahn-Teller distortion71. In a long series of articles (as examples numbers 27,31,39 and 44 are mentioned in the reference G . Eaton and S. Eaton and coworkers published J-values of a large variety of spinlabeled metal complexes. The series includes tens of different spinlabels and various S = 1 (VO(1V), Cu(I1) , Ag(I1)) and S > (Mn(I1)) 2 metal ions. The exchange parameters are obtained by computersimulation of the liquid solution or single crystal spectra, including the Zeeman interactions and the metal and nitroxyl-nitrogen nuclear hyperfine interactions. The J-values are used to monitor changes in electron spin delocalization due to changes in the coordinated metal, in the metal-nitroxyl linkages and in the ligand conformation.

$

3.3.4 ENDOR. - Although we restrict ourselves almost entirely to metal-metal dimers, an exciting paper by Kirste, KrGger and Kurreck should be m e n t i ~ n e d ~These ~. authors measured the nitrogen hyperfine splitting, aN, in a series of mixed galvinoxyl/nitroxide biradicals and found from the AB-type of ESR spectra that J varies from I JI < I aNI to I J [ > I aNI , depending on the length of the connecting bridge. The splittings in the ESR spectra do not represent the hyperfine coupling constants when J has about the same magnitude. The ENDOR technique, however, allows direct measurement of these couplings. Moreover, the sign of the exchange constant can be determined with ENDOR whereas the sign determination with ESR is restricted to favorable cases and requires an elaborate analysis77 and susceptibility measurements are very difficult (if not impossible) if IJI Q kT. For these systems the coupling is ferromagnetic. 3.3.5 Undiluted Compounds with Small Exchange. - Commonly when examining undiluted monomeric compounds, no hyperfine splittings are observed due to the neighbouring magnetic dipoles causing splittings to such an extent that the hyperfine structure is lost and a broadened single line results. On the other hand, if there is strong electronic exchange over a large (infinite) number of paramagnetic centres, the hyperfine structure will still be lost, but now line

I: Spin-Spin Interactions

15

narrowing rather than broadening will result (both situations will not be discussed in this contribution). A less common situation is that in which the electronic exchange interaction between adjacent paramagnetic centres are comparable to, or slightly less than, the nuclear hyperfine interaction. This situation has been observed in cis-[ VO (pbd) 178 (pbd = 1-phenylbutane-1,3-dionate) where the molecules occur as pairs, in the one-dimensional exchange system [ N-nBu4 I,[ Cu(mnt) 17' (mnt = maleonitrile dithiolate) in the twodimensional exchange systems [VOX2(tmu)2]80 (X = Cl,Br, tmu = N,N,N' ,N'-tetramethylurea) and L[ VO(ma1onato)2H20 l.2H,081'82 (where L = N,N' -dimethylpiperazinediumgl or N ,N,N ' ,N ' -tetramethylethylenediaminediumg2) and in the system Na6Cu (P2071 2. 16H2062. The spectra in reference 79 were interpreted on the basis of a one-dimensional system in which the combined isotropic exchange and dipolar interactions were slightly smaller than the copper nuclear hyperfine interaction and were treated as perturbations upon the zeroth order hyperfine levels. Neighbouring copper sites in the chain are magnetically inequivalent (although crystallographically equivalent) if MI(i) # MI(i+l) and thus the chains may be broken down into 7 segments. This treatment was extended to the case of vanadium (I= $ I involved in exchange within a two-dimensional layer in referencesgo, In Na6Cu(P207)2.16H20 the spectra were simulated with ring models where the two terminal ions of an n-membered chain are connected by an imaginary dipolar interaction of the same magnitude. This serves to remove some extra lines. The exact magnitude of the exchange interaction could not be determined but it was inferred that it is quite small and probably antiferromagnetic. Similar spectra were observed in an undiluted crystal of [ MB+ 12[ Cu(mnt) 1 (MB+ = methylene blue cation)83. However, in this system the molecules are dimerized with a singlet-triplet separation (singlet-ground state) large enough to create a magnetically diluted crystal at low temperatures. The hyperfine splitting is resolved below 5 K. A small exchange between the triplets gives rise to satellite transitions which can be quantitatively accounted for.

'u2.

:-

-

3.3.6 Heteronuclear Dimers. - Buluggiu" derived a spin-Hamiltonian formalism €or a pair of exchange coupled paramagnetic ions, operating within the single S-multiplet of total spin and in which coupling among the different multiplets is taken into account by a second order perturbation approximation. The formalism is valid for 20 arbitrary S1 and S2. It is similar to the one by Scaringe et al. but the latter authors used a first order treatmerit (J very large).

16

Electron Spin Resonance

Although this paper is mentioned under this heading, the formalism of Buluggiu can be applied to homonuclear dimers as well, of course. We confine ourselves to a listing of references and the kind of dimers that were studied without going into many details: Ni-Co, NiCu (and paramagnetic Cu-diamagnetic Zn)85 ; Ni-Cu and Ni-Co (one mono87 mer has orbital degeneracy, the other one not)86; Ni-Co and Ni-Cu (the pure Ni lattice acts as the host lattice), both dimers can be described with a S = spin-Hamiltonian, the exchange is antiferro23 magnetic: Mn-Cu and Ni-Cu ; Mn-Cu22r88 (the pure copper lattice acts as a diamagnetic host lattice because of the strong antiferromagnetic coupling within the Cu-Cu dimers, compare also references 21,83,89); Cu-NigO (A simple method is applied for the calculation of the g and hyperfine tensor of the ground Kramers doublet of Cu(I1)Ni(I1) coupled pairs as a function of the spin-Hamiltonian parameters of the two isolated ions and of the exchange parameter. The model is applied to reinterpret the spectra of Cu(I1)-Ni(I1) pairs): Fe-Cugl (Two different models are used to calculate the effect of the quasi degenerate ground state of low spin Fe(II1) on the exchange interaction with an orbitally non-degenerate ion. In general, the four lowest levels are not grouped in a singlet and a triplet). 3.3.7 Ferromagnetic Coupling. - It is clear from equations (11) and (17) that ferromagnetic coupling can be obtained if the overlap between the "magnetic orbitals" of the monomer is really zero. This orthogonality can be strict, in case the two MO's belong to two different representations of the molecular symmetry group, or it can be accidental if they have the same symmetry but the overlap is zerog2. Accidental orthogonality was found in a series of squareplanar-based copper(I1) systems (coordinated in a macrocyclic ligand) where bridging occurred axially via one bridge. The resulting (super) exchange is still antiferromagnetic but very weak ( I JI < 1 cm-') . The reason is the orthogonality of the magnetic and the bridging orbitalsg3. Other examples are planar dimers with two bridges. In di-p-hydroxo copper(I1) dimers the coupling is ferromagnetic for bridging angles smaller than 97.5 0 g 4 . Also in di-p-chlorog2 95 copper (11) and nickel (11)92 9 6 9 7 dimers accidental orthogonality is found. Examples of strict orthogonality are heterodinuclear Cu-VO dimers where the coupling is very smallg8 or large (118 cm-l) and ferromagnetic99 .

'*

3.3.8 Cluster Systems. - Besides the dimeric systems, which are and

1: Spin-Spin Interactions

17

have been studied extensively and in great detail, more and more articles are appearing which report studies of small clusters or of infinite systems in which dimers can bedistinguished. Magnetic properties of trimeric copper complexes and possible modifications were summarized by Jothard et al.’”. A theory of EPR spectra of trinuclear antiferromagnetic clusters with near trigonal symmetry was described by Rakitin et al.lol. The theory is applied to interpret the spectra of trinuclear iron acetate. Again, without going into many details, we list some references and the type of system studied: CU-CU-CU’’~, a linear trimer with two C1 and one adenine bridge per Cu-Cu, the exchange mechanism is described in terms of the orbital pathways and the single-ion ground-state wave functions; CU-CU-CU’~~, a linear symmetric trimer; since no information is available on the g-tensors of the individual copper-ions the interpretation is difficult, the g-tensor is temperature dependent probably because of the (de)population of excited doublet and quartet states; C U ~ ~ ’a~ , triad without symmetry consisting of metal adducts of Schiff-base Cu(I1) complexes; CU~VO’’~, a tentative analysis of a powder spectrum, susceptibility shows that a doublet and a quartet are close in energy, one interaction between Cu and V is ferromagnetic and large (in correspondence with reference 99) , the other two interactions (Cu-Cu and Cu-V) are almost zero; C U ~ ’ ~a ~more , or less trigonal cluster with three bridging Schiff-base ligands (bridging via 0) and a central hydroxo group, the framework is similar to a structure with bridging pyraz~lates’’~, in EPR doublet and quartet transitions are observed; Cuio8 , consisting of two antiferromagnetically coupled inner ions and two outer ions with a much smaller coupling with the inner ones. Very recent studies are those on CdCuio9 and a comparison of three similar trimers Cu-Cu-Cu, CuZn-Cu and Cu-Ni-Cu with all atoms in the bivalent state”’. A nice example of the complementarity of the magnetic and the EPR techniques was found by Journaux et al. In a (CuNi) bis

’”.

heterobinuclear compound the susceptibility is dominated by the CuNi interaction, resulting in a ground state doublet and an excited quartet 35.4 cm-’ above it. The doublet-doublet interaction between the two Cu-Ni pairs gives rise to a triplet state which dominates the EPR spectrum. The mechanism of the exchange interaction in a linear chain Cu(11)-imidazolate compound was studied by Bencini et al. ’12. The same authors published a study of a linear chain of weakly coupled copper-nitroxyl radical (TEMPOL) pairs’ 1 3 . The Cu-ON coupling is ferromagnetic. In a subsequent paper the ratio between the intra-

Electron Spin Resonance

18

pair and inter-pair exchange was found to be J J' = -0.004114. 3.3.9 Paramagnetic Host Crystals. - The theory of exchange interactions in non-Kramers' paramagnetic host crystals with iron-group impurities has been developed by Moriya and Obata115, and St. John and Myers117. A review of the g-shift which is caused by the interaction between a lattice of triplet molecules with a large positive zerofield splitting and a doublet molecule was given by Mehran and Stevens118. The g-values of the doublet are increased if the exchange coupling is ferromagnetic, they are decreased if the coupling is antiferromagnetic. We do not go into details and cannot be complete on this subject. However, we want to draw special attention for a beautiful thesis119 that has not been published in the open literature, as far as we know. The author (Hulliger) studied nickelocene doped with various spin-doublet sandwich compounds like bis-benzene-vanadium, cyclopentadienyl-cycloheptat r i e n y l - v a n a d i u m , c o b a l t o c e n e , vanadocene and bis-benzene-vanadium in diamagnetically diluted nickelocene. Other studies of this type were carried out in the FeSiF6.6H 0 host lattice, where the exchange coupling between Mn2+-Fe2+12 , Ni2+-F'e2+l2l and Cu2+-Fe2+123 was studied via the g-shift. In the paramagnetic CoNbOF5.6H20 host lattice, the broadening of the sharp Mn(I1) lines below 120 K was interpreted in terms of the random modulation of the dipolar interaction between the guest Mn2+ ions and the host Co2' ions by the fast spin-lattice relaxation (T1,co) of the latter124. An estimate of T l r C owas made from the linewidth of Mn(I1). The temperature dependence of the Mn EPR parameters indicates a structural phase transition, in accordance with the structural aspects of the isomorphous CoSiF6.6H20125.

8

3 . 4 Calculation of J. - Although a little bit out of the scope of an "EPR Specialists Report'' we want to pay some attention to this subject because a real understanding of the experimentally determined exchange constants should be obtained from theoretical calculations. The problem with these calculations is, however, that configuration interaction treatments are necessary for a quantitatively correct result and for extended, many-electron,systems such a treatment is not completely possible. In an all-electron ab-initio calculation on the 02-O2 dimer the monomer €unctions were calculated at the level of the Hartree-Fock limit (no CI) 35'36. Subsequently, the dimer €unctions were

I : Spin-Spin Interactions

19

constructed by spin-projection of the fully antisymmetrized product of the two 1 ' , monomer functions in order to represent the three possible spin states of the 02-O2 dimer ( S = 0,1,2). The nonorthogonality problem was handled in a second-quantized hole-particle formalism. The interaction energy €or each of these states is obtained by taking the expectation values of the full dimer Hamiltonian over these wavefunctions and subtracting the O2 monomer energies. Broer and Maaskant126, on the other hand, did a direct CI calculation but restricted themselves to simple model systems €or planar dichloroand difluoro-bridged Cu(I1) dimers. In these models the terminating ligands are replaced by a negative point charge or a fluorine ion. The singlet-triplet splitting is found to be strongly dependent on the angle 9 between the copper ions and the bridging ligand and shows a maximum for IP between 900 and 1000. In this region, the triplet state is lowest. The magnitude of the splittingdependsvery much on the level of configuration interaction treatment but the v -dependence is qualitatively the same for the two levels which they used and agrees with the dependence as calculated with the Anderson model. Although no attempt was made to compare the calculated splittings with observed splittings, it was noted that the v-dependence is the same as observed for series of doubly bridged Cu dimers47'127r128. De Loth, Daudey and coworkers129'130r131 developed an ab-initio second order perturbation theory for the calculation of J, that can be applied to extended systems like, €or instance, copper dimers. However, for the ligand atoms the orbital set was necessarily restricted to a minimal basis. The computation scheme has been applied to a variety of problems. One of them is the "HatfieldHodgson relationship". These authors found for 9 out of 11 known bis(p-hydroxo)-bridged copper(I1) complexes a linear relationship between J and the Cu-0-Cu angle, @ 47,94,132. EST = -74.53 (cm-l deg-l)4

+

7270 Cm-l

(The compounds that do not obey this rule have two perchlorato bridges in addition to the two hydroxo bridges). The overall agreement between the calculated singlet-triplet splitting by Daudey et al.130 and the experimental one for a real complex is fair. The variation in the calculated J, which is obtained by varying only 4 (and the Cu-Cu distance accordingly),and fixing all other structural parameters, is a little smaller than predicted by the HatfieldHodgson relation. The result of the calculation is the understanding that the variation of J with

is caused by a variation in the

Electron Spin Resonance

20

kinetic (Anderson nomenclature) or antiferromagnetic exchange (equation (11)). The direct exchange (or potential exchange in Anderson's nomenclature), which is the ferromagnetic contribution (equation (11)) has a minor effect on the variation of J but dominates for 4 angles larger than 950. This conclusion is exactly the one of Hay et a1.45 which authors gave a rationalization of this relationship with a semi-empirical extended H k k e l method, based upon the variation of the splitting between the bonding and antibonding singly occupied orbitals (paragraph 3.3.2).

-

The extended Hcckel model was also used by Leuenberger and Giide1133. They found good agreement with the experimental J-values in the series Cr2Xi- ( X = Cl,Br,I). The observed trends in the series M2C1z- (M = Ti,V,Cr) were reproduced semiquantitatively. The model was not able to account for the large increase of the exchange 35couplingbetweenCr2C19 and V2C19 (same d-electron Configuration). A very interesting and complicated class of dimers are those with azido-bridges. Azido can bridge either in a symmetrical end-toend (SEE) fashion or in an end-on (EO) fashion134 or in an asymmetrical end-to-end (AEE) fashion135. Many studies of crystal structures and magnetic properties of these dimers have appeared. It turns out that the SEE bridged dimers are antiferromagnetic, the EO dimers are ferromagnetic and the AEE ones have almost no interaction (or the ferro- and antiferromagnetic contributions cancel, which is different from having no interaction!47). Papers proposing the mechanism of the interaction have appeared previously136 but only recently a full account was given of the properties of all three types of bridges 46 . Especially the role of the NJ orbitals must be taken into account which means that one has to go beyond the approximation of the (two electron) models which are normally used for the quantitative description of exchange-coupled dimers. In an ab-initio calculation of a model system (two Be+ ions bridged SEE or EO by N; bridges) quantitative agreement is found with the observed couplings but only after an extended configuration interaction. Whether this means that the simple models, which can be used to predict magnetic properties, are out of date is not clear yet. Perhaps, the azido is an exception among the bridges. 4 The Zero Field Splitting Tensor

There are two main contributions to the symmetrical part ofthe

1: Spin-Spin Interactions

21

spin-spin interaction tensor (equation (2)): the dipole-dipole interaction which is a through space, direct interaction between themagnetic moments of the two ions monomers). It is a first order contribution, i.e. it acts between the three functions of the (ground state) triplet, and generally it is the most important contribution to the zero field splitting tensor; the spin-orbit contribution which is sometimes also called "pseudo-dipolar interaction" or "anisotropic exchange" (Sometimes (a) + (b) are defined as the anisotropic exchange. Although this definition is formally correct, if judged from the spin Hamiltonian (2), it is absolutely incorrect if judged from the physical mechanisms). For this contribution to be non-zero, it is necessary that an exchange interaction is operative in the excited states of the system, i.e. there must be a singlettriplet separation in the excited states. If this condition is fulfilled, the spin-orbit coupling gives a second order contribution to the zero field splitting tensor. two contributions (a) and (b) cannot be distinguished experimentally. Only a theoretical calculation of one of them (mostly the dipole-dipole interaction although the accuracy of such a calculation is questionable) yields the magnitude of the other one by (tensor) subtraction from the experimentally determined tensor. 4.1 Analytical Expressions for

Eq.

4.1.1 The Dipole-Diple Interaction -+ = 3 %gi.Si of two ions is pi =

zi

-

between the magnetic moments,

(GT

where is the g-tensor of ion i is its transpose) and ;12 is the distance vector between the two magnetic moments. Comparing (21) with the spin-Hamiltonian S1.Es.S2 yields €or the elements of the kstensor: =T = =T -+ =T -+ (g1.g2)a8 - 3(g1.r12)a(g2'r12) 6 2 I dJT > (22) D p = UB < 3 5 r12 r12 where I$, > is the orbital part of the triplet functions. If the gtensors are isotropic, expression ( 2 2 ) reduces to:

Electron Spin Resonance

22

(23)

The computation of these tensor elements necessitates the accurate knowledge of the orbital function However, if the distance between the two ions is large and if delocalization of the unpaired electrons can be neglected, the dipole-dipole interaction is =proximated with a point charge calculation:

12 If the coordinate system is defined such that the z-axis is along + the inter-ionic vector, r12, the result is an axial and traceless + tensor with the axial axis along r12:

12 4.1.2 The Spin-Orbit Contribution - is a second order effect thatis non-zero only if an isotropic exchange interaction is operative in the excited states. The effect was already mentioned by Bleaney and and it has been discussed in many reviews and textbooks12'13'139r140. Following Morya, who was the first to derive an analytical expression of the effect by expanding Anderson's formalism with spin-orbit coupling, the effect is commonly described as a third order perturbation by the combined interaction of spinorbit coupling and isotropic exchange. However, we prefer a description as a second order perturbation of the spin-orbit coupling after the exact calculation of the effect of the isotropic exchange. The advantage of this procedure is that one is not tempted to describe the singlet-triplet-splitting in the excited states with a single two-electron exchange integral. As was discussed in section 3 for the ground state, only the ferromagnetic term in the singlet-triplet splitting is a "simple" exchange integral, the antiferromagnetic term contains one- and two-electron integrals. This was correctly taken into account by Morya but in recent literature

I : Spin-Spin Interactions

23

there is a tendency to neglect the antiferromagnetic term in the excited state splittings. We start from ground and excited states in which the exchange interactions have been included to the highest accuracy. The resulting singlet-triplet splittings are defined ZJ,, where Jn is the effective exchange constant in the spin-Hamiltonian: En + 2Jn

S

>

=

>ISo

>

In this diagram, the Ji are positive but the derivation is valid for any value of Ji. It is assumed that the splitting between the > are the orbital triplet functions may be neglected.l+g > and parts of the singlet and triplet functions,ISO>and ITi > are the spin functions ( 4 ) . As in the derivations of the expressions for the isotropic exchange, the model is an effective two-electron model: the core electrons are assumed to influence only the energies and delocalization (In an all-electron model, the separation of the wave-functions into an orbital and a spin part would be impossible). The perturbation is written:

where gi is the one-electron spin-orbit coupling constant. The second order energy correction for the ground-triplet state is:

c c < *Ti 1 5 , i l Z , +5,f2Z2

> < $1

5 ,I,-8,+c2f2 .b,

> (28)

E(2) = n

E~-E:

where the summation over u runs over all (four) spin states of the excited states n. Separation of orbital and spin matrixelements, + + using the Hermiticity of 1 and s operators, the permutation symmetry of the singlet and triplet functions and the fact that matrixelements +. of 1 are purely imaginary for orbitally non-degenerate states, yields for E"):

Electron Spin Resonance

24

With the closure relation

and the following matrix relation which is easily proven for S1 = 1 s2 = -2'

+

6 a s 6 ~ i ~ j

E ( ~ )is rewritten:

- + -

Comparison with the spin-Hamiltonian elements :

Sl.6

.z2

yields for the tensor

In (33) an isotropic contribution is omitted:

therefore, the spin-orbit coupling gives tion to the singlet-triplet splitting in changing the energy of all three triplet provided that Jn # 0 . The last condition

in second order a contributhe ground state by €unctions simultaneously, holds also for the

1: Spin-Spin Interactions

25

anisotropic contribution: it vanishes if there is no singlet-triplet splitting in the excited states. Assuming Jn En, equation (35) may be rewritten:

which expression is equal to the expression obtaiced with a third order perturbation treatmentl4l. It should be stressed that Jn is not a simple exchange integral142 but instead (half) the singlettriplet splitting in the excited state. dd 4.2 Experimental. - As stated before, the dipole-dipole, (Ds ),and the spin-orbit, (DZo), contributions cannot be separated experimentally. A very nice example that shows the influence of the latter contribution is the case of Cu(I1)-Cu(I1) and Ag(I1)-Ag(I1) dimers in [ Zn (dtc) I2l7'l8 (dtc = N,N-di-ethyldithiocarbamate or N,N-diisopropyldithiocarbamate). The dimers are obtained by growing crystals with about 25% of Ag(dtcI2 or Cu(dtcI2. The EPR spectra show signals of "monomers" (i.e. Ag(I1)-Zn(I1) or Cu(I1)-Zn(I1) dimers) and dimers. The experiments show that the g-tensors of the triplet species are almost equal to those of the doublet species, and that the metal hyperfine tensors of the triplet species are about half those of the doublet species. This proves that the electronic structure of the M-M (M = Cu,Ag) dimers differs only slightly from that of the M-Zn pairs. The Cu and Ag dimers are expected to have similar geometrical structures with the Ag-Ag distance larger than the Cu-Cu distance. Since the delocalization in the Ag dimers is stronger than in the Cu dimers112, it is reasonable to expect that the dipolar interaction in the Cu dimer is larger than or equal to this interaction in the Ag dimer. However, the experiment shows that the D-tensor of the Ag dimer isstrikingly different: ~

[ Ag (dtc)

[ CU (dtc)2 12

exp. Ds

7184 f f

92 92

calculated Dzd DEo

-208

-188

+

90

+118

exp . Ds

l2

calculated DZd DZo

-25

T684

-659

-188

-514

+ 81

+11

+155

+317

+

81

+218

+lo7

+14

+529

+342

+lo7

+296

26

Electron Spin Resonance

In order to get an understanding of the difference, the Ds-tensors were calculated in the following way: the dipolarcontribution was computed with equation (23) from spin-densities as obtained from extended Hcckel calculations and they were supposed to be equal for both dimers. The spin-orbit interaction of the Cu-dimer was calculated with equation ( 3 5 1 , using again the results (energies and MO’s) of the extended Hiickel calculation andan average singlettriplet splitting in the excited states (assumed tobeequal to the ground-state value). The results are listed in the table, the agreement with the experiment is fair and certainly good enough to make a decision about the sign of the tensor elements. For the Ag dimer, it was found that the singlet-triplet splitting is larger than in the C ~ - d i m e r l ~and ~ , a factor of 4 was assumed for this calculation. With the much larger spin-orbit parameter (1830 cm-l for Ag, 828 cm-l for Cu) the values in the table were obtained. Again, the agreement with the experiment is very satisfactory. The above example seems to be a unique case where the influence of the spin-orbit coupling is so apparent. Mostly, the electronic structure of the dimer cannot be compared with that of a monomer (or of a dimer with the paramagnetic and a diamagnetic monomer) because a monomer is not available. Also the variation in DEo can normally not be accomplished because a variation in metal ions is notpossible without severe structural changes. In these cases, the spin-orbit contribution is very difficult to assess. However, one gets an indication of the presence of a sizable EEo term from the directions of the principal axes of the Es-tensor: if the unpaired electrons tensor would be completely localized on the metal atoms, the would be axial and directed along the metal-metal axis (paragraph 4.1.1). Large deviations from axiallity and from this direction are therefore used as indications for large spin-orbit contributions. These deviations were found in a very large number of metal-metal dimers. Mostly the magnitude of the zero field splitting tensor deviates severely from the values which are expected on the basis of the point-dipole approximation. Furthermore, in almost all systems the largest c o m p o n e n t o f E s i s n o t o n l y n o t oriented along the metalmetal vector (as expected in the point charge model) but, instead, it is perpendicular to it! This was observed for a large variety of bridges: C1144t145, F ~ o ~with , GH 146’147r148, 0 with pyridine-N-oxide and water8’, OCNi4’, end-on azido15’, end-to-end azido135’151, N-N-N of benzotria~olate’~~ , N-N of 3 ,5-bis (pyridin-2-yl)153, NO of TEMPOL

d:E

1: Spin-Spin Interactions

27

.

(not a metal-metal dimer but showing the same ZFS-tensor behaviourf13 Also the ZFS-tensor in the [Ag(dtc)Z12 dimer deviates very much from the Ag-Ag direction”. As pointed out above, it is not possible to separate the dipoledipole and the spin-orbit contributions unless one is able to calculate one of them. Since a calculation of the latter contribution necessitates the knowledge of the excited states, it seems much easier to calculate the dipole-dipole interaction. However, in order to do that one should have an accurate knowledge of the spindensity distribution in the dimer. If there is a sizable delocalization of the unpaired electrons into the ligands and, of much more importance, into the bridges, then it is not sufficient to calculate the dipole-dipole interaction in an approximation of localized point charges, even a model of several point charges (one €or each atom, the charge being the calculated spin density) is in principle not suffient. Instead, one should compute all one-centre (metal, bridging atoms and non-bridging ligands) and two-centre (metal-metal but certainly also metal-bridging atoms) integrals over the electronic wavefunctions (three- and four-centre integrals are expected to be small). This procedure was followed for a fluorinebridged copper d i ~ n e r ~but ~ , in a rather approximate way: the spindensities were calculated with an extended HGckel molecular orbital method. The two-centre integrals were obtained by numerical integration. As compared to the localized point-charge approximation the longest principle axis does hardly change: it remains along the Cu-Cu vector and its principle value does not change as well. However, the asymmetry in the plane perpendicular to the copper-copper vector increases very much. This means that, in principle, a large value perpendicular to the metal-metal direction could be obtained from the dipole-dipole contribution. This calculation is, however, too approximate to draw definite conclusions. Most other authors use the point-charge approximation for the calculation of This tensor is then subtracted from the experimental. one and the difference is attributed to the influence of the spin-orbit coupling (anisotropic exchange)89,135,145-153. The danger of this procedure is obvious and drawing definite conclusions from these results seems to be rather risky as long as better calculations of the dipole-dipole contribution are not available. Since there is a relation between the bridging angle and the isotropic exchange constant in OH-bridged dimers (paragraph 3.4) , Banci et al.148 investigated whether there is also a relation

zdd.

28

Electron Spin Resonance

between J and the spin-orbit contribution to the zero-field splitting tensor. It turns out that such a relation does not exist. + - +

Whereas J varies from - 1 8 0 to +245 cm-l (spin-Hamiltonian -2JS1.S2), DEo varies from - 0 . 8 4 6 , via -0.940 to -0.747 cm-l. Bencini et al. 1 4 6 r 1 4 9 did find a relation between EZo and the Cu-Cu distance in a series of oxygen bridged copper dimers. The pyridine-N-oxide and the methoxo bridges have the larger Cu-Cu distances and the smaller ES0 contribution. The systems with the smallest metal-metal distances and the largest spin-orbit contribution are the hydroxo and alkoxo bridged ones. The relationship was found to be DZo = 5.31(0.03)

-

1.57(0.01)r

(hence a linear relationship in r ) but an exponential relation is as good. Recently, Charlot et al. 154 tried to rationalize the spin-orbit part of the zero field splitting in the bis-hydroxo bridged copper dimers with a topological approach. From the results of an extended Hiickel calculation the exchange integrals j XY 1 xY and jxy ,x2-y2 were computed as a function of the bridging angle. The reasoning is that for bridging angles close to 90° the first integral stands €or the singlet-triplet splitting in the ground state (where a relation between the S-T splitting and the bridging angle was found, see paragraph 3 . 4 ) . The second integral is used in the formula (35), instead of (in our opinion) the singlet-triplet splitting in the excited state, Jn. Although €or both splittings the antiferromagnetic contribution was neglected, a fairly good agreement with the experimental trend in the DZo values was obtained. 5 Antisymmetric Exchange + +

-b

This term of the spin-Hamiltonian (2), d. S1xS2, was first proposed by D ~ i a l o s h i n s k ion ~ ~symmetry ~ arguments. (It is sometimes a l s o called "skew symmetric exchange"). As mentioned in section 2, the requirement €or this term to exist is the absence of an inversion centre. There are two contributions to this part of the spinspin interaction tensor: (a) the dipole-dipole interaction gives a first order contribution if the q-tensors of the two electrons are different, (b) the spin-orbit coupling gives a second order contribution.

1: Spin--Spin Interactions

29

+

5.1 Analytical Expressions for d. 5.1.1 The Dipole-Dipole Interaction

-

(equation (22)) can be written:

where dk are the direction cosines of :12. It is quite clear that D U B = D B u only when the dimer consists of similar ions and has an inversion centre. Otherwise this will yield a first order contri+ bution to d. 5.1.2 The Spin-Orbit Contribution - was derived by Morya together with the contributions to the zero field splitting tensorl3'' 14'. His approach was a perturbation treatment with localized, orthogonal (Wannier) functions and as perturbations the interaction between the unpaired electrons and the spin-orbit coupling. As in section 4 , we prefer as the starting point the singlet and triplet states. It is, therefore, assumed that the interaction between the electrons is taken care of, leading to singlet-triplet splittings in the ground state (2J and in the excited states (2Jn) (see diagram (26)). 0 Again, an effective two-electron model is used, neglecting the influences of the core-electrons. Provided that excited states are high in energy as compared to the magnitude of the spin-orbit coupling, the effect of this interaction can be computed by calculating and diagonalizing the four by + + + + four matrix of the operator < l l l . ~ l + 1. Here the zero field splitting arises from hyperfine and quadrupolar interactions, and two recent papers describe the observation and analysis of such spectra from the vanadyl ion in ammonium sulphate2' and in Tutton salt.22 In each case z.f .r. has provided accurate values for the hyperfine tensor and quadrupole terms and the significant finding is that there are differences between these and the earlier e.s.r. data. The differences were put down to errors in t.he orientation of a crystal in a field; measurement of the field; and in the approximations in using perturbation expressions for the resonant fields, factors which of course do not arise in z.f.r..

42

Electron Spin Resonance

Theory.- Several pseudotetrahedral copper(I1) compoundsI in particular the [CuCl4I2- ion, appear to have, in comparison to what is expected from their g shifts, anomalously low A, hyperfine values. Sharnoff has ascribed this to a significant contribution of metal p orbitals in the ground state; later workers have concluded that P and in particular K are substantially reduced in such compounds from their normal values; while Bencini et a l . have suggested that high covalency of the metal bonds is responsible for the discrepancy (see ref. 23 €or references). Hitchman however has recently pointed out that in fact there is no anomaly once the minor d-components (dx,, dyzI dx2-y2 for the 02d point group) are properly included.23 This may be done from a knowledge of a2 (from ligand hyperfine struct$ 2 and y 2 (from reasonable estimates); and E x 2 - y 2 , E x , , y z ure); (from electronic spectroscopy). These contributions are quite small for planar compounds but become rapidly large as distortion towards tetrahedral geometry causes the relevant excited states to drop in energy. It must also be remembered that formulae in which these excitation energies have been eliminated by inclusion of g shifts will be in error if a g contribution arises from the ligand. This is likely the case when strong covalency takes the unpaired electron on to a heavy ligand atom with its relatively high spin-orbit coupling constant. The way in which the d orbital contributions in low symmetries may be estimated has been discussed by Fogel. In D2,, symmetry, the mixing coefficients a l and b, for dX2-,2 and d,2 for the ground state 2 A g extended orbital are related simply to the ratio of the ionic radii in the x y plane while that of b 2 (the amount of d,2 in the 1z2> extended orbital which is the excited state comprised mostly of the d,2 orbital) is related to the ionic These thoughts become particularly radius in the z direction.24 relevant because a number of molecules of biological interest have a low A3 value; the low value is likely not a direct result of its stereochemistry but is rather due to low lying excited states. Relatively few aquo ions have been thoroughly characterized within the second and third transition metal series. In the paper by Daul and GoursotZ5 the electronic structures of the two low-spin d5 ions [Ru(H20)6I3+ and [Ru(NH~)~]~+ are derived using MS-Xa and extended Hdckel molecular orbital calculations. By comparison of the experimental and calculated g and a values, the former ion is analysed requiring D3d symmetry with a strong trigonal field (about 2500 cm-l) imposed by restrictions in the relative orientations of the water molecules. In the latter, a free rotation of the ammonia molecules is suggested by the good agreement between the experimen-

2: Transition-metal Ions

43

tal hyperfine parameters and their calculated values assuming O h symmetry. Surprisingly, the orbital reduction factor k has the greater reduction from unity in the hexaquo complex, revealing a greater covalency between metal and oxygen than between metal and nitrogen. The mechanism of covalent reduction of both the hyperfine coupling constant and the orbital angular momentum part. of the g value of 3d impurities in silicon has been discussed by KatayamaYoshida and Zunger using self-consistent Green's function calculations.26 For high-spin configurations, measurement of n and E leads directly to information on the symmetry of the transition metal ion, though since there are many factors which together contribute to the magnitudes of 0 and E , this information is at t.his stage largely qualitative. Many authors have attempted to calculate, on electrostatic and other grounds, these various contributions to D with varying amounts of success. Recently the superposition model of Newman and Urban has been gaining support particularly for the S state ions Mn2+ and Fe3+. Lehman et a l . , working with host lattices of known structure, obtained good agreement between experimental and calculated zero-field splitting obtained by application of t.his theory for Mn2+ in triclinic zinc(I1) bis (pyridine-3-sulphonate). ~ and ~ for0Fe3+~in CsGaC14.28 ~ This group has also applied the same technique to an S = 3 / 2 system, Cr3+ in tris-(acety1acetonato)-galli~m(II1)~~. Sunandana and Jagannathan in their study of Fe3+ in A calcium boroaluminate glasses worked from the other direction.30 correlated e.s.r. and M6ssbauer study yielded an estimate of t.he Fe3+-02- distance which, together with an assumption of the appropriate ligand and angular parameters required by the Superposition Theory, led to the conclusion that the FeOq tetrahedron suffers an

4

angular distortion up to 30' from a perfect tetrahedron as the iron doping is increased. The effect on zero-field splitting of the metal-ligand distance has also been the subject of a paper by Xiong et a l . 3 1 . In particular , these authors applied high-order perturbation theory to the d 8 ion in a trigonal field and interpreted the pressure dependence of the zero-field splitting in NiSiF6.6H20. The ferredoxins, which contain [2Fe-2S] clusters in which each iron atom is bound to two bridging and two terminal sulphurs, have long been recognized by a gav of 1.96, but some metalloproteins of the Rieske type and also their model compounds in glass-forming solvents have more recently come to be characterized instead by gav GaYda and his collaborators have had considerable in applying ligand field theory to the ferredoxins and now have = 1-91-32

44

Electron Spin Resonance

applied a similar technique to this new class of binuclear clusters. Basically the reduced cluster is assumed to contain high-spin tetrahedral ferric and ferrous ions which couple antiferromagnetical.ly to give an S = 1 1 2 ground state but t.he spread of g values within each of these two classes is related primarily to a distortion of the ferrous tetrahedron. The first-principle L.C.A.0.-Xa calculations of Noodleman and Baerends support t.his contention.3 3 The main difference between the two classes noted above is a significant increase, for the gav = 1.91 class, in the splitting between two of the excited states d x z and d y z which stems from a greater inequivalence theory

between the bridging and the terminal ligands. Thus the is consistent with the ENDOR and ESEEM finding that probably

two nitrogenous ligands are bound in certain of these clusters.34 Model compounds containing ligands with phenolic,3 5 1 36 or carboxylic oxygen37 as well as those containing nitrogenous l i g a n d ~ 3~8 ~ 'are providing in fact convincing evidence from e.s.r., n.m.r., Raman, visible and MClssbauer spectroscopy that several combinations of incomplete sulphur ligation must be considered. In recent years it has been recognized t.hat a satisfactory understanding of lineshapes may only be found when a d i s t r i b u t i o n of spin Hamiltonian parameters is allowed for. This is frequently the case for proteins some of which are sufficiently flexible as to be best described by a range of ligand field strengths and symmetries leading to a range of values of, for example, g , D and A spin Hamiltonian parameters. It is also now realized that many low molecular weight molecules also may be flexible enough t.o warrant special consideration of this kind; indeed a review on t.he non-rigidity of central metal atoms of various transition metal complexes has appeared.39 Thus it is with interest t.hat we look at a statistical theory for powder e.s.r. in which t.he dominant broadening of the e.s .r. spectrum arises from a distribution of g values (g strain) . 4 0 Hagen et a l . assume that while g , as usually defined is satisfactory for a single molecule, the usual concept of geff must be substituted by < Y > , an expectation value for an ensemble of molecules within which individual members may have slightly different physical properties method tensor

and g

correspondingly slightly different 9 tensors. heir introducing this concept requires that the conventional be redefined as

where

p

is another three dimensional tensor, whose principal elem-

Of

2: Transition-metal Ions

45

go and p are not necessarily collinear ents are random variables. and R ( a , $ , y ) is the three dimensional rotation which transforms t.he p principal axis system to the go principal axis system. An expression for g is derived and from this, partial derivatives of g with respect to p deduced, from which later emerges a polynomial which is fourth order in the direction cosines of t.he field and which represents the expectation value and variance of y . The application of this theory to lineshape simulations is noted in t.he next section. Analysis o f Spectra, Computing. The usual method of determining the principal components of g and D for a single crystal is to run spectra in three mutually perpendicular planes and convert the data thus gained to the principal axis system by a method based upon perturbation theory. But such a technique breaks down when t.he Zeeman term is comparable in energy with the zero-field splitting term. Kobayashi overcomes this problem using a direct search method which uses a two-axis goniometer.41 According to the flow diagram of actions required in t.his method, the first task is to vary the two goniometer angles to find a maximum (or a minimum) in the resonance field; a sterographic net is then used to define the plane normal to this direction and selected sets of angles ( 9 and + ) are read off the net for conversion to the goniometer angles s o t.hat t.he plane may be searched experimentally. Turning to powder, or frozen solution spectra, the most reliable method of extracting spin Hamiltonian parameters is by computer simulation of t-he spectrum. Computer programs for s = 1 / 2 systems may be based upon algebraic equations derived using perturbation theory and have been very successful, though limited in application. Very general, matrix diagonalization procedures have been described for more complex cases which contain quartic spin terms, quadrupole terms and so on, but t.heir main disadvantage seems to be one of computer time. One answer to the problem of computer t.ime is to take the theory pertinent to a special case, even one where perturb-ation theory is not applicable, and develop specialized algebraic equations which then may be used for very rapid computer solutions. A case in point is the simulation of signals in t.he geff = 4.3 region where D is not assumed t.o be large with respect t.o gfiu. McGavin and Tennant42 fit the angular variation of geff to an expression of the form

46

Electron Spin Resonance

where g:ll represents the usual ellipsoidal variation when I D 1 > > gpH and giix arises from the mixing in by magnetic field of the Kramers doublets into one another. The method requires an initial estimate of n and E using readily available plots, from which exact diagonalizations yield plots of geff in the principal planes of t.he D tensor. From these plots, components g : i x may be estimated and these are used in a least-squares matrix refinement program t.o find improved estimates of n , E , etc. and the process is repeated; it usually converges within two or three loops of this kind. The method was developed for spectra where more than three features are seen in the geff = 4.3 region. The saving of computer time was also

of much concern to Baranowski et a l . 4 3 in their study of vibronic effects in a copper(I1) complex (see next section). They found in their S = 1 / 2 system t.hat lineshape was best represented by a linear sum of both Lorentzian and Gaussian contributions as opposed to the sum of squared functions used earlier. Furthermore, since the main contribution is from the central part of the spin packet, acceptable fits were found when its width was truncated at dist.ances of less than twice the peak-to-peak linewidth from the centre. Second order corrections also were omit.ted in their case. The statistical theory of Hagen et a l . 40 mentioned in the previous section also may be programmed t.o c0mput.e lineshapes. In a second paper , Hagen et a 1 . 44 describe a numerical analysis which incorporates g strain based upon t.he following steps: ( 1 ) tabulate the lineshape function: ( 2 ) compute t.he rotation matrix of equation (1) and t.he coefficients in t.he equations for the resonance position, intensity and width; ( 3 ) integrate in g space over t.he surface of a sphere (integration in H space is asymmetric and takes twice as long); (4) transpose into H space as an absorption spectrum; (5) transform to Fourier space, chop out high frequency components to reduce mosaic rippling, differentiate in Fourier space, and finally back t.ransform. They illustrate the use of t.he program with reference to s = 1 / 2 having g but no A . Much of t.he work done in powder ENDOR has been undertaken only at turning points and has not made full use of the angular selection which is provided by anisotropy in t.he e.s.r. spectrum. Hurst et al.45 show t.hat when frequency modulation of t,he swept radio frequency radiation .is employed, one is able to obtain ENDOR spectra throughout. the e.s.r. spectrum thereby increasing the range of angular orientations which are selected. A t.heoretica1 development of the equations for ENDOR shifts in randomly oriented samples is also given because the traditional equations, although they yield

2: Transition-metal Ions

47

nearly correct answers in some limiting cases, are generally incorrect, because they have been derived assuming that the electron and nuclear spins are quantized along the same axis and that the spinonly g value of the electron can be used to determine t.he dipolar field at the nucleus. The new equations are general, and can be used to determine local geometries of ligand nuclei near transition metal ions in a variety of types of metal complexes but the specific examples chosen in this and in the following paper46 refer to a copper(I1) complex. In those cases where the transition metal complex gives a broad spectrum, overlapping in multicomponent systems prevents a straightforward analysis. For example the hydrolysis of the cupric complex of 4,7,10-triazatridecane dinitrile in aqueous NaOH at room temperature creates two new species and the concentrations of all three change with time. The e.s.r. spectrum is thus a poorly resolved four line spectrum which also changes with time. G m t ~ p phas ~ ~ analysed this system by the following method. The whole data set of several spectra all recorded at the same rate are written as a matrix. Each row represents a spectrum at a given time while each column is a kinetic curve taken at a particular field. This matrix of experimental data can be represented by a calculated matrix whose rows are a linear combination of the unknown spectra of the complexes present in solution and whose columns are represented by an expression which involves the rate constants. A least squares analysis yields the rate constants and the spectra from which the spin concentrations may be calculated for each complex, and the species distribution as a function of time, may be plotted. In this case the data were analysed in terms of stepwise hydrolysis of the two nitrile groups of the original complex. Jahn-Teller Effects.-

A

detailed treatment has been given

by

Ber-

suker4' of the E x E Jahn-Teller coupling in which the E g electronic ground state is vibronically coupled with the E~ vibrational mode to give the potential surface which takes the form of a Mexican hat. Deeth and Hitchman discuss, for six coordinate copper(I1) and lowspin nickel(II), the additional effects of anharmonicity in the vibrational potential, d - s mixing and second-order electronic corrections.49 Their general conclusion is that for all reasonable sets of input parameters, the potential surface is warped by these additions in such a way as to stabilize the elongated tetragonal geometry primarily because the first two of these corrections favour an outward displacement of the ligands.

48

Electron Spin Resonance

Single crystals of the perovskite-related A2B2-xCux(N02)6 [A = group 1 metal, B = group 2 metal] obtained over a wide range of x enabled t.he study of several isolated copper polyhedra, in which a nine-line hyperfine interaction from t.wo pairs of nitrogen atoms could be seen, and of exchange interactions in the semi-diluted and concentrated orthorhombic

samples.50 Even in the diluted crystals there is an powder-type spectrum due to C u N 6 polyhedra which are

statically Jahn-Teller distorted even at 298 K. The departure from axial symmetry arises because dZ2 is mixed into the d x 2 - 2 ground state. There is also an isotropic signal in t.hese dilute samples whose intensity diminishes t.o zero as the temperat.ure is lowered to 4.2 K and this characterizes the dynamic averaging over t.he t.hree potential minima. In the less dilute complexes, exchange-coupled signals due to antiferrodistortive Cu2'-Cu2+ pairs with a -7 value of about 0 . 6 cm-' additional.ly appear. Mehran et a . Z . 5 1 question descriptions of this kind and,with part.icular reference to T12PbCu(N02)6, 1.e. with x =: 1 , conclude that both phases I1 and I11 must be regarded as ferrodistortive and with an almost pure d Z 2 copper(11) configuration. Baranowski et a l . 4 3 studied a complex in which an undiluted octahedral copper(I1) i.on was effectively diluted sufficiently by its four neighbouring copper(1) ions to see copper hyperfine structure. The linewidth variation of t-hese hyperfine lines was used to investigate the vibronic interactions rather in the way that it has al-so been used to study the rate of t.umbling in solut.ion. This sort of treatment, has not been possible previously with pure compounds because of dipolar and exchange effects. Both

staticfi2 and d y n a ~ n i c ~Jahn--Teller ~-~~ effects were also noted in CsCuC13 ; 5 3 and (NH4) 3Hot,her copper (111 syst.ems viz : a-LiI03 ; 52 (S04)2.54 Among the d 1 configurations, most of the earlier e.s.r. st.udies on metal tetraoxyanions are confined t.o Cr0;- and MnO2- in a variety of lattices with symmetry less t.han cubic. Sekhar and Bill therefore chose to study ReOz-, produced by X-irradiation at 77 K from Re04 which substitutes for t.he halide in KC1, KBr and KI lattices because i.r. and Raman spectroscopy clearly exhibit T d symmetry of the unirradiated perrhenate ions.55 The e.s.r. spectra have tetragonal symmetry (compressed ion with d,2 ground state) and experiments with applied stress prove that the cluster is capable of reorientation. The results are consistent with a strong E x E JahnTeller effect. By contrast, in similar experiments within the same lattice, the related MnOi- was found to be intermediate to weak.5 6 Agrest.1 et a l . on the other hand note anomalies in the tetrahedral d' system because t.he strongly reduced anisotropy of g and

A

2: Transition-metal Ions

49

deduced from the e.s.r. spectra cannot be explained by consideration of the E x E coupling within the ground stat.e alone.57 Here also the role of second order effects have been analysed and it was shown that the pseudo Jahn-Teller effect, which mixes the ground state with the first excited state, is effective in some tetrahedral d' systems; particular reference was made to VC14. The Pd3+ ion has been doped into an octahedral site in calcium oxide. Here it experiences a strong ligand field and is low-spin d 7 like other 4d and 5 d ions. In O h it is 2 E g ( t ~ g e ~and ) subject to a Jahn-Teller distortion. At 1.65 to 4.2 K three tetragonal spectra are superimposed. At temperatures over 77 K the spectrum is isotropic at the average value of the low temperature spectra. This is quite a clear case of a static Jahn-Teller effect becoming dynamic as the temperature is raised.58 Ramakrishna e t a l . have reported the e.s.r. data on the mixed sandwich relatives of cobaltocene such as [Co(cp)(Bz)]+ and some of its ring methylated derivatives.59 These contain cobalt(I1) in the d7 configuration with the unpaired electron in e l g having largely metal (d,z,yz) character and give well resolved spectra only at helium temperatures. Spin Hamiltonian parameters were derived by computer simulation and used via an iterative process to deduce the unknowns in the equations for the static- and vibronically-coupled limits. There is clear evidence for a dynamic Jahn-Teller effect from the characteristic host-lattice dependence of the e.s.r. parameters. By varying ligand substitutions, rat.her than the temperature, they were able to cover examples from the dynamic to the static case. Methyl substitution increases covalency, and methylation of the cyclopentadienyl ring has a larger effect than methylation of the benzene ring. Mixed [ (NH3

Valence Complexes.- The properties of the mixed

valence

ion

5Ru ( p-pyrazine )Ru (NH3)5 ] 5 + have generated considerable debate with regard to its electronic structure. The experimental status of this ion has recently been updated6' with the conclusion that even now the evidence does not allow one to decide the question whether the odd electron is trapped on one ruthenium atom or delocalized over both. More recently, Dubicki e t a 1 . 6 1 have favoured a delocalized coupled pair model which incorporates polarized electronic absorption spectra as well as the e.s.r. data which, only now that three g values have been found, has been fully described.60,61 Dubicki and Krausz have used a similar approach for the trigonal mixed valence dimers such as [ (NH3)3RuX3Ru(NH3)312+ in order to

50

Electron Spin Resonance

explain the e.s.r. spectra of the trichloro ( X = C1) and t.ribromo (X = Br) compounds.62 Complexes such as O ~ ~ ( h p ) ~ (hp C l ~= 2-hydroxypyridinate) may be reversibly reduced by one electron to give the novel O s 2 5 + core. Magnetic susceptibility measurements indicate, from a magnetic moment of about 2 . 7 B.M., the likely existence of molecular orbital or 027r4626*2n*I . The e.s . r . configurations such as 0 2 ~ 4 5 2 6 * spectrum is consistent with such a spin quartet. if a large zero-field splitting between 1*3/2> and 1i1/2> is assumed.63 When hp is l replaced by chp (6-chlor0-2-hydroxypyridinate)~ O ~ ~ ( c h p ) ~ Cis formed. Here, a reduction has already taken place and one of the original axial chlorides is absent, the vacant site being blocked by the four chlorine atoms of the chp ligands. This is the first neutral complex of t.his kind which contains t.he core as confirmed by a similar e.s . r . spectrum.64 Delocalization over two metal atoms is assumed in two further e ~ a m p l e s . ~ ~ The , ~ tetra~ sulphur-bridged molybdenum dimers of the type [(C~)MO(LIS)]~S~CH~ display an extensive reaction chemistry with hydrogen and unsaturated molecules while extended Htlckel molecular orbital calculations have provided a very useful framework for understanding this. In the monoanion which formally contains Mo3+ and Mo4+, the e.s.r. spectrum shows that one electron is delocalized over two equivalent molybdenum atoms (Robin and Day class 111). Not surprisingly no hyperfine structure is seen from t.he sulphur nuclei but indirect evidence is obtained for spin density on the sulphur by observing a four-line splitting from the Na' counter ion in tetrahydrofuran solution in which solvent tight ion-pairs evidently are formed. The assumption that Na+ is interacting with the sulphide ligands is consistent with the description of the reactive b 2 s frontier molecular orbital . 6 5 The binuclear anion [ tCH3N(PF2)2}3C02(CO)2]- which formally contains Coo and Co-, but which more accurately is described as a 19-electron system with its unpaired electron in a a * molecular orbital, may be similarly classified. Here the e.s.r. spectrum indicates complete delocalization over the two cobalt atoms even at 4 . 2 K , by its isotropic spectrum which shows rich hyperfine structure from two equivalent cobalt, six equivalent phosphorus and 12 equivalent hydrogen atoms.66 Systems in which the hopping frequency is slow and in which the unpaired electron is said to be localized on the e.s.r. timescale are represented by the polynuclear copper cluster43 described in the previous section (Cu'. and Cu2+) and by the binuclear complexes of the type [Mn2OqL4I3+ (L = 2 , 2 ' -bipyridine and 1 , 10-phenanthrol-

51

2: Transition-metal Ions

ine) ;67 the latter contain antiferromagnetically coupled Mn3+ and Mn4+ centres and at low temperatures only the ground doublet state is appreciably populated. This gives a 16-line e.s.r. spectrum with almost isotropic g and A tensors but with unequal hyperfine structure from the two manganese nuclei. Major activity has come about in the field of the platinum blues as a result of the discovery of the powerful antitumour activity of the blues prepared from ~ i s - [ P t ( N H ~ ) ~ [ H ~ 0 )and ~ 1 ~pyrimidines. + The crystal Structure of a-pyridone blue is known to be a P t 4 unit and with an overall charge of +5, the formal oxidation state is 2.25. The g values are appropriate for a d Z 2 hole along the mean axis of the Pt4 chain. Woollins and Kelly have tabulated e.s.r. data for blues which on the basis of e.s.r. and other spectroscopic evidence are thought to be similar to this.68 Unusually well-resolved hyperfine structure was observed in two platinum blue complexes obtained using ligands containing the -C(O)-NH- group and an analysis was favoured here in terms of two pairs of platinum ions whose hyperfine constants require appreciably different spin densities." Unusually small g anisotropy was found in another.70 The molybdenum blues also have been reviewed71 and classified in terms of localized- or delocalized-valence. The simplest isopolymolybdate yields a oneelectron blue C M ~ ~ O whose ~ ~ Ianalysis ~ has been in terms of an isolated Mo(V) in an axial field, whereas the heteropolyblues, or more highly reduced species, e . g . , [ PMe 2040]4- are more delocalized.7 1 Phase Transitions.- The general application of e.s.r. to phase transitions has been reviewed' while a more specific application to transition metal fluorides, mainly d 9 cations, also has appeared.72 In using transition metals as dopants, the choice of ion is not

'

always straight forward. For example Jerzak and FuJimotoc13in their study of tris (sarcosine)calcium chloride found that Fe3+ or Cr3+ dopants, when the crystals were cooled in the paraelectric phase towards the ferroelectric transition, showed a marked line broadening. With Mn2+ the effect was very small while with Cu2+ it was barely recognizable. Extensive measurements were made also on the system M,TiS2 (M = V,Cr,Mn,Fe,Co,Ni; 0 < x 5 These metals go into the octahedral intersheet sites of TiS2 which has a simple C d 1 2 layer structure. The motivation here was to learn about the intercalation process of different transition metals. Unfortunately Fe and Ni gave no e.s.r. signal but the other metals gave some informative results. Vanadium gave a single line arising from V2+ or V3+

52

Electron Spin Resonance

whose maximum intensity occurred at x = 314. Chromium gave a broad line which got broader due to dipole-dipole interactions as x rose to 113, then narrower for x > 113 due to exchange interactions even though the interlayer spacing was expanding. Manganese gave an asymmetric lineshape due to skin effect, while cobalt gave a single line only if 0.175 < x < 1/3 and in a narrow band of temperatures. This last ion became ferromagnetic on cooling below 140 K with the onset of considerable anisotropy which is not observed in the high temperature (paramagnetic) state. Lead copper fluoride (Pb2CuF6) also is ferromagnetic at low temperatures but in this case, e.s.r. suggests antiferrodist.ortive ordering of the dx2-y2 and dZ2 Cu2+ orbitals above the transition temperature.7 5 Discontinuous changes in the linewidth and the shift of the resonance field are observed as a function of temperature when single crystals of CsCuC13 are heated through the structural phase transition point at 420 K. In the structure of this compound, CuC16 octahedra form infinite chains within which the exchange coupling the chain is 102-103 times stronger than b e t w e e n t.he chains. along Below 420 K all the octahedra are elongated and the long axes form, in succession, a helical chain along the c axis. Above 420 K , as judged by the lineshape, the magnit.ude of the linewidth and its angular dependence, the octahedra are still distorted and the exchange coupling along the chain remain strong. The discontinuity probably arises from a change in the magnitude of t.he isotropic exchange interaction.53 In the system [CO(NH,)~]X~ (X = Br,I) a similar abrupt change in linewidth occurs. In this case the cause is a sharp rotational entropy change due to onset of rotat.ion of t.he total hexammine complex.7 6 When doped into the cadmium host lattice this same probe again provides drastic changes in the e.s.r. spect.ra which are evidence of a structural phase transition.7 7 This time the cobalt hyperfine structure was monitored and registered a change from isotropic, above T c , to anisotropic, indicating tetragonal or trigonal symmetry, below it. Several groups have paid special at.tention t.o the slowing-down behaviour in the critical region of temperature close to but above a transition temperature. Kaziba e t a 1 . 7 8 observed line broadening in the tranthe paraelectric Rb2Zn(Mn)C14 as it is cooled towards T I , sition to an incommensurate phase. They used a Mn2+ probe, because it correctly fit.s t.he Zn2' site of the host lattice, and looked at its hyperfine sextet in this fast. fluctuation regime. The lineshape is pure Lorentzian and associated with the broadening is an upfield shift of the centre of the hyperfine group. The analysis is based

53

2: Transition-metal Ions

upon a slowing down of the rotation around c of t.he Zn(Mn)C14 tetrahedron. The tris- (sarcosine)calcium chloride mentioned above73 showed a similar line broadening; this was due to fluctuations in In the case of NHqAlF4, in which Fe3+ the sarcosine orientations. substitutes for A13’, a single e.s.r. line is seen above the orderThis, it is postulated, is the disorder t.ransition at about 175 K. result of jumping between several sets of t.wo--dimensional ordered domains. On cooling through T c a doublet grows and the single line diminishes in intensity. The presence of the doublet is as expected for the fully ordered three dimensional phase in which A13’ occupies a site of 2/m symmetry. The lineshapes were computed over the critical region using the Kubo-Anderson theory and the variation of domain lifetime with temperature fitted to an Ising The dynamics of spin-state interconversion for spin-crossover complexes also have been studied. In the ferric ion, spin crossover from S = 1/2 to S = 5/2 involves transfer of two electrons from t 2g orbitals to eg orbitals. Bond length changes should therefore be anticipated and the solid must be able to accommodate these if both states are seen in the same lattice. In the case of CFe(3-OEtSalAPAI2]X (X = C104- or BPh4-; and 3-OEt-SalAPA is the monoanionic Schiff base derived from 3-ethoxysalicylaldehyde and N-(3-aminopropyl)aziridine), a g 4 2 (rhombic low-spin) signal gradually increases in intensity at the expense of a g 4 4 (high-spin) signal as the temperature is lowered. Distinct high- and low-spin spectra were seen at the same time from which it was deduced that slow interconversion was occurring on the e.s.r. timescale ( < 10” s-’) though rapid on the Mbssbauer time scale ( > l o 7 sell.8o The suggestion was made that rapid solid state reorganizations are responsible for these rapid interconversions. An analysis of the low-spin signal led to the conclusion that the unpaired electron is in the d,,, orbital and that the gap between the ground and first excited Kramers doublets is * 5000 cm-’, a value much larger than thermal energies. Consequently, the intensity of t.he low-spin state is expected to be temperature-independent so the observed temperature dependence can be attributed solely to spin-crossover origins. Spin state crossovers have also been observed in the family of compounds of the general type C~(H~(fsa)~en)L, where H2(fsaI2en2is the ligand shown in ( 1 ) and ( 2 ) on the next page and L is a substituted pyridine. 81 The e.s . r . spectrum of the five-coordinate CoN302 compound ( 1 ) with L = 3-hydroxypyridine, is dominated by the S = 3/2 isomer (geff = 6.33, erature is raised, only the

3.44 and 2.22) at 9 K but as the temp2.22 signal remains and this is

g =

Electron Spin Resonance

54

(1) n = l

(2) n = 2

assigned to S = 1/2. Such an equilibrium is difficult to follow as a function of temperature because e.s.r. intensities vary also as a result of line broadening through relaxation phenomena. However in this case the peak-to-peak line width of the g = 2.22 signal remained constant over the range 50 to 150 K s o the variation of intensity with temperature, at least in part, reflects a conversion from low-spin to predominantly high-spin molecules. The six-coordinate CoN402 complexes (2) with L = 4-methylpyridine or 4-t-butylpyridine display similar behaviour. Paramagnetic Ligands.- Diamagnetic transition metal complexes or ions may be made amenable to study by e.s.r. by reaction with a paramagnetic ligand. Semiquinones provide such a route.8 2 1 8 3 Nitric oxide is in some ways more useful since the nitrogen nucleus has a spin thus rendering the ligand a useful diagnostic marker.84J85 Alternatively, redox reactions may be used. For example, binuclear carbonyls in which either Cr(0) or Mn(1) atoms are bridged by pyrazine have been studied as t.heir radical ions with the purpose of describing the bridging molecular orbitals to evaluate the metalmetal interaction.86 The N-methylpyrazinium cation cannot bridge in this way but it has been used as a paramagnetic ligand in this case with the purpose of describing the molecular orbital parameters of the metal-ligand bonding.87 Where the transition metal ion is paramagnetic, then the possibility of spin-spin interaction between the metal and its ligand also exists. It may be observed as a broadening or even a splitting and may be used to infer distances between the paramagnetic centres or the mode of interaction. Much progress in deciding the importance of elkctron-electron exchange interaction relative to dipolar interaction has been made. 88-92 There is much interest in finding complexes which have a high affinity for binding dioxygen without undergoing autoxidation. In

2: Transition-metal Ions

55

this respect we are much concerned with where the unpaired electron is situated after a redox process, something for which the e.s.r. experiment is eminently suited to tell us. Chavan et a l . have studied a new family (L) of so-called lacunar complexes i.e., complexes with ligands which wrap around in such a way as to leave a void, or lacuna, in one of the coordination positions.93 They used nickel (I1) and cobalt (I1) to form [ML] 2+ which was then electrochemically oxidized to [MLI3+ the nature of which was dependent upon the extent of axial ligation which, in turn, was dependent upon the size of the lacuna. Principally from the g anisotropy they were able to show that the unpaired electron in [NiLI3+ resides on the metal (i.e., as [Ni(III)L]3+, low spin d7 with g1 > g , , ) in those cases where a six-coordinate complex can be formed by addition of two monodentate solvent ligands. In contrast, in those cases where the lacuna is too small to allow a six-coordinate complex to form, or at higher temperatures when the sixth ligand is more labile, the unpaired electron rests on the ligand (i.e. as [Ni(II)Ltl3+ with a single isotropic line). Likewise with cobalt the complexes formed were found to be [CO(III)L]~+ or low-spin [Co(II)L!13+. Dioxygen was shown to react with the former. Ichimori et a 1 . 9 4 found a similar effect in their study of the simpler cobalt tetraphenylporphyrin as the dication [CO(III)TPPT]~+. Here H2TPP is the free acid and TPP2- has been oxidized to TPP-. In this case the nitrogen and cobalt hyperfine structure rather than the g anisotropy were found to be strong indicators of axial bonding when, with a series of coordinating bases or solvents, a greater spin density was found on nitrogen or cobalt for the stronger axial ligand. Last in this section we come to the long-running battle between the HSAB (hard and soft acid and base) description v s that of the E and C equation. Bilgrien et a l . 9 5 in their support of the latter, have sought to establish a correlation between the g value of the nitroxide TEMPO and the donor strength of the base B in the carboxylate-bridged di-rhodium dimer which has TEMPO bound at one end and B bound at the opposite end thus:- TEMP0.Rh(carboxylate)4Rh.B. The results were satisfactory provided the effects of the base were transmitted directly through the metal-metal bond only i.e., by atype interactions. Where n-back bonding was possible, presumably via the carboxylates, deviations from their model resulted. Binuclear and Oligonuclear Complexes.- The study of spin-coupled systems continues to be of great interest. A review of the fundamental t.heory for the interpretation of their e.s.r. spectra has

Electron Spin Resonance

56

appeared’ but before going on to quote recent experimental work, brief mention will be made of a recent theoretical paper which gives exact expressions for the g and A tensors in spin-coupled systems in terms of the parameters from the isolated spins for S, = 1 , 3 1 2 , 2, 5/2 and s2 = l/2.96 The spin Hamiltonian used assumes both isotropic exchange ( - 7 ) terms and axially symmetric zero-field-splitting ( D ) terms in varying proportions. The new thing about this approach is that the effect of interactions between different spin manifolds i.e. when s is not a good quantum number, is specifically included. This has not been done before except for S 1 = 1, S2 = 1/297 and the main conclusion is that such effects should not be neglected, even when 1.71 > > I n ! , an extreme situation which is frequently assumed as a justification for their neglect. The experimental D tensor is given by the sum of contributions from DdiP and Dex. The former may be calculated using the point dipole approximation if the structure is known, hence Dex the anisotropic exchange contribution may be extracted. If the mechanism of isotropic exchange is currently well understood, the same cannot be said for the anisotropic exchange and to correct t.his situation, the Florence group has been very active recently, mostly with single crystal studies, in measuring Dex and discussing the theory which explains its magnitude. They have noted a trend to a decrease in this parameter on increasing the Cu---Cu distance in planar biand relate this to the two-electron exchange bridged complexesg8 integral which involves the overlap between the xy-type orbital of

’’

one ion with the x2-y2-type of the ~ t h e r . ’ ~Conversely, since D;~P is less dramatically dependent upon t.he Cu---Cu distance (which is along the x direction) than is DZ;, the observation of the largest component of D roughly orthogonal to the equatorial coordination planes in such complexes, is indicative of the presence of a dominant exchange contribution. This line of reasoning has been applied also in several other cases.’Oo-’O3 The same exchange pathway is not always operative however and in a pyridine oxide-bridged complex with a very long copper-copper distance, a large, exchangedetermined Dyz component was postulated to account for the rotation of D , , away from the expected direction.lo4 A l s o , in the case of a binuclear copper oxamidato complex, the largest component of D is along the Cu---Cu direction indicating that , in this instance, DdiP dominates.Io5The generalization was made that when two x2-y2 magnetic orbitals are coupled, a fairly large exchange contribution, and consequently a fairly large zero-field splitting in the e.s.r. spectra can be anticipated if the bridge is monatomic; but the

2: Transition-metal Ions

57

exchange contribution is substantially quenched if t.he bridge, as in this case of the oxamidato complex, is an extended one. I o 5 A similar approach was used by Ozarowski and Reinenlo6 in an investigation of hydroxy-bridged copper(I1) and vanadium(1V) dimers. direction Here the largest component of D is again along the M---M even though the largest component of Dex is D f Z and exchange is strong. In fact it is dominant for the copper dimer (d9-d9) leading to a large D but almost equally opposed by D$:p for the vanadium Thus there is as yet no simple dimer (dl-d’) for which D is small. analysis for anisotropic exchange and we can anticipate much more work in t.his area. Satellite lines in the Re(abt)3 spectrum (see section on 92x1valent rhenium) are assumed to arise from dimeric species where there are both weak exchange and dipole-dipole interactions between the paramagnetic centres. Computer simulations indicated a Re---Re distance between 9 and 10 Angstroms. Exchange at such a distance without a direct line of bonds is rare and in this case possibly arises through the so-called edge-to-face or face-to-face contacts between the benzene rings of neighbouring molecules by consequence of the extreme delocalization in these molecules.I o 7 Among the bis(p-hydroxo)- and bis(p-ch1oro)-bridged complexes, strong correlations have already been established. Thus a smooth curve results when 2 J is plotted against 9 (the bridge angle) for The p-OH or against b / R for v-Cl ( R is t.he metal-metal distance). dimeric unit [cu2C18l4- in [Co(en) 3]2[cu2c18]c12. 2H20 for example obeys the latter relationship and to test it further Hoffmann et a l . Io8 have substituted cobalt in the cation, with ions of larger ionic radii, Rh(II1) and Ir(II1). What they found instead was t.hat neither hyperfine nor fine structure were resolved and that the spectra are partially averaged by interdimer exchange. Values of the interdimer exchange integrals were determined from spectral simulation and were shown to decrease with decreasing temperature due to decreases in overlaps between the atomic orbitals in the superexchange pathway. The opposite trend was found with [ C ~ ~ ( d i e n ) ~ C121 (ClO4I2 as a result of thermal lattice contraction.lo’ However, returning to the correlations between 2J and 0 or 0 / R , [Ph4PI2[cu2c16] and C P ~ ~ A S I ~ C are C U found ~ C ~ to ~ ~obey the rule“’ but there must always be exceptions and the [Cu2(2-methylimidazole)4(OH)21(C104)2.2H20 is a case in point.” It is noted that no smooth curve results with p-bromo complexes either. lo Clearly more research is needed here to seek out the additional factors which must be involved.

Electron Spin Resonance

58

It is well over 30 years since Bleaney and Bowers first recognized the triplet state e.s.r. spectrum of copper acetate and solved the s = 1 Hamiltonian but variants of this molecule continue to attract attention. Thus Barba Behrens et a l . have substituted two of the bridging groups using a substitut.ed naphthyridine which replaces bridging oxygens by bridging nitrogens. And rather than change the bridging groups , Sharrock and Melnik' have concentrated on

t.he end

groups in their study

of

C C U ( C H ~ C O ~ ) ~ . C H ~ C Oand ~H]~

[ C U ( C H ~ C O ~ ) ~ . C H ~ C O ~ H .among H ~ O ]related ~ molecules in the

complete series of products of copper acetate solvated with either water or acetic acid or both, and their dissociat.ion products in the dimermonomer equilibria. Many analagous compounds t.o these in which N atoms are the donors in the bridging units additionally are known, as also are a few of those in which the four bridges each contain one N and one 0 donor atom. Goodgame et a 1 . l 3 provide u s w j fh a recent example of the latter which at the same time demonstrates the great advant.age of measuring spectra of this type of compound at more than one frequency. In their case, the features at R Z 2 and Bxy2 overlap at Q band (34 CHI.) but not at X ( 9 GHz) while R,,, R x y , and the AM^ = 2 transition are not very clearly resolved at X band while they are at Q. The dicopper complexes of six linked-cofacial diporphyrins have been studied as their frozen solutions. l 4 Since there is no Cu--Cu bond, it was felt reasonable to assume an isotropic exchange that is sufficiently weak that anisotropic exchange may be neglected, and to use the point-dipole approximation to calculate copper-copper distances. In this study, computer integrations and simulations were heavily relied upon. The interspin distance, r , was obtained from the intensity of the half-field transition relative to the allowed

'

transition and E , the angle between the interspin vector and the gz (and A z ) direction, was obtained by simulation, again of the halffield line. These values of I' and E were then used as the starting Parameters for the simulation of the allowed transitions which in turn led t.o a second estimate of r . The results for three of these compounds were in good agreement with values from X-ray crystal structure determinations. Intensity measurements have been used by Ranci also to study dicopper and other dimetalloporphyrins but in a quite different way. ' I 5 In this case, the concentration of a solution of monomeric metalloporphyrin was varied and the room temperature e.s.r. intensity of the monomer related to dimer formation, the intensity of t.he aggregate signal being assumed negligible. In a similar way, hetero-

2: Transition-metal Ions

59

dimer formation was studied but only where one of the partners was fast-relaxing in order to achieve simple e.s.r. spectra; the inten-sities of copper, silver or vanadyl porphyrins were monitored as they diminished with increasing amounts of added ferric porphyrin chloride. In this way, formation constants for planar copper(I1) and silver(I1) porphyrins have been estimated and for the first time, homo- and heterodimer formation constants involving square pyramidal porphyrins have been estimated. Turning now to exchange in trimeric clusters, a review has appeared wherein are discussed details of isotropic and anisotropic exchange interactions in relation to struct.ura1 features.l o The same four authors have given also the single crystal theory of such exchange including antisymmetric interactions. Triangular clusters have been much studied in order to determine how exchange interactions reflect chemical. bonds between the atoms. Studies of homopolynuclear clusters have dominated in t.he past but by way of contrast, two examples of heteropolynuclear t.rimers are reported here. Thus a Cu-Cu-Fe triangular complex has been studied not only to answer magnetochemical questions but also because cytochrome oxidase possesses Cu(I1)-Fe(II1) heterodimers. The copper(11) ions are equally coupled antiferromagnetically to the ferric ion but not to each other. The resultant S = 3 / 2 state was shown to be split by quite a large zero field splitting, by observation of e.s.r. signals in the g 4 and 2 regions, corresponding to the M s =

-

* 1/ 2

Kramers doublet. l 7 The tetradentate Schiff base complex of Bencini e t a l . however, in which two cupric ions and one vanadyl ion are coupled, is an example of the much less common ferromagnetic exchange as judged by its spin quartet e.s.r. spectrurn.ll8 It is well established that metal-metal bonds can have a profound effect upon the energy states of solids containing infinite linear chains of metal ions. Thus we have a reason for studying linear trimers since these are simply truncated chains. Pal et a l . have described a new class of mixed-valence homo- (Fe(I1) Fe(II1) Fe(I1)) and hetero- (Fe(I1) M(I1) Fe(I1)) trinuclear clusters. With M = Co or Nil the complexes are e.s.r.-silent presumably because of rapid relaxation but with M = Cr(II1) or Mn(II1, strong e.s.r. signals are seen, enabling the zero field splitting and other parameters to be measured. Curiously , the cobalt or nickel compounds could each be doped with manganese thereby acting as a paramagnetic host, without excessive broadening of the manganese signal. In the linear Cu-Cu-Cu trimer wherein adjacent copper atoms are bridged by bis(N,N'-bis(3-aminopropyl)oxamido), there is strong

60

Electron Spin Resonance

antiferromagnetic coupling between neighbours and of course a correspondingly weaker ferromagnetic coupling between terminal copper atoms. The expected energy level scheme is a doublet, a doublet and a quartet in order of increasing energy. The e.s.r. temperature dependence confirms the doublet ground st-ate and the observation of a much reduced anisotropy in which the effect of local g tensors is averaged out, also confirms the strong exchange. The reduction in anisotropy was negated as expected by replacing the central copper(11) by diamagnetic zinc(I1) .I2’ The main results found by Lippert et a1.l2’ for the linear Pt-Cupt trimer in which adjacent ligands this time were bridged by pyrimidine nucleobases, also came from the nature of the anisotropy in g, and of A . No exchange int.eractions are expected of course. The copper(I1) ion resides in a tetragonally elongated octahedron with the platinum(I1) ions behaving, despite their formal positive charges, as o-donor ligands. This is in keeping with earlier work in which the magnetic properties of t.he central ion were probed by ions with high-spin d 5 electronic states. 22 The electronic and e.s.r. spectra of the copper(I1) compound have been analysed using an angular overlap which shows that t.he filled 5d,2 orbitals on platinum interact with t.he 3d,2 on copper slightly raising it but not so much that it becomes semi-occupied and favoured as t.he ground state over d x 2 - 2. We finish this section with reference to two larger clusters. The first, [ F ~ ~ ( ~ ~ - s ) ~ ( Pconsists E ~ ~ ) ~ of I ~an + octahedron of iron atoms with the sulphur ligands t.riply bridging all the octahedral faces. Each metal is terminally bonded to a phosphine. Thus each metal is bound to four sulphur and one phosphorus atom in a distorted square pyramid. Formally, each iron is t.rivalent, and bound to t.his type of ligand is expected to be low-spin. Therefore we have six S = 112 ions and the best description, admittedly crude, is that t.here is a weak interaction to give s = 3 . Agresti et a l . find for the powder spectrum of t.his cation,1 2 4 among several other resonances, fairly sharp lines at g = 2 and g = 4.9; the latter disappears at temperatures greater than 200 K. They interpret their results rather tentatively in terms of an axial Hamiltonian ( E = 01, and with a small D value ( 0 . 2 cm-’1. Your reviewer feels that t.his work could benefit from being repeated and perhaps with more rigour with respect to the assignment as s = 3. The second is [ W @ 1 4 1 - . A s compared with the corresponding dithe tungsten-tungsten bonds are much longer, which is consistent with the removal of an electron from a metal-metal bonding

2: Transition-metal Ions

61

orbital. The e.s.r. in frozen methylene chloride solution at 9 K has the appearance of a broad perpendicular feature for which the parallel part is unresolved or unrecorded; at t.emperatures above 2 5 K, the signal is lost. Such behaviour is attributed to a 2 E ground state and comparison is made with [ M O ~ C ~ , ~ ]whose spectra are anisotropic indicating axial compression relative to [Mo6C114] 2, The spectrum of [W6BrI4]- however is described as only slightly anisotropic125 which implies an absence of such compression, an observation which is in keeping with the lack of compression revealed by the X-ray structure.

3 s = 112 d’ Configuration.- Tervalent Titanium, Zirconium and Hafnium. Reaction of ( 3 ) , in the scheme below (tmen = N,N,N’,N’-tetramethylethylenediamine), with (cp)2MC12, (M = Ti, Zr, Hf, Nb; cp = cyclopentadienyl) has yielded the corresponding meso-metalloindans, ( 4 1 , below. Reduction of the Ti, Zr, or Hf compounds at a platinum

SiMe3

SiMe3 I

Li(tmen)12 SiMe3

(3)

SiMe3

(4)

electrode or with sodium dihydronaphthylide, Na(CIOH8) converts them t.o the monoanions which, along with (4) (M = Nb), gave e.s.r. specCharacteristic metal hyperfine splittra typical of d 1 complexes. ting was seen for all of these except the hafnium compound. No hydrogen hyperfine structure was seen in any of t.hem and though this is normal for Nb(1V) and Hf(II1) it is unexpected for Ti(II1) and Zr(II1). The metal hyperfine structure was correlated with the angle at t.he metal subtended by the ligand.126 A similar reaction in which (cpI2MCl2 (M = Zr, Hf) reacts with dimethylaminobenzyllithium gave either a mono-alkylated monochloro, or a di-alkyl-ated derivative depending upon stoichiometry. After sodium amalgam reduction, such compounds are reduced t.o (cp)2Zr(CH2C6H4NMe2) or [ (cp)2Zr ( CH2C6H4NMe2 ) 2]- which gave e.s . r . spectra in which t.he Zr hyperfine structure was quite well resolved, at least in the second derivative presentation, (the reduced hafnium compounds were not

62

Electron Spin Resonance

reported). Related compounds also were studied, €or example that in which NMe2 is replaced by PPh2. In all of these compounds the methylene proton hyperfine structure is seen as anticipated though only in full if essentially free rotation is permitted. If rotation is slow as in ( C ~ ) ~ T ~ ( C H ~ C ~ H a~ N doublet M ~ ~ ) of , doublets obtains but only the larger coupling is observed.127 A related reduction has been carried out on ( c ~ ) ~ T ~ ( N C S12') ~ . In this case, after electrolysis in THF and CH2C12 solution, the reduction product gives an intense quintet showing the presence of two equivalent nitrogen atoms; the appropriate 47Ti and 49Ti satellites also were seen. In DHF solution however no e.s.r. is seen due to diamagnetic dimer formation, unless pyridine is present, when a paramagnetic species giving no ligand hyperfine structure is seen. Among the more inorganic examples we note that Ti(CH2C6H5I4 has been introduced to a silica surface with the subsequent formation of These species formed ( si-O)T13f(CH2C6H5) 2 and (SiO)2Ti3+(CH2C6H5) . a site for adsorption because reversible changes in the e.s.r. spectra were seen in the presence of nitrogen.12' Zirconium(II1) was shown, by the use of isotopically enriched "Zr, to be responsible for the parasitic signal previously observed in iron-doped ScP04. The related LuP04 and YP04 were similarly studied but in the latter, v-irradiation at 77 K was necessary before Zr3+ was produced. Site symmetry in all of these orthophosphates is tetragonally distorted eight-fold cubic and the ground state as judged by g anisotropy is d,2-y2. I3O octaThe hafnium ions in Cs2HfF6 are located at L I sites ~ ~ in an hedron of six fluoride ions. Again v--irradiationat low temperatures was used to create the d ' configuration, Hf3+, recognized by its g values which were reduced from 2.0 and by its interaction with In this case, the g the hafnium nucleus and with the six F- ions. anisotropy is indicative of trigonal distortion with an A l (dZ2) ground state. The large 177Hf hyperfine constants existence of a very large 6s involvement.13'

implied

the

Vanadium, Niobium a n d T a n t a l u m . The single crystal e.s.r. spectra of V 0 2 + in ZnS04.7H20 have been r e e ~ a m i n e d . ' ~ ~The vanadyl ion substitutes for a-Zn-H20 pair and since the six water molecules are inequivalent in this crystal, in principle, six different e.s.r. spectra could result. Bramley was able to distinguish four of these and confirm the previously noted correlation that the most intense spectra were associated with populations in which vanadyl had substituted the shortest Zn-H20 distance. Accurate

Quadrivalent

2: Transition-metal Ions

63

values of the hyperfine tensor were determined by zero field e.s.r. and corrections to two earlier publications on this system were proffered. The crystal field splitting of the d orbitals is derived for the nine-coordinated, capped antiprism structure said to be important because this arrangement is found in various monazite analogues (LnP04, where Ln = La, Ce, Pr, Nd, Sm, Eu or Gd) which have been proposed as possible hosts for the long-term storage of actinide wastes. The regular structure should have a degenerate d x y , d x 2 - y 2 ground state but distortion will cause this to split into two singlets. Doping of V4’ into CeP04 leads to a well resolved spectrum These observations, coupled with with gll< gL < 2.0 and All > Al. theoretical arguments, confirm that the site in this case, is distorted by rotation of one pyramidal base with respect to the other, ground state.133 and has a d,2-,2 Other conventional vanadyl studies include its substitution for the metal-water pair in a trigonal bipyramidal thiosemicarbazidodiacetate e n ~ i r o n m e n t ’and ~ ~ in a tetragonal K2Cd(S04) 2. 6H20 environment. 1 3 5 Where the related K2Co ( Se04 1 2. 6 H 2 O acts as a host however we have a much less conventional approach. The interesting thing about this system is that when vanadyl is doped in, well-resolved e.s.r. lines at room temperature become less well-resolved and eventually broaden into obscurity as the temperature is lowered towards 100 K. This is the reverse of t.he usual behaviour but can easily be understood when it is noticed that, containing cobalt(I1) as it does, the host is paramagnetic and is relaxing rapidly at the higher temperatures. Indeed the relaxation time of the C o 2 + ion was estimated over a temperature range as a function of the vanadyl linewidth.1 3 6 The porphyrin niobium dimer [(OEP)Nb1203, where OEP is octaethylporphyrin, may undergo up to three electrolytic oxidations and three reductions in one-electron-transfer steps. Free radical e.s.r. signals are seen in each case but the best clear example of a Nb4+ species is that attributed to [ (OEP)NbO12- which is generated on reduction at - 1 . 5 V and is third in time sequence after two free radicals. On reoxidation of this species at - 1 . 0 V, neutral [(OEP)NbOl is formed and gives a similar e.s.r. signal.1 3 7 Nb(OMeI4, also was formed by electrolytic reduction, this time from NbC15 in dry methanol. In solution at room temperature it gives an e.s.r. signal with a ten line hyperfine structure from 93Nb as expected, but it also gives a half-field line. The alkoxide is poorly soluble in most solvents by which it is not hydrolysed; this

64

Electron Spin Resonance

is an indication of its probable polymeric nature and the g 9 4 line is attributed to a AM^ = 2 absorption resulting from the presence of dimers. This is claimed to be the first characterization of niobium alkoxide dimerisation observed by e.s.r. The presence of t.rimethy1phosphine or other such donors yields only weak superhyperfine structure, an indication that t.his alkoxide, in keeping with other early transition metal alkoxides, tends to self associate rather than form complexes with donor ligands. In this respect its chemistry is shown to be similar to that of niobium(V) compounds.138 Reference to niobium bistrimethylsilylxylenediyl biscyclopentadienyl, a niobium(1V) metallaindanla6 has been made in the previous section in relation to related group(1V) compounds. Reduction of LiTa03 under argon creates oxygen vacancies which, on bleaching, transfer an electron to the metal to form a Ta4+ ion.139 The e.s.r. study of this system is incomplete but the immediately noticeable observation is the very low g value of 1.2 The species is rather unstable when the field is along the c axis. at ordinary temperatures since the spectrum disappears above 77 K. Apart from the rather large "Ta hyperf ine splitting and increased g shift, this Ta4+ e.s .I. spectrum is not unlike that of Nb4+ in the same lattice. Hovnanian et a l . have recently described the synthesis and properties of TaBr3 (PMe2Ph)2. 140 Your reviewer sensed a keen interest in this compound since, the existence of a monomeric Ta3+ compound with S = 1 would be very surprizing. No e.s.r. spectra were given but a gav of 1.55 was quoted for the solid and a g of 1.81 with an eight--linehyperfine structure for the methylene chloride solution, both at room temperature. These data convey a slight sense of unease since they could equally well describe a Ta4+, s = 112 sysIndeed t.hey have proposed a tem. Cotton e t a l . felt the same.14' reformulation, using X--raycrystallographic and spectroscopic arguments that the compound has in fact the approximate formula TaC12Br2(PMe2PhI2 and therefore belongs, as far as we are concerned, in No doubt we will hear more about it in the this sect.ion on Ta4+. future. Quinquevalent Chromium a n d M o l y b d e n u m . The nature of CrOI-, its ground state and its relationship to similar ions such a s MnOi-. continue to attract considerable attention. When K2S04 doped with

CrOi- is subjected to X irradiation at 7 7 K , a well-resolved spectrum, observable at helium temperatures, is seen. Analysis shows that this arises from CrOi- which is not perturbed, as was the anion

2: Transition-metal Ions

65

in an earlier irradiation at room temperature, by the presence of a As such it may now be compared with Mn0;- in the nearby proton. same lattice. The two were found142 to have different g anisotropies and different ground states being largely dZ2 and d X Y respecThe CrOa- ion together with CrO;, have been postulated as tively. active sites in chromia-alumina and chromia-silica catalysts but their identity is difficult to confirm and a previous assignment of CrOj is in disagreement with ligand field calculations. Maple and Dalal feel that a tricoordinate species such as the latter should exhibit a longer T I than a tetrahedral species such as CrOi- and this should be reflected in the spectral linewidths provided that the lines are homogeneously broadened, i.e., the dominant linebroadening mechanism is spin-lattice relaxation. CrOi- should thus be observable with a larger signal linewidth which should sharply decrease with decreasing temperature and the difference in expected behaviour should be sufficient to distinguish this ion from Cr03. Their findings'43 were that the so-called C centre from r-irradiated K2Cr207 is in fact CrOi- in agreement with theory and not CrO; as Similar reported; conversely, the A centre is CrO;, not CrO:-. Cr(V) species may be formed in which the chromium is not introduced as an oxochromium species. For example, KH2P04 doped with Cr3+, after r-irradiation, yields two spectra, both analysed as from Cr5+. One of these has g values close to those of CrOa- and in all probability results from Cr5+ surrounded by oxygens.144 In the area of non-ionic chromium(V) coordination chemistry, only a few reactive chromium(V) complexes are known; see Mitewa and Bontchev for a review.1 4 5 However useful advances have now been reported by the complexation of chromium(V) with lactic and thioglycollic acids146 and by the preparation of the [O=Cr(salen)]+ cation [salen = N,N'-ethylenebis(salicylideneaminato)], and its reaction with donor ligands.147 The cation itself is easily recognized by its isotropic e.s . r. spectrum. Well-defined I4N hyperfine splitting from two nitrogens and all four 53Cr satellites also may be seen. Adduct formation is accompanied by a decrease in the I4N splitting which signals an increased spin density on the 0x0 ligand. Several adducts were studied but the effect of fluoride was unique. It appears that the addition of one equivalent of fluoride leads to no observable e.s.r. signal, though the magnetic susceptibility indicates one unpaired electron per chromium still. Upon the addition of a second equivalent of fluoride a new e.s.r. spectrum is seen whose simulation requires the coupling of two nitrogen atoms but only o n e fluorine. Clearly this reaction needs further investigation.

66

Electron Spin Resonance

Molybdenum(V) chemistry also has been actively followed as exemplified by the complexes with 2 , 3-dimethyl-2,3-butanedioi , 146 salicylic acid146 and p-tolylimide.148 The last mentioned of these are dominated by a strong MOEN triple bond and electronically are quite similar to the more familiar oxomolybdenum(V) species. Sexivalent Manganese, T e c h n e t i u m a n d R h e n i u m . A KBr single Crystal doped with MnOz- which substitutes for the bromide, has been subjected to uniaxial stress. In the e.s.r. spectrum, all six members of the hyperfine structure sharpen and increase in intensity when the stress is applied. This behaviour is interpreted in terms of a reduction of random static strains acting within the vibronic ground doublet of E symmetry.56 Similar experiments with ReO$- yield a much stronger (static) Jahn-Teller effect though under stress the tetragonal axis of the anion is capable of reorientation.55 The use of technetium in radiopharmaceuticals has stimulated interest in the physical and chemical properties of technetium in general. The trigonal prismatic Tc(abt)3 where abt = 2-aminobenzenethiolato(Z-)-S,N tends to be polymeric in concentrated solutions as judged by the gradual appearance of hyperfine structure with progressive dilution. The best resolved e.s.r. spectra were found with a

2 x M solution of CHC13 to which a coordinating solvent such as DMF had been added for disaggregation purposes. Since the spectra taken at several frequencies were almost identical, it was concluded that what features there were must come from hyperfine structure, which is both isotropic and small, or from weak zero field splitting, and that g wa,s almost isotropic. Thus the species in solution under these conditions is monomeric with a strongly delocalized unpaired electron.I o 7 Similar results were found with Re(abt)3. Again there is a strong tendency to aggregation as shown by an isotropic e.s.r. line but the shoulders either side of it represent Both of these moleca monomeric species with isotropic g and A . ules then are assigned to the same (2ai) configuration proposed earlier by Mowali and Porte, a configuration in which the electron density is spread well away from the metal and onto the ligands. d 5 Configuration.- Z e r o v a l e n t V a n a d i u m a n d U n i v a l e n t C h r o m i u m . Previous e.s.r. studies of the d 5 sandwich compounds dibenzene vanadium, in various solutions and solid matrices, h a v e yielded g and A tensors and sometimes the ring proton hyperfine tensors, but at room temperature the rotation of the benzene rings is so fast that all twelve protons are magnetically equivalent and thus only an average

67

2: Transition-metal Ions

hyperfine tensor is obtained. In the paper by Wolf et a1.14' low temperature e.s.r. and ENDOR data of V(bzI2 doped into single crystals of 2,2-paracyclophane are reported. The ENDOR experiment provides good resolution of the ring proton hyperfine structure and surprizingly, ten lines were seen. The principal axis system of g and 5 1 A reflect the D6,., symmetry of the molecule but the isotropic part of the proton coupling tensor classify the protons into three sets of equivalent nuclei whereas from the symmetry of the host only two were expected. A lowering of the site symmetry from D 2 h to D 2 was proposed to explain the spectra, the temperature dependences of which, were related to reorientation processes of the aromatic rings in the following paper. I5O E.s.r. has been invaluable in following the chemistry of some novel Cr+ species. (cp)CrNO(L)(X) [X = halogen, L = PPh3, P(OPhI3, P(OEtl31 for example are 17-electron species whose e.s.r. is indicative of one free electron interacting with one phosphorus nucleus and, in the case of L = PPh X = C1 but only in this case, additThis latter observation is not exionally vith one nitrogen. plained but it presumably arises because the maximum electronegativity in X and maximum electron release in the phosphorus ligand, combine to convert Cr+NO+ to something more like Cr2+N0 thereby shifting spin density onto the NO group. Well resolved spectra showing ligand hyperfine structure also were seen when trans-Cr(L)2(dmpe) 2

'"

[L or

N2 or C2H4; dmpe = 1,2-bis(dimethylphosphino)ethane] in CH3CN CH3CH2CN were protonated by CF3S03H.152 The main product is t r a n ~ - [ C r ( N C R ) ~ ( d m p e ) ~ ] ~(R + = CH3 or CH3CH2) , which unexpectedly gives sharp e.s.r. spectra at both room temperature and below. The satellites from 53Cr were observed in a single crystal but the most informative spectra were those taken in mobile solution at room Here a quintet of quartets was seen in CH3CN as soltemperature. vent; a quintet of triplets in CH3CH2CN and a quintet of singlets in CD3CN. Deuteriation of the CF3S03H did not affect the spectrum. The conclusions were that the spectra came from a trace impurity, [Cr(NCR)(dmpe)2]+ (R = CH3, CH3CH2) as shown in ( 5 ) below for R = CH3, formed by competitive oxidation of the starting material. =

68

Electron Spin Resonance

B ~ v a l e n tM a n g a n e s e , T e r v a l e n t I r o n , R u t h e n i u m a n d O s m i u m . Conditions which control the high-spin/low-spin behaviour of d 5 complexes of iron in macrocyclic ligands are relevant to the function of many haemoproteins where it is now established that a hydrogen-bonded network between the axial ligands and the protein is probably important in regulating the strength of the ligand. Two types of hydrogen bonding are recognized. The metal-bound ligand acts as a hydrogen donor in the first type (M-L-H---XI, but as a hydrogen acceptor in the second (M-L---H-X). Haemoproteins with proximal histidyl imidazole and low-spin bis-imidazole complexes are typical of the first and while in fact several examples of the second are known, a new example, the low-spin dimethoxo(tetraphenylporphinato1ferrate(III), [Fe(TPP)(OCH3)2]-, has now been announced by Otsuka e t a l . 1 5 3 In the presence of the (CH3)2SO/CH30H solvent system, three low-spin spectra are observed; these are interpreted as resulting from different degrees of hydrogen bonding between the iron-bound methoxide and the solvent CH30H. A s the concentration of the latter

increases, so hydrogen-bonding proceeds stepwise, and the three species are assumed to result, from zero, one or two CH30H molecules hydrogen bonding to the CH30- ions. Furthermore the greater degree of hydrogen bonding is the reason for the weaker crystal fields at the ferric ion as judged by the usual tetragonal and rhombic parameters reflected by the g values. This is anticipated since both the a and the IT donor strengths of the axial ligands are reduced as negative charge is removed from the oxygen of methoxide to the hydrogen of CH30H. Such modulation of charge it is argued may well be responsible for the electronic control of autoreduction in haemoproteins. Similar studies have been made in a related macrocycle containing the isoelectronic Mn2+ ion. 154 For example manganese (11)t - e t r a s u l p h o p h t h a l o c y a n i n e yields, in the presence of dimethylformamide/water, three low-spin species though in this case they convert to high-spin with time. The conversion process is accelerated for one of them by the presence, additionally, of hydrogen peroxide. The analysis of haem reduction potential was also examined by Schejter e t a l . this time by actually varying the axial ligands. At the same time, these authors draw our attention to the correlation which ought to exist between the axial and rhombic distortion parameters on the one hand, with optical absorption in the infrared region on the other; it.155

reassuringly they then proceed to demonstrate

Turning now to the second and third transition series, dissolves in the rUtlle structure (Ti02) as Ru(IV) but on

ruthenium reduction

2: Transition-metal Ions

with

69

hydrogen yields Ru (111) as well as lower oxidation states. 5 6

Several impurity ions are likely to exist in a host oxide such as this and there are often difficulties in establishing the metal which gives rise to a given e.s.r. spectrum. In this case, experiments at helium temperatures, with two frequencies and using enrichment with lolRu, gave a positive identification of ruthenium(111). No detailed e.s.r. analysis was given but since an average g value below 2.0 was noted, this was regarded as meeting the critThe conventional lowerion that all g values should be negative. spin d 5 theory was used in other cases, to measure the axial and rhombic ligand field parameters A and V and the orbital reduction Applications to [Ru (H20) J 3 + and L R(NH3 ~ 6 13+ have parameter k . been noted earlier.25 Other studies include the ruthenium complexes of N,S- and C,N,S-coordinating azo ligands;157 and the dithiophosphinates of ruthenium and osmium. 5 8 Q u i n q u e v a l e n t P l a t i n u m . The oxidation of [PtL2], (H2L = 1,2-diphenylethene-1,2-dithiol) by chlorine was studied by e.s.r. spectroscopy.159 The first-formed product, whose lifetime is only a few seconds, is judged, on the basis of a simple four line superhyperNo such evidence is fine structure from chlorine, to be PtL2C1. available for the second product, but by comparison with earlier work with t.he corresponding bromides is thought to be [PtL2C12J-. The unpaired electron is assigned to d,, which can form an out-ofplane bond with two sulphur atoms. This being the case, donation to the metal from the ligands must lower the charge on the platinum and render the label Pt(V) less real.

d 7 Configuration.- U n i v a l e n t Iron a n d B i v a l e n t C o b a l t . Reduct.ion of the nitroprusside ion [Fe(CN) has been accomplished by electrolytic, photolytic and chemical means to give one of two principal products, both of which may be described as radicals. But there has been much discussion as to whether these are best described as iron(1) compounds with NO+ or as iron(I1) compounds with neutral NO ligands. We now learn from Eaton and Watkinsl" that the related pentacyano(4-nitro-imidazolato~ferrate(II~ complex, [Fe(CN) 5NIM14-, may be reduced by dithionite (actually by SO;) to give, as its first reduction product, a paramagnetic species. From the g value, which is higher than those of the radicals above, and from the nitrogen hyperfine structure, which is much reduced, this species is assigned as an iron(1) complex, the pentacoordinated [Fe(CN)4(NIMH)]3- in which the imidazole ligand has been protonated. Likewise, chemical

70

Electron Spin Resonance

(or electrochemical) reduction of tetraphenylporphyrin ferrate(I1) also yields a paramagnetic species,16' again assigned as iron(1) and with t.he unpaired electron in the d,2 orbital of the iron atom. The CoN302 complex (formula ( 1 ) discussed in t.he section on Phase Transitions but with L = 4-methylpyridine) is assigned as low-spin d7 by its strong signal at g = 2.32.81 It is rather curious that this should be the case when we consider that the corresponding compound with two 4-methylpyridine ligands is, as we have seen, in

spin-dependent equilibrium between high-spin and low-spin. One would have expected the compound with only one strong 4-methylpyridine ligand, on this basis, to be high-spin. The oxygen-carrying capability of cobalt(I1) complexes in square coordination is well established, while that of cobalt(I1) in pentadentate stereochemistry has so far received only minor attention. Recent examples include bis(2-hydroxy-l-naphthylidene-3-propyl)am~ne cobalt(I1). E.s.r. is seen only in the oxygenated species where it has been used to show that the reaction is not completely reversible but that at least the rate of decay of the oxygenated complex into non-paramagnetic products is considerably slower than that of some other pentadentate species. 62 The n-arene complexes of cobalt(I1) continue to be a rich source of material for e.s . r. studies.5 9 f 163-165 The mixed sandwich complexes [Co(cp) (Bz)]' have already been mentioned.5 9 With [Co(hexamethylbenzene) 2 1 2 + , helium temperature spectra show orthorhombic distortions much larger than observed hitherto in comparable systems. 63 The cyclopentadienylcobalt tetraazabutadienes are claimed to be the only 19-electron systems containing the cyclopent.adieny1cobalt moiety for which isotropic room temperature e.s.r. spectra are known. 164 And t.he complex (ri6-toluene)bis(q1-pentafluorophenyl)cobalt(II) is shown by a single crystal study t.o have a 2 ~ 1 ground state corresponding to a d J Z configuration. 65 Strong COvalency is assumed in some of these examples.1 6 3 ,165 T e r v a l e n t N i c k e l , P a l l a d i u m and P l a t i n u m . Nickel(II1) Complexes are now well known. A recent catalytic study for example has shown the presence of coordinatively unsaturated and distorted octahedral Ni3+ ions at t.he surface of mixed nickel/magnesium oxide. 1 6 6 And many of its complexes are both stable and isolable, as witnessed by the t.riazacyclononane nickel(II1) dithionate compound whose single crystal study by e.s.r. and X-ray diffraction has recently been publish-

ed. 1 6 7 The g anisotropy is compatible only with a 2 ~ l g ( d , 2 ) ground state and a tetragonally elongated NiN6 octahedron. The crystal

2: Transition-metal Ions

71

structure shows that there are six nickel-nitrogen bonds which form two sets with two longer axial bonds and four shorter equatorial bonds in nice confirmation of the e.s.r. data. However, even if nickel(II1) is well known, substitution reactions at the nickel atom are not and this has been the interest of Fairbank and McAuley who, starting with molecules of the type CNiL(OH2)2]3+, where L is one of three tetraaza-macrocyclic ligands, followed their substitution reactions with the halides, chloride or bromide.'68 The first step is the substitution of one water molecule by halide and appears to follow a dissociative mechanism. There is a second, unimolecular, step independent of both halide and nickel complex concentrations. This is believed to be removal of the second water to form a five-coordinate species presumably [NiLXI2+ since hyperfine structure from one halide is observed. This five-coordinate complex is unexpectedly neither truly square pyramidal nor trigonal bipyramidal since the g anisotropy is lower than axial, Tervalent species of the higher congeners in this group also are known at least as intermediates in solution or as trapped ions in solids. The e.s.r. spectrum of a single crystal of CaO doped with PdC13 for example, showed the presence of Pd3+ in the crystal as grown, i.e. not irradiated. The spectrum has already been discussed in the section on Jahn-Teller Effects . 58 We mention here however that anomalous effects in the Io5Pd hyperf ine structure were caused by a relatively large quadrupole interaction which is strong enough that exact diagonalization was required rather than use of the usual perturbation theory formulae. A s with Ni3+ above, the g anisotropy indicates an elongated octahedron though in this case there was a positive g shift for which a second order contribution from an excited energy level due to the t 2 3 g configuration arising mainly from 4 7 1 was invoked. A comparison may be made with other d7 configurations. Rh2+ 4 d 7 for example has a lower charge and therefore weaker ligand field; the weaker splitting of the electronic energy levels therefore causes greater mixing and greater positive g shift. Such a trend should continue on going to 5d7 (Pt3+ and Ir2+) where the well known increase in crystal field strength leads to a negative g shift for these ions. A further example of platinum(II1) has been given by Nizova et a l . 16' When photoinduced by light from a high pressure mercury lamp, [ptC16l2- reacts with arenes, alkyl derivatives of tin and germanium, olefins and saturated hydrocarbons to give u and IT complexes of Pt(I1) and Pt(1V) apparently via intermediate complexes of

72

Electron Spin Resonance

Pt(II1). The reaction with acetone for example gives a frozen The proposed mechanism solution e.s . r . spectrum typical of Pt3+. involves electron transfer from acetone, in its enol tautomeric form to the quadrivalent platinum of [PtCl6I2-. Configuration.- B L v a i e n t C o p p e r . Of all the transition metal ions, Cu2+ is the one configuration about which more is written than any other. For this reason I will be correspondingly brief in my comments. A short review has appeared; It focuses attention mostly on square and distorted tetrahedral CuC14 complexes. Turning first to papers which have some bearing on the structure d9

of the environment around the Cu2+ ion, we note that the X-ray crystallographic structure of CuLL1.6H20, where Is and L1 are tridentate ligands, determines the copper environment to be distorted octahedral, and that e.s.r. indicates this symmetry to be preserved in solution.170 Three examples of the relatively uncommon tetragonal compression which leads to a d Z 2 ground state, are mentioned. These are: CCu(phenanthro1ine) 202CCH3]+; l 7 copper-doped ammonium 173 The first iodide;1 7 2 and the hexafluorocuprate ion, [CUF~]~-. of these becomes axially elongated at low temperature. Stach et a 1 show, by the non-alignment of g and A with the crystal two-fold axis, that the planar bis(maleonitriledithio1ato)cuprate ( I1 ) anions [ Cu (mnt)2]2- , do not adopt the tetrahedral structure of the [Zn(mnt)2]2- host. Rather an angle of 30' between ligand planes is suggested by e.s.r. and confirmed by ENDOR.174 While for [CuL2I2+ ( L = 2,2'-biquinoline N,N'-dioxide), in spite of the clear indication from the g anisotropy, of a dx2-,2 ground state, corresponding to square, square pyramidal or tetragonally distorted octahedral geometry, the value of A , , at 1 0 5 gauss is only two thirds the value of the related biquinolyl derivative, and a pronounced tetrahedral distortion is suspected.'75 [CuL2C11+ on the other hand with its similar ligand, L ( = 2,2'-bipyridine), is found to have a nearregular trigonal bipyramidal stereochemistry by X-ray analysis, though the g tensors of this, and related molecules, were consistent with either triganal bipyramidal or square pyramidal structures.176 CuL3Br2 (L = benzoxazole) 77 and CuLL2. (CH3)2Co.H20 (H2L2 = iminodiac@tic acid)'70 a l s o are found to have trigonal bipyramidal stereochemistries though the latter is not preserved in solution. copper has been studied also in the distorted antiprismatic site of strontium formate dihydrate where, a s expected, the and A tensors do not coincide, in conformity with the low symmetry there.I78

2: Transition-metal Ions

73

The location and coordination of Cu2+ in various alkali metal-X zeolites was shown to be dependent on the replacement of alkali metal by thallium. It seems that the presence of T1' forces the copper into the a cage to form the hexaaquo species while the presence of Na+ and K+ results in the formation of triaquo or monoaquo species.17' Long chain hydrophobic molecules possessing hydrophilic head groups typically dissolve in bilayers with their head groups at the outer surface; such molecules may be described as amphiphilic. In this way the amphiphilic molecule comprised of a long chain hydrophobic azobenzene moiety with a macrocyclic tetramine (cyclam) as its hydrophilic head group, dissolves in t.he bilayers formed by a long chain quaternary ammonium bromide. Under these conditions, copper may complex with the cyclam part and when it does is, as judged by the observation of g anisotropy and of hyperfine structure, monomeric, with no evidence of stacking of the macrocycle ligands. In the absence of the quaternary ammonium salt however, the amphiphile itself forms bilayers as shown by the single broad line indicative of strong spin-spin interaction between the Cu(cyclam) head groups. Coordination of Cu(I1) with two different N3 macrocycles has been studied; each ligand forms CuLH, with x = 1 , 2, 3 . Stability constants and spectra were calculated from the e.s.r. titration data by the method described in the earlier section entitled A n a l y s i s o f S p e c t r a , C o m p u t i n g . '*I

The stability of the copper-sulphur bond is put t.o good use in the dithiocarbamate-functionalized copolymer which has been studied as a reagent for the concentration and separation of precious metals from base metals.182 An incidental finding is that at 1% loading of copper, the preferred site is five-coordinate, but at a higher loading (about 1 5 % ) , the coordination is CuS2N2 and the species responsible is claimed to be one of the closest models yet found for the "blue" copper proteins. The greater stability of the L L or D D diastereoisomers over their mixed counterparts, the L D or D L diastereoisomers, has earlier been noted with respect to the copper complexes of amino acids and dipeptides. Bonomo e t a 1 have now made careful e.s.r. measurements and have shown slight differences in the Hamiltonian parameters for several copper(I1) dipeptides. The perpendicular region was usually complicated by the appearance of nitrogen superhyperfine structure but nevertheless, good values of g1 and A~ were calculated by exploiting the presence of overshoot peaks as explained in previous

Electron Spin Resonance

74

papers.

A

slightly larger

gl,

and smaller

AI,

were usually observed

for the L D dipeptides and although the reason for this might be through a stronger interaction with apical solvent molecules, in fact, a small distortion from square towards tetrahedral was the favoured explanation. We turn away now from structural information and consider a few papers which describe the use of e.s.r. to follow the progress of a chemical reaction. Stach et a 1 have a particular int.erest in the ligand exchange reactions between copper(I1) and nickel(I1) chelates of various sulphur- and selenium-containing ligands. The rearrangement of Cu[Se(S)CNEt2I2 at elevated temperatures has been followed by e.s.r. and mass spectrometry. The selenium donor atoms in the CuSe4-nSn coordination sphere give a linear contribution to g and A values with the result that several mixed ligand complexes may be recognized. Thus the conversion of CuSe2S2 to CuSe3S and CuSeS3 was followed by observation of the 63 65Cu and 77Se hyperf ine structure. Stopped flow e.s .r. measurements with a time resolution of 1 ms later were used t.0 study ligand exchange between copper dithiolenes, or copper dithiocarbamates, with copper diselenocarbamates; simple second-order rate laws were observed. . The mechanism of the formation of copper phthalocyanine from phthalic anhydride and urea, also, has been followed, in this case by specific labelling with I4N, 63Cu and I3C. Analysis of the superhyperfine structure detectable by e.s.r. confirmed a reaction mechanism that includes the formation of phthalonitrile as an intermediate product. l e 6 In experiments with another macrocycle, one of the two acidic hydrogens in tetraphenylporphyrin (H2TPP), was replaced by a methyl group. The resultant ligand HL formed a complex CuLC1 which, not surprizingly, was recognizably different, through its e.s.r. parameters, from CuTPP. Of more interest perhaps was the spontaneous demethylation of the CuLCl complex which, by virtue of these different parameters, could then be followed as CuLCl was converted to CuTPP. Increasingly, double and triple resonance experiments are being reported. ENDOR for example was used to evaluate the hyperfine tensors of all 3C nuclei in the copper (11) bismaleonitrile anion while the signs of the couplings were obtained through TRIPLE resSimilar experiments have been performed on bis (dithioonance. car barn at^)-^" and bis(g1ycinato)-copper(I1) using I4N ENDOR; on

”’

bis (dithiophosphato)- and bis (dithiophosphinato)-copper(I1 using ’H and 31P ENDOR;’” and a bis(glycinato)copper(Ir) using 2H ENDOR. The hyperfine couPlin9 Constants of donor nitrogens in a wide range

2: Transition-metal Ions

75

of copper(I1) complexes have also been found using I4N ENDOR, and correlated according to the donor set. Thus N4, 03N3, cis--02N2and t r a n s - 0 2 N 2 donor sets could be separately grouped, and unknown metal binding sites thereby determined. 9 3 Zerovalent Cobalt, U n i v a l e n t N i c k e l . Zerovalent cobalt forms mononuclear complexes of composition Co(olefin)(PMe3)3 or Co(dio1efin)(PMe3I2. The thermal stability of the latter are expected to be enhanced by the chelating effect offered by the diolefin. With norbornadiene however this is not the case since it forms the monoand bis-exo complexes (6) and (7) below. The monomeric nature of (6)

is shown by its 2 4 line e.s.r. spectrum while in (7) it appears that a through-space interaction between the norbornadiene TT "systems results ir. a strong antiferromagnetic coupling of the two cobalt(0) centres and no observable e . s . r . 9 4 The cyclopentadienyl compound (cpd)C0(C0)~ reacts with the neutral thiadiborole L (a five-membered ring comprising two carbons, two borons and one sulphur atom) to give (cpd)CoL which is easily reduced to [(cpd)CoL]-. The general appearance of the e.s.r. spectrum of this anion, and in particular the g values, are similar to that of (6) above. A similar reaction takes place starting with (cpd)Ni(C0I2 though in this case (cpd)NiL is not reducible. It is however isoelectronic with [(cpd)CoLl- and gives a similar e.s.r. spectrum though of course without the eight-line hyperfine structure. 95 Univalent nickel is commonly produced from the bivalent state by ionizing radiation; three examples are given here. Ni2+ may be doped into CaF2 and after X-irradiation, Ni+ is found, but it is not at the Ca2+ substitutional site. A single crystal study has shown a tetragonal Ni+ centre whose e.s.r. angular variation shows an offcentre position for the defect. Using I9F ENDOR, interactions with up to seven fluorine-neighbour shells were measured and the angular variation of these confirmed the suggestion from the e.s.r. study that the Ni+ centre is displaced from the cubic Ca2+ site towards

76

Electron Spin Resonance

the face of t.he fluoride cube so that it is almost, but not quite, A five-line hyperfine structure shows the expected square planar. interaction with four equival.ent fluoride ions. 9 6 Ni+ is also found in fresh grown AgCl crystals. This impurity, along wit.h others (Co2+, C u ' ) has t.he effect of suppressing the ability of the silver halide matrix to darken by using up some of the photoelectrons which otherwise would be used to build up the silver met.al specks on exposure to light. Its e.s.r. features which are typical of the d x 2 - y 2 ground state, are best resolved at 3 5 K and are lost above 160 K. The centre is also produced together with it seems that a Cu2+ defect by ultra violet irradiation at 50 K ; the Ni2+ traps the photoelectron while Cu' traps the The reader is referred forward to the section on S = 3/2 for reference to Co2+ in this same lattice. with the dx2-,2 ground state also is formed when the NiS202 Ni'

hoto oh ole.'^^

complex bis(2-thiopyridine N-oxide)nickel(II) is irradiated at 77 K by y rays. Here an interesting comparison is made with the corresponding copper(I1) complex which has smaller g shifts even t.hough it has a larger spin-orbit coupling parameter. The reason for this is understood by the analysis of the 61Ni and 63Cu hyperfine structure which reveals a greater delocalizat.ion of spin density, hence a lower k value, for the copper. Estimates of the 3d orbital separations also were made, from the g components. Smaller separations for the nickel complex were derived by this method which is in keeping with the higher spin density on the nickel, smaller positive charge and consequent weaker bonding. 98 Controlled potential electrolysis has been used to reduce nickel(11) and palladium(I1) species. In the case of the mixed ligand complex [Ni (dpe1 (sacsac1 1' (dpe = 1 , 2-bis (diphenylphosphino)ethane and Hsacsac = lI3-dithio-p-diketone) the e.s.r. spectrum of the oneelectron reduction product shows a triplet from the two phosphorus atoms while the anisotropy of the g and A tensors indicate about 75% localization of the unpaired electron on the nickel. Such a complex may properly be regarded as a nickel(1) species. The corresponding [Ni(sa~sac)~]- anion and its analogue [Pd(~acsac)~]-however both show less anisotropy, indicating a greater delocalization away from the metal and onto the ligands so the label "univalent" is here less good. The reason for the different behaviour was sought in terms of stabilization of the ligand IT orbitals through interaction between orbitals on different ligands, but not properly understood.

77

2: Transition-metal Ions 4 s = 1

Configuration.- U n i v a l e n t C o b a l t a n d B i v a l e n t N i c k e l . The cobalt complex [n-B~~N][Co(bds)~](bds = benzene-1,2-diselenolate) formally contains the cobalt (111) oxidation state with a d 6 configuration. Earlier magnetic susceptibility studies had revealed a triplet ground state but no e.s.r. was seen at that t.ime. An e.s.r. signal has now been observed below 10 K however and is interpreted also in d8

terms of a triplet ground state.200 Extended Hflckel molecular orbital calculations suggest that though there should be two unpaired electrons, and that the orbital for one of these is based on the metal, that for the other electron is spread over all four selenium atoms, thus accounting for a considerable degree of delocalization. Perhaps the complex should better be regarded a5 d 8 cobalt(1) therefore. The pressure dependence of zero field splitting in NiSiF6.6H20 has been related to high order perturbation formulae. The conclusions were that the sign of n indicates whether expansion or that contributions contraction along the C3 axis has taken place; to D arise not only from effects of low symmetry crystal field and spin-orbit interaction, in t.riplets as suggested earlier by Gxiffith, but also from spin-orbit interaction with singlets. A detailed pressure-dependent wave function for the Ni2+ 5

S =

d

orbit is given.3 1

312

Configuration.- T e r v a l e n t Chrarniurn a n d Q u a d r i v a l e n t R h e n i u m . Chromium(II1) is a commonly studied ion. Doped into KH2P04 it has been used to display the working of a two circle e.s.r. goniometer4’ and doped into alexandrite, where it substitutes for A13+, emission from one site leads to resonant reabsorption in the mirror site thereby shifting the sublevel populations in the ground state. Interest in this material arises from the four-level tunable lasing action on one of the phonon sidebands of the Cr3+ optical emission spectrum.20’ A s the acetylacetonate complex, Cr(acacI3, doped into the isomorphous gallium, aluminium or cobalt(II1) hosts, showed very little difference in its g and D tensor^.^' Thus, in contrast with ionic lattices where considerable variations in D parameters are observed in isomorphous compounds, the geometry of the paramagnetic ion in these molecular crystals, seems to be little affected by changes in d3

78

Electron Spin Resonance

the size of the host ion. The zero field splitting pattern seems to be largely caused by the first coordination sphere of oxygens alone. Rhenium is notorious for its propensity to give strong e.s.r. lines in the places expected for forbidden transitions; the allowed transitions, or some of them at least, having been partly robbed of their intensity, are then correspondingly weak. Harris and Tucker found, in their study of Re4+ doped into (NH4)2PtC16 that the Am = * I transitions were not only comparable in magnitude to the allowed Am = 0 transitions but a150 were shifted relative to the positions expected on the basis of Q band spectra (35 GHz) . 202 With Bufaical they attributed such features to the presence of random strains by adding the term S.d.S to the Hamiltonian; d is traceless and its components are related to components of strain.203 Configuration.- Zerovalent Manganese, Univalent Iron and Bivalent Starting with MnTPP (TPP is tetraphenylporphyrin), which has a d 5 configuration with S = 5/2, adduct formation with nitric oxide produces [MnTPP(NO)] which could be regarded as having Mn+NOif it is considered that the odd electron on the nitric oxide ligand is transferred to the metal. Gamma irradiation in 2-methyl-tetrahydrofuran at 77 K transfers a second electron on to the metal to give [MnTPP(NO)]-, and a d7, S = 3 / 2 state is finally suggested by Warming rethe typical manganese sextet in the geff = 4 region. verts to the MnTPP with its characteristic geff = 6 feature.204 Various Fe+ centres in NaCl are known which, though S = 3 1 2 , have g values like S = 1/2 and are therefore described as S ' = 1/2. Gamma irradiation of the Fe2+-doped NaCl yields two spectra of comparable intensities: Fe+(ortho) assigned to an orthorhombic centre; and Fe+(tetrag), to a tetragonal centre. The two differ in the way the original Fe2+ impurity was charge-compensated by a nearest, or a next-nearest Positive ion vacancy respectively. The second grows at the expense of the first at 1 7 0 K while both give way at 220 K to Fe+(cub), a centre with a cubic spectrum, in which the positive ion vacancy has now migrated far from the Fe' ion. These observations were reported independently by two groups205 206 though one group prepared the centres simply by irradiating nominally pure sodium chloride,205 while the other group describe their preparation in terms of a heat treatment at 800 K followed by d7

Cobalt.

1

quenching to room temperature immediately before irradiation.206 Exactly the same types of centre are found in cobalt(I1)-doped In this case, since Co2+ ions are silver chloride as grown.I g 7 always present as an unintentional impurity in this lattice, the

2: Transition-metal Ions

79

interest is in their ability to use up some of the photoelectrons in the conversion to Co', thereby reducing the efficiency of the silver halide to darken. The relative intensities of Co2+(ortho) , Co2+(tetrag) and Co2+(cub) are again reversibly a function of temperature though this system needs a higher temperature (-. 300 K) to convert to Co2+(cub) which, incidentally is the only one to be photoreduced to Co' on ultra violet radiation at room temperature. Relative to the Ni+ defect discussed earlier (see section on S = 112, d 9 ) , this Co+ impurity is thermally less stable and its parent Co2+ impurity has therefore less ability to suppress darkening than the corresponding Ni2+ impurity. [ C O C ~ ~ ] ~as - the cinchonium salt was studied as a dopant in the isostructural zinc compound.207 Low temperature e.s . r . spectra gave no hyperfine structure but the widespread g values were said to be 1-1/2> transition in a typical of those from the A M s = 11/2> ---> pseudotetrahedral environment, on the basis of similar earlier work by Bencini et a 1 . Below 50 K t.hree groups of eight lines may be seen in the spectrum of cobalt(I1)-doped NH41. They arise from three mutually perpendicular centres in the crystal which in turn arise from the need for charge compensation by a positive ion vacancy. The cobalt enters the NH; site, and the vacancy is an ammonium ion at the body centre of one of the adjacent unit cells. Such vacancies exert strong axial fields which would be expected t.o compress the cobalt That they do is confirmed by t.he strong increase of g,, from site. the octahedral field (cubic) value of about 4.3 (for s ' = 1/21 to t.he observed value of 6 . 6 9 . 172 The range of magnetic behaviour achieved by the C~(H~(fsa)~en)L, compounds has been mentioned in the section on S = 1/2, d7 and in the section of Phase Transitions where the formulae ( 1 ) for n -- 1 and (2) for n = 2, are given.81 The six-coordinate compounds, formula ( 2 ) but with L = 3-methylpyridineI 3-aminopyridine or 3 , s dimethylpyridine, are shown clearly by their e.s.r. spectra to be high spin S = 3/2 but with 5' = 1/2 as also is t.he five-coordinate compound, formula (1) but with L = 2-methylpyridine. The weak axial ligand field strength in the former compounds is attributed to a tilting of the pyridine moiety which reduces TI back bonding from the cobalt to the ligand. 6 S = 512 d5

Configuration.- The 6 S state ions Mn2+ and Fe3+

display

e.s.r.

80

Electron Spin Resonance

spectra which are often extremely sensitive to the environment of the ion and are correspondingly rewarding to study though sometimes complex to intepret. The spin Hamiltonian parameters are frequently measured , sometimes in detail , and related to stru~ture~'~-~' while in other cases measurements over a temperature range have been made 5-2 Their measurement is one for a variety of reasons. 72 thing, while the fundamental reasons for their magnitudes and signs However the empirical is another, which in general escapes u s . superposition theory can be regarded as an intermediate stage which relates the zero-field splitting parameters to structure, and a steady flow of papers27f28r30r220is building up a bank of data here which is bound to be helpful for future theoretical work. It is remarkable, that nearly thirty years after the first reports of e.s.r. in manganese-doped zinc sulphide, there is still something new to report. In this case, Backs has studied platelets (rather than needles) of perfect hexagonal structure (rather than mixed cubic and hexagonal structures) which gave e.s.r. lines sharp enough to permit, at certain orientations, a splitting in the spectrum arising from the two magnetically non-equivalent sites in the wurtzite lattice.221 From these he was able to calculate the cubic fine structure parameter, a , directly (rather t.han the difference a - F ) . Under strong saturating conditions, microwave-induced multiple-quantum transitions also have been seen in this system;222 from their anisotropy, their power dependence and their magnitudes relative to one-electron transitions, it is concluded they are due to double and triple transitions. Over a very small power range, a phase-invert.ed line also appears. These effects are only poorly understood however and observed in selected crystals only. Manganese doping has been used also in ZnTe whose structure has been probed by measurement of a , this time in an D i v a l e n t Manganese.

electric field because the lattice has piezoelectric properties.208 The results were reported in terms of the component R I 4 of the third rank tensor which describes t.his effect. A strong correlation was found between R I 4 and a for the four cubic compounds Zn(Mn)S, Zn(Mn)Se, Cd(MnlTe and Zn(Mn)Te; further, both of these parameters correlated strongly with covalency in t.his series. The results provide additional confirmation of earlier theories. Turning now to some manganese-doped dif luorides, when Mn2+ substitutionally replaces Pb2+ in the superionic conductor pbF2, a strong transferred hyperfine structure ' ' A exists between the Mn2+ and the "F nearest neighbour nuclei. Madrid et have now

2: Transition-metal Ions

81

resolved an earlier discrepancy in which e.s.r. above 77 K gave a smaller value for this hyperfine structure than did n.m.r. at 2 K. In this new study over a wide temperature range, it appears that at about 50 K, a random crystal distortion starts to produce a broad background and loss of resolution while below 2 0 K sharp lines reappear, from the A M s = 11/2> to 1-1/2> transition only, with the correct, larger, spacing. A likely explanation is that a local contraction of the Mn-F nearest neighbour separation occurs on cooling, which causes first an increase in the I9F transferred hyperfine interaction and second, a strain which broadens all lines to first order in D so t.hat only the (1/2> to 1-1/2> transition is seen, the other four lines making up the broad background. A similar variable temperature study, but of Mn2+ in CdF2 notes an increase in the manganese hyperfine splitting constant A , together with g shifts, as the temperature is lowered.216 Decreased vibrational amplitudes of the ligands are responsible for increased spin-orbit interaction, increased 4s admixture and hence larger A , as the temperature is lowered. The lattice vibrations do not affect g but thermal contraction does though the effect is very complex, influencing as it does the coefficients of the 3d wave function in the molecular orbitals, and no simple explanation could be found. A qualitative analysis showed that g could be represented as a power series in temperature however. The same 8-fold cubic coordination obtains when Mn2+ enters CaF2 or SrF2. Annealing at high temperatures (1100 K) causes l o s s of fluoride, gain of vacancies and/or oxide to form low symmetry species. In SrF2, by this method, is seen a new centre with trigonal symmetry.217 The new signal has five groups of six lines typical of S = 5/2, high-spin. In some directions, doubling from interaction with o n e fluoride ion is seen and D is found to decrease with decreasing temperature. The reader is referred forward to the section on S = 3 to see what happens when such a sample is X-irradiated. As for the Cu2+ and Co2+ dopants discussed earlier, Mn2+ in NH41 displays spectra indicative of similar, compressed, mutually orthogonal complexes related to Mn2+ ions , which having substituted for NH4+ ions , have neighbouring NHi vacancies for charge compensation.172 Rather unusually, Mn2+ has been studied as a dopant in paramagnetic single crystals of [CO(H~O)~]MX~ where MX6 = SiF6, SnF6 or PtC16. Not surprizingly, the manganese linewidths are larger than usual; they also broaden on lowering the temperature. These observations are naturally attributed to the random modulation of the

82

Electron Spin Resonance

dipole and exchange interactions between host and impurity ions resulting from fast spin-lattice relaxation of the host and from the slowing down of this relaxation on lowering the temperature. They are put t.o good use, at least for the SiF6 salt whose structure is known, to estimate the cobalt T I which is found by this method to be s in good agreement with other measurements.218 about 3.1 x The manganese probing of lattices such as those above gives very useful information about the e.s.r. behaviour in known geometries. Inorganic and organometallic complexes, often of unknown geometry may t.hen usefully be probed, whether in pure form or by use of manganese as dopant, as the following examples show. Alkylation of MnBr2(dmpeI2 [dmpe = 1,2-bis(dimethylphosphino)ethane] with MgBu: produces the new four-coordinate complex MnBuk(dmpe). This 13-electron species was shown, by its characteristic high-spin manganese(I1) spectrum, and by computer fitting of this spectrum, to belong to the class of rhombically distorted tetraBy contrast, a diamagnetic manganese ( I ) hedral geometries.209 species is formed, as is indicated by absence of an e.s.r. signal, on alkylation with MgEt2. The methylated porphyrin described in the section on bivalent copper also binds manganese(I1) to form complexes of t.he type MnLCl where HL is the methylated tetraphenylporphyrin. Such complexes give spectra with features in the geff = 3.2 - 5.2 region for which D > > h v and the symmetry parameter A = E / D z 0.3. The spectra are solvent-dependent indicating that the manganese is five-coordinate, the sixth position being blocked by the methyl group. The sensitivity of the e.s.r. spectra to axial coordination permitted the following of titrations such as t,hatwith di-n-butylamine (DBA) in which the amine displaces the fifth ligand to form [MnL(DBA)]+. As for the copper compoundr raising the temperature causes demethylation and formation of Mn(TPP) (DBA).187 The e.s.r. spectrum of the seven-coordinate, pentagonal bipyramidal complex of pure Mn(DAPSC)(NCS)2r where DAPSC = 2,6-diacetyl-pyridine-bis(sernicarbazone), has been reported together with its computer-simulated spectrum. The same compound has been doped into Ni(DAPSC)(NCSI2.2H20 where it displays a better resolved spectrum but with slightly different Hamiltonian parameters. The differences are explained in terms of the structures, one of the main reasons being a more asymmetric coordination geometry in t.he latter.210 Mabad et a 1 have reported the initial results of their systematic study of Schiff base manganese complexes as models of the photosystem I1 manganese site.223 The chosen Schiff bases are potent-

83

2: Transition-metal Ions

ially capable of penta- and hexadentate coordination ii'lcluding N203, N302 and N402 donor sets. The e.5.r. spectra were remarkably sensitive to stereochemistry about the manganous ion. For example, of four complexes of the type N302, two were described, by reference to their e.s.r. spectra, as near-axial through a A value close to zero, one as dimeric because a characteristic 11-line hyperfine structure was seen, and the fourth as pseudo-octahedral with coordination by the solvent. Nine other related complexes were also described. Margaret Goodgame and her group continue to be active in using manganese(I1) as a structure probe, concentrating recently on halide-bridged polymeric complexes of the type CdL2XZ2 l l and CdLX22l (L = pyridine or substituted pyridine; X = C1 or Br). Measurements of D and A at both X and Q band frequencies, and often backed up by computer simulation, are used to gauge the influence of donor set, steric and electronic factors. Thus, for the first mentioned group above, D values in general are high when t.he pyridine ligand is a good a-donor, while steric hindrance by the substituent reduces the D value.21 For the second group, somewhat greater A values are found, in keeping with the double-stranded chain structure which is proposed in which t.he halide ions are not all equivalent. Manganese hyperfine structure could be sufficiently well resolved for the chloro complexes that t.he sign of D could be determined from the lt5/2> mean spacing of the hyperfine components in the 1*3/2> transitions. The negative sign thus established corresponds to an octahedron compressed along an approximate 4-fold axis in these complexes. Bivalent manganese occupies a zinc substitutional site when doped into hexaimidazole zinc(I1) chloride. Since there is no need for charge compensation, t.he environment is close to cubic, as witnessed by quite small D , a and F parameters. The observation of two sets of 30 lines, rather than one, was attributed to twinning of the crystals. This twinning always occurred with manganese doping but not with Fe3+ (see next section) or with mixed, ants.2'3

Fe3+ and Mn2+

dop-

Tervalent Iron. When the above-mentioned zinc(I1) site is occupied by iron(II1) the octahedral field of the six imidazole ligands is disturbed by a low-symmetry contribution presumably arising from the need for charge-compensating vacancies. As is frequently the case

for the increased charge, D at 4 . 7 cm-' is much larger, but not so large as to prevent observation of signals from all three Kramers doublets. In principle, a temperature variation should have con-

84

Electron Spin Resonance

firmed that signals in the geff a 4 region arise either from the middle Kramers doublet according to the theory of Castner et a 1 or In fact no such in the lowest doublet according t.o Kedzie et a l . distinction could be made, but using other arguments, the former theory was favoured.224 Although such earlier papers as those referred to above have given a detailed understanding how the geff = 4 . 3 resonance c o u l d occur, examples of detailed analyses in known site symmetries where it d o e s occur are relatively rare. McGavin and Tennant dismiss those examples where the g tensor is assumed isotropic (even if possibly nearly so) because it must be orthorhombic, and for a site of monoclinic or lower symmetry, those examples where g is assumed coaxial with D and also those which omit fourth-order crystal-field parameters. They then proceed to discuss iron-doped calcium tungstate in terms of a very general spin Hamiltonian appropriate to the The e.s.r. transitions for all lowest (ci) Laue symmetry.225 three Kramers doublets were followed and, as expected, those from the upper and lower doublets are extremely anisotropic. Substitutional sites at tungsten and calcium are considered with various forms of charge compensation as also are two interstitial sites. The favoured proposal is that Fe3+ substitutes for Ca2+ in the lattice, with an associated univalent metal ion, M+, which acts as a charge compensator or, with an associated vacancy, as one of the four adjacent calcium positions. This second possibility requires further compensation by a (remote) M+ ion. None of these sites postulated conform to the special symmetries proposed by Griffith: octahedral MA3B3 and MA4B2 (both c2,,) or MA4B2 (n2,,); or tetrahedral MA2B2 (C2,,) all of which are sites of mmm Laue symmetry. Clearly, even a site of the lowest symmetry, c i , can give the completely rhombic geff = 3017 signal. The production of Fe+ centres by 7-irradiation of Fe2+-doped NaCl has been mentioned in an earlier section.205f206 Nistor et a 1 used crystals which were freshly quenched from 800 K to room temperature immediately before irradiation at 7 7 K.206 Using the same quenching technique but irradiating instead with X-rays at room temperature, produced Fe3+ centres however.226 These were shown by their cubic e.s.r. spectra, which contained also superhyperfine structure, to be FeC1; in tetrahedral interstitial sites neighbour cation sites vacant. Some lower were also seen but these would appear to be a ones since the latter grow at the expense of above 3 2 0 K.

with all four nearest symmetry Fe3+ centres precursor to the cubic the former on

warming

85

2: Transition-metal Ions

ZnSe doped with both iron and copper gives spectra attributed to The the lowest and middle doublets of the high-spin d 5 system. multitude of spectra and their anisotropy result from a strong distortion of the tetrahedral ZnSe crystal field due to chargecompensating Cu+ on a next nearest cation site.227 Perhaps the centre should really be described as Fe3+-Cu+ because a four line hyperfine structure is seen on some of the lines. Contrast t.his with the hexakis(ant1pyrine) manganese-doped cadmium perchlorate where as many as 50% of the Ca2+ ions may be replaced by system2" Cu2+ with the introduction of negligible exchange between Mn2+ and cu2+. Iron chelation with DHB (2,3-dihydroxybenzoic acid) or itoic acid (2,3-dihydroxy-benzoyl glycine) is easy to follow because the complexes are intensely coloured. At pH 2 the iron is ferrous but on raising the pH to 4 a change of oxidation state occurs because an 4.2 appears. This signal is typical of e.s.r. signal at geff high-spin ferric in an orthorhombic crystal field, but Bagyinka e t a l take their analysis further.2 1 4 By comparison with other complexes they claim that the degree of anisotropy in such a signal is an indicator of the stereochemistry; small anisotropy arises from distorted octahedral systems with 0 and N ligands, while large anisotropy is typical of distorted tetrahedral centres containing sulphur. On this basis, their two complexes belong to the first category. While your reviewer is somewhat dubious as to the validity of this as a general supposition, there is no doubt that the modulation of the redox state by pH is most interesting and could be the key to a mechanism whereby iron may be scavenged and passed through a biological cell wall in a highly stable ferric form yet, in a more acid environment inside the cell, be easily released in its ferrous form. f

7 s = 3 U n i v a l e n t M a n g a n e s e . As grown, manganese-doped SrF2 contains Mn2+ in a cubic 8-fold environment of fluoride ions. X-irradiation at 77 K yields two e.s.r. signals. One is a known VK centre; the other is Only the outer lines of the cubic Mn+ having S = 3 (3d54s). spectrum were seen, the remainder being masked by the original Mn2+ The same experiment on manganese-doped SrC12 yields spectrum.228 the corresponding centre with Mn+ in the centre of a cube of chloride ions. A superhyperfine structure from these chlorine nuclei is seen when the field is along certain directions.229 This same

86

Electron Spin Resonance

ground state may also be present in a quite different system, that of manganese-doped Bi12Ge020 or Bi12Si020.230 After visible light illumination of such material, an isotropic signal is seen and attributed to Mn2+ in an accurately four-fold cubic field. This implies that before illumination, the manganese was in the Mn3+ ( d 4 ) form or the Mn+ ( 3 d 5 4 s ) form. Optical evidence was given supporting the latter, which alone also is tetrahedral though the former exists too in an orthorhombic site. No e.s.r. was reported but perhaps it would be worth looking for.

2: Transition-metal Ions

87

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3 Metalloproteins B Y G. R. HANSON AND J. R. P I L B R O W

1 Introduction A general review of the e.p.r. properties of transition ions in metalloproteins, and the underlying theoretical basis, has been presented by Palmer.’ Beinert2 has recently given a summary of the history of e.p.r. in biology, particularly emphasizing the crucial role played by e.p.r. in the development of an understanding of the proteins in the respiratory chain. The article includes a particularly instructive table which compares the information content of e.p.r. and Masshauer effect data on iron sulphur proteins. Beinert also notes the importance of e.p.r.-related techniques such as ENDQR, ELDOR, electron spin echo envelope modulation (ESEEM) and the linear electric field effect (LEFE) in refining the data from metalloenzymes. Kamin et al.3 give a brief

summary of the e.p.r. results for multicomponent electron transfer systems while the Sir Hans Krebs Lecture by Williams4 provides a very readable account of some of the general issues concerning metal and protein function in metalloenzymes. An overview e.p.r. of copper proteins has been given by Boas.

93

of

the

[ F o r references see p a g e 130

94

Electron Spin Resonance

Type

2.1. ____

1

Copper

E n z y me s. -

characteristic

A

deuterium

m o d u l a t i o n p a t t e r n was o b s e r v e d i n t h e e l e c t r o n s p i n - e c h o e n v e l o p e s f o r s t e l l a c y a n i n , a z u r i n , laccase and d e c u p r o laccase Type 2 copper h a d b e e n r e m o v e d ) i n w h i c h From t h e d e c a y r a t e o f

a g a i n s t 2H20.

from a q u a n t i t a t i v e

analysis

of

concluded t h a t t h e Cu(I1) sites accessible (c.2.)

to

solvent.

e.p.r.

and

a z u r i n . 7 The f o r m e r

in

Visible

spectra

techniques

modulation

these

exhibited

pattern

and

it

was

directly

are

circular

to

of

which

exchanged

depth,

proteins

used

analogue

(from

been

modulation

absorption

were

L-selenomthionine-containing

the

the

had

H20

dichroism

characterize

the

Pseudomonas

a e r u g i n o sa

a

to

selenium

copper

c h a r g e t r a n s f e r b a n d a t 1 8 , 0 3 4 ~ m - ~ A. r o l e f o r t h e m e t h i o n i n e a s

a

m o d u l a t o r of t h e C u ( I 1 ) - C u ( 1 ) r e d o x p o t e n t i a l b y m e t a l - l i g a n d

back

bonding is

from

d i s c ~ s s e d .T~h e

direct

incorporation

[Cu(I)(thiourea)3]C1, a s t r u c t u r a l analogue apo-stellacyanin

occurred

of

both

of

Cu(1)

Cu-thionein,

into

aerobically

and

a n a e r o b i c a l l y . O x i d a t i o n o f t h e reduced s t e l l a c y a n i n g i v e s rise Type 1 e.p.r.

spectra

characteristic

s i m i l a r method h a s been used

to

a p o - p l a ~ t o c y a n i n . ~A m e t h o d

for

protein

stellacyanin

with

r e p o r t e d . SmalJ. d i f f e r e n c e s

the

native

incorporate

Cu(1)

reconstituting

both in

of

63Cu

the

e.p.r.

into

the

and

r a t e s o f s t e l l a c y a n i n by r a p i d f r e e z e e . p . r .

denitrificans,

of

been

two

the

self-exchange

methods."

d e n i t r i f i c a n s s y n t h e s i z e s a T y p e 1 h l u e copper P.

copper

has

spectra

When g r o w n o n m e t h y l a m i n e a s s o l e c a r b o n i n t h e periplasms of

spinach

hlue

65Cu

isotopic forms h a s been used t o measure t h e e l - e c t r o n

to

protein.8 A

source,

Paracoccus_

protein,

localized

which

mediates

electron

t r a n s f e r b e t w e e n m e t h y l a m i n e d e h y d r o g e n a s e a n d c y t o c h r o m e c. E . p . r . spectra c o n f i r m t h e p r e s e n c e of a

Type

1

'blue'

copper

protein

which is d i f f e r e n t from a z u r i n . Nitrous bacterium,

oxide

reductase

Pseudomonas

isolated

perfectomarina,

from

the

contains

denitrifying

two

identical

s u b u n i t s w i t h a b o u t e i g h t copper atoms p e r m o l e c u l e . A ' p i n k '

form

o f t h e enzym e r e s u l t s f r o m a e r o b i c p u r i f i c a t i o n , w h i l e a much

more

active 'purple' form p u r i f i c a t i o n . Although the

was e.p.r.

p r o d u c ed spectra d u e

by to

anaerobic 'pink' and

' p u r p l e ' f o r m s were s i m i l a r , t h e l a t t e r was b e t t e r r e s o l v e d . T h e e . p . r . s p e c t r a e x h i b i t e d s e v e n l i n e s i n t h e g,, r e g i o n w i t h a h y p e r f i n e c o u p l i n g of 38.98 x

cm-l,

indicative

of

Type

1

3: Metalloproteins

95

copper centres.

s p e c t r a f r o m T y p e 2 c o p p e r were a b s e n t . 1 2

E.p.r.

2 . 2 Type 2 C o p p e r Enzymes.- E l e c t r o n i c , c . d . and spectra of a s c o r b a t e o x i d a s e i s o l a t e d from g r e e n z u c c h i n i (Cucurbita

pepo

medullosa)

r e p o r t e d . Type 1 and Type

e.p.r.

in

2

phosphate

copper

buffer

were

centres

s p e c t r o s c o p y a n d c o m p u t e r s i m u l a t i o n . Of

atoms i n t h e enzyme, f o u r a r e e.p.r.

to

r a t i o o f Type 1 t o Type 2 c o p p e r s w h i c h , i n in

symmetry

from

the

eight

pseudo

a

by

copper

decrease

part,

may

in

to

tetrahedral

the

involve

t e t r a g 0 n a 1 . l ~ E.p.r.

e v i d e n c e f o r i r r e v e r s i b l e damage o f action

the

been

identified

oxidase

by

squash

have

i n a c t i v e , t h r e e a r e Type 1 and

o n e i s Type 2 . Aging o f t h e p r o t e i n l e a d s change

e.p.r.

a

pseudo ascorbate

of

L-dihydroxyphenylalanine and is g i v e n . I t is c h a r a c t e r i z e d by a 3,4-dihydroxycinnamic acid p a r t i a l i r r e v e r s i b l e l o s s of t h e e . p . r . s i g n a l from Type 1 c o p p e r centres i n ascorbate

oxidase

during

secondary

catechol

a c t i v i t y . A c o p p e r s p e c i e s c h a r a c t e r i z e d by a w e a k , b a n d b e t w e e n 3 0 0 a n d 400 nm a n d a n e . p . r .

at

signal

cucumber p e e l i n g s electronic, c.d.

by

reduction

of

ascorbate

hexacyanoferrate(I1)

has

the

early

oxidase

been

spectroscopy to t r y t o

and e . p . r .

c.d.

intermediate

f i e l d s b e t w e e n T y p e 1 a n d T y p e 2 s p e c t r a is d e t e c t e d i n s t a g e s o f ~ a t a 1 y s i s . lThe ~

oxidase

positive

from

studied

by

light

on

shed

t h e e l e c t r o n t r a n s f e r m e c h a n i s m s o f t h e c o p p e r c e n t r e s . The T y p e

copper c e n t r e i s t h o u g h t t o b e h a v e a s a m e d i a t o r

in

t r a n s f e r c h a i n . 1 5 R e o x i d a t i o n o f t h e r e d u c e d enzyme h a s a l s o been reported.16

One of t h e t h r e e Type

1

electron

with

dioxygen

copper

centres

a n d t h e T y p e 2 c o p p e r c e n t r e were r e o x i d i z e d more r a p i d l y t h a n o t h e r Type

1

c o p p e r s . The

principal

active

2

the

site

of

the

ascorbate

o x i d a s e i s c o n s i d e r e d t o c o n s i s t o f o n e T y p e 1, o n e T y p e 2 a n d

two

Type 3 c o p p e r s s i m i l a r t o t h o s e i n l a c c a s e and c e r u l o p l a s m i n . T h e i n a c t i v a t i o n o f b e e f plasma a m i n e o x i d a s e b y s u l p h i d e h a s been d e s c r i b e d . E.p.r. significantly

from

parameters f o r t h e sulphide those

of

native

amine

complex

oxidase

differ

(Type

superhyperfine s t r u c t u r e , a t t r i b u t a b l e to coordinated

2),

nitrogen

is

c l e a r l y r e s o l v e d . R e d u c t i o n o f c o p p e r ( I 1 ) a s a f u n c t i o n o f t i m e was examined u s i n g k i n e t i c s , v i s i b l e a b s o r p t i o n and e . p . r .

s p e c t r a . The

a m i n e o x i d a s e a z i d e c o m p l e x was i n a c t i v a t e d by s u l p h i d e

much

s l o w l y t h a n i n t h e r e s t i n g enzyme. S i n c e a z i d e b i n d s t o

copper

t h e e n z y m e , i t was c o n c l u d e d t h a t t h e enzyme-bound site

of

sulphide

i n h i b i t i o n . 1 7 Extended

s t r u c t u r e (EXAFS) s t u d i e s o f

copper(I1)

X-ray in

copper

is

absorption

bovine

plasma

more in the fine

amine

96

Electron Spin Resonance

o x i d a s e confirmed t h e r e s u l t s from e . p . r . ,

that the

set c o n s i s t e d of n i t r o g e n s and oxygens; i n imidazole

nitrogens

and

one

water

fact

primary

donor

are

three

there

molecule.’*

et a d 9

Suzuki

r e p o r t t h a t , as a r e s u l t of e . p . r . and o t h e r e v i d e n c e , b o v i n e s e r u m a m i n e o x i d a s e s h o w s o n l y o n e a c t i v e s i t e i n w h a t o t h e r w i s e appear

to

be

two

identical

copper

s u b u n i t s . The

depleted

enzyme

is

i n a c t i v e . One o f t h e most i m p o r t a n t r o l e s o f t h e copper i o n s i n t h e enzyme i s t o r e t a i n t h e c h r o m o p h o r e i n a f a v o u r a b l e

geometric

and

e l e c t r o n i c s t r u c t u r e f o r b i n d i n g t h e s u b s t r a t e . l9 C o m p l e x e s o f

pig

k i d n e y amine o x i d a s e w i t h a z i d e , t h i o c y a n a t e and c y a n i d e h a v e

been

c h a r a c t e r i z e d by e . p . r . establish that

the

and

copper

c.d.

spectra

s p e c t r o s c o p y . 2o E . p . r .

anion

are

complexes

s u b s t a n t i a l anion-induced s h i f t s i n t h e e.p.r. f o r t h i o c y a n a t e and c y a n i d e c om pl e xe s .

tetragonal

parameters

Superhyperfine

but

occurred

splittings,

a t t r i b u t a b l e t o endogenous c o p p e r ( I 1 ) l i g a n d s , probably

imidazole,

a r e found f o r t h e s e c y a n a t e a nd c y a n i d e complexes2’. of

The r e a c t i o n w i t h s u b s t r a t e

plant

e i t h e r Euphorbia l a t e x , or l e n t i l s e e d l i n g s ,

amine in

oxidases

the

c y a n i d e , l e a d s t o t h e a p p e a r a n c e of a f r e e r a d i c a l t y p e spectrum with w e l l resolved hyperfine s t r u c t u r e due to a n aromatic r i n g .

The

presence

a p p e a r a n c e of t h e r a d i c a l e . p . r .

of

is

copper

signal, but

of

to is

signal

of

e.p.r.

protons

essential

this

d e r i v e d f r o m t h e s u b s t r a t e . Type 2 c o p p e r e . p . r .

from

presence

in the not

also

s p e c t r a were

o b s e r v e d . 21 G l y c e r o l o x i d a s e from A s p e r g i l l u s j a p a n o n i c u s c o n t a i n s 1 m o l o f p r o t o h e m e a n d 2 g atoms o f c o p p e r

per

enzyme

molecule.

E.p.r.

s p e c t r a were more r e m i n i s c e n t o f a r a d i c a l s p e c i e s a s s o c i a t e d t h e c o p p e r i o n , r a t h e r t h a n d u e t o e i t h e r a copper or a

with

s i g n a l . Weak s i g n a l s a t g = 4 . 3 a n d 4 . 7 a r e i n d i c a t i v e

heme

of

heme

the

chromophore. 2 2 Chemical and s p e c t r o s c o p i c d a t a a r e r e p o r t e d f o r t h e

of (

several

carboxylate

[ ~ C U ( I I ) - O ~ ~, -mI e) t

competitive

( [ 2 C u ( I I ) I)

to

inhibitors

and

half-met

(

d e r i v a t i v e s of t h e b i n u c l e a r c o p p e r a c t i v e s i t e i n t h e competitive i n h i b i t o r s , unusual e.p.r.

binding the

oxy

[CU(II)-CU(I)]) tyrosinase.

For

f e a t u r e s a r e found which

r e l a t e t o d i f f e r e n c e s i n t h e geometry of t h e b i n d i n g t o t h e c o p p e r i o n . T h e poor i n h i b i t o r s g i v e r i s e t o

of

substrate

normal

e.p.r.

s p e c t r a t y p i c a l of t e t r a g o n a l c o p p e r i n a s q u a r e p y r a m i d a l g e o m e t r y w i t h t h e c o p p e r a t o m d i s p l a c e d 0 . 0 3 nm t o w a r d s t h e a x i a l l i g a n d . I t

is

suggested

that

the

protein

pocket

appears

to

assist

in

s t a b i l i z a t i o n o f t h e b i n d i n g o f t h e c o m p e t i t i v e i n h i b i t o r s . 2 3 EXAFS

3: Metalloproteins

97

studies of the coupled binuclear copper site of tyrosinase from Neurospora crassa show coordination in the first shell to two nitrogen and two oxygen atoms but not to sulphur. 24 Broad, weak e.p.r. signals associated with met-hemocyanin originate from a small fraction of the sites where the endogenous bridge has a lower stability constant. The bridge can be uncoupled at low pH resulting in a zero field triplet e.p.r. spectrum of dipolar copper ions where the bridging ligands determine

interacting the Cu-Cu

separation. For the azide bridged case a distance of 0.5 nm was found. 2 5 The question of the stoichiometry of copper bound to dopamine 0-hydroxylase, and the number of copper atoms required for maximum activity, was monitored by titration of the enzyme with copper.26 It was shown that the nuclear spin lattice relaxation time of solvent water protons determined by n.m.r., and the amplitude of the g = 2 e.p.r. signal increased linearly up to a Cu:protein ratio of 8 : l . It was concluded that the enzyme binds 8 g atoms of copper per tetramer. Fitzpatrick and Villafranca 27 provide e.p.r. evidence for the inactivation of dopamine 0-hydroxylase by phenyl, phenethyl, benzyl and methylhydrazine. An almost complete absence of copper e.p.r. signals was found after r e d ~ c t i o n . ~ ’Since the Type 2 copper e.p.r. spectrum of the dopamine 0-hydroxylase-Cu(I1)-tyramine-cyanide is distinct from or the that of either the dopamine 6-hydroxylase-Cu(I1)-tyramine dopamine 0-hydroxylase-Cu(I1)-cyanide complexelit is suggested that a quaternary complex involving bound copper in dopamine 0-hydroxylase , tyramine and cyanide exists. 28 Antholine and Taketa2’ report that 2-f ormyl-pyrid i nemonoth iosemicarbazone copper(I1) is readily taken up by red blood cells and is initially bound to glutathione and hemoglobin. E.p.r. spectra at both X- and S-band show evidence of coordination of copper ( I1 to two nitrogens, and this is confirmed by computer simulations. It is suggested that two sulphur atoms complete the in-plane coordination. 29 2.3 Multi-centred Copper Enzymes.- A number of investigations have been concerned with the properties of Rhus vernicifera laccase. In an investigation of spectro-electrochemical, c.d. and kinetic measurements on native and Type 2 copper depleted enzyme to determine the effect of Type 2 copper removal on the tertiary structure, the blue copper reduction potential and

Electron Spin Resonance

98 e l e c t r o n - t r a n s f e r r e a c t i v i t y of t h e

enzyme,

spectra

e.p.r.

were

u s e d t o c h a r a c t e r i z e t h e s t a r t i n g m a t e r i a l . 3 0 An EXAFS s t u d y o f t h e

same s y s t e m

is

relevant

in

it

that

provides

evidence

a

for

h i n u c l e a r s i t e i n n a t i v e laccase t h a t is s i g n i f i c a n t l y p e r t u r b e d by Type 2 c o p p e r removal.31

The c h e m i s t r y of Type

2

copper

depleted

Rhus v e r n i c i f e r a l a c c a s e h a s b e e n i n v e s t i g a t e d w i t h r e g a r d b i n d i n g o f p e r o x i d e and

the

ability

r e d u c t i o n and r e o x i d a t i o n . 3 2

of

the

Multifrequency e.p.r.

studies

d e r i v a t i v e o f Rhus v e r n i c i f e r a laccase c o n t a i n i n g o n e t h r e e c o p p e r a t o m s were r e p o r t e d a t -15OOC. t h e mercury d e r i v a t i v e , that

a

Type

2-like

and copper

centre,

is p r e s e n t . A t S-band,

broadening, Type

2

site

copper

upon

signal

of

establish

exhibits

g-strain

structural

occurs

a

and

studies,

ligand hyperfine

t h r e e n i t r o g e n atoms is r e s o l v e d . A the

which

of

mercury

The e . p . r .

fluoride-binding

to the undergo

to

enzyme

coupling

from

of

reorganization

fluoride

b i n d i n g . 3 3 The

c o o r d i n a t i o n e n v i r o n m e n t o f t h e p a r a m a g n e t i c c o p p e r i n J a p a n e s e and

laccase

Vietnamese

was

examined

room

by

temperature

s p e c t r o s c o p y . A t low t e m p e r a t u r e s , t h e e.p.r.

e.p.r.

p a r a m e t e r s o f Type

s p e c t r a d u e t o J a p a n e s e l a c c a s e show more m a r k e d c h a n g e s

than

t h e T y p e 1 copper.34 L a s e r i r r a d i a t i o n i n t h e 450 nm r e g i o n a b o u t i r r e v e r s i b l e c h a n g e s i n t h e c o p p e r s i t e s o f Rhus

laccase

and

its

Type

2

depleted

s p e c t r a o f t h e enzyme b e f o r e a n d appreciable

after

differences. Stellacyanin

apparent

ultraviolet

irradiation

is

brings

vernicifera

derivative. No

r e a r r a n g e m e n t of t h e p r o t e i n b a c k b o n e o c c u r s , a s

2 for

do

c.d.

not

show

to

insensitive

laser

r a d i a t i o n a t a n y ~ a v e l e n g t h .E~. p~. r . s p e c t r a o f T y p e 1 a n d Type 2 c o p p e r ( I 1 ) c e n t r e s i n V i e t n a m e s e and J a p a n e s e l a c q u e r t r e e laccases show t e m p e r a t u r e d e p e n d e n t s h i f t s

associated

with

conformational

c h a n g e s upon f r e e z i n g . D e t e c t i o n o f 19F s u p e r h y p e r f i n e s t r u c t u r e a t

low

temperatures

upon

addition

of

fluoride

indicates

direct

c o p p e r - f l u o r i d e b i n d i n g . 35 Redox

potentials

two

of

c e r u l o p l a s m i n were d e t e r m i n e d t o

Type be

1 370

Cu(I1) and

ions

390

of

mV.

bovine

were

They

d i f f e r e n t i a t e d d u r i n g a n a e r o b i c r e d u c t i o n of o x i d i z e d c e r u l o p l a s m i n and

the

reoxidation

absorption, c.d.

of

and e . p . r

completely

reduced

ceruloplasmin

by

s t u d i e s . The C u ( I 1 ) i o n which i s r e d u c e d

f a s t e r a n d r e o x i d i z e d more s l o w l y t h a n t h e o t h e r i s t h o u g h t t o b e w e l l away f r o m t h e a c t i v e s i t e , w h i l e t h a t w h i c h i s r e d u c e d more s l o w l y and r a p i d l y r e o x i d i z e s i s supposed t o i n t e r a c t w i t h ( n o n - b l u e ) and Type 3 ( e . p . r .

silent)

copper

c e n t r e s . The

s i t e i s t h o u g h t t o i n v o l v e o n e T y p e 1, o n e T y p e 2 a n d

two

Type

2

active Type

3

3: Metalloproteins

99

copper ions.36 Evans et al.37 review the status of the Type 2 copper e.p.r. signal normally found in ceruloplasmin preparations and conclude that it is an artifact of the purification procedure. Giugliarelli et al. 38 report Monte Carlo computer simulations for the two Type 1 and one Type 2 copper e.p.r. spectra from human ceruloplasmin and determine the stoichiometry for Type 2 : Type la : Type lb to be in the ratio 0.39 : 0.27 : 0.29. The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. Analysis of both optical and X-band e.p.r. spectra showed that there are five distinct paramagnetic copper ions, two of which ( X and Y ) are not involved in enzymatic activity but are chelated to the enzyme. Type 1, 2 and 3 copper e.p.r. spectra were observed. In addition diethyldithiocarbamate acts as a reducing agent for the two Type 1 copper ions when added in excess.39 The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate. In particular its effect on ceruloplasmin was studied. E.p.r. evidence supports reduction of Cu(I1) to Cu(1) by thiomolybdate and suggests covalent binding by sulphide to ceruloplasmin copper. 40 3 Iron Proteins 3.1 Non Heme Iron Proteins.- A detailed study of the exchange of Fe(II1) between pyrophosphate and human serum apo-transferrin-CO 2- revealed the formation of a distinct e.p. r. 3 active pyrophosphate Fe(II1) transferrin - carbonate intermediate during the rapid first phase of the reaction.41 The binding of a number of paramagnetic metal ions to transferrin has been the subject of a number of e.p.r. N.m.r. relaxation studies of the solvent protons in solutions of copper and vanadyl substituted human transferrins have been examined. 4 2 The results show that the protons are approximately 0.35 nm from the ions and are in rapid exchange with the solvent. Gadolinium(II1) binds to human serum apo-transferrin at the two Fe(II1) sites as well as to non-specific sites at pH < 7.43 The observed e.p.r. spectrum (g = 4.96) was interpreted using similar crystal field parameters to that required €or Fe(II1) in the native enzyme. The binuclear cluster of uteroferrin in the reduced and enzymatically active pink form is inhibited by orthophosphate, as indicated by the disappearance of the gav=1.74 e.p.r.

100

Electron Spin Resonance

signal.44 Vanadate also oxidizes the protein to an e.p.r. inactive form but itself yields a vanadyl e.p.r. spectrum centred around g = 2. Uteroferrin and semi-hemerythrin, which possess spin-coupled binuclear iron centres, exhibit large linear electric field effects in their mixed-valence, e.p.r. active states, indicative of non-centrosymmetric paramagnetic centres. 4 5 The largest effect occurs for the gmin feature. Ligation of a histidine ligand to Fe(II1) is suggested. Exposure of met-hemerythrin from Phascolopsis gouldii to sulphide under anaerobic conditions results in a one electron reduction and replacement of the p-0x0 bridge between the irons with a single sulphide bridge. E.p.r. spectra in the 9-2 region are similar to those reported for the 'Rieske' [2Fe-2S] iron sulphur cluster.46r47 It is proposed that the histidine imidazoles are ligated to the iron atoms in this centre. Addition of iron (0.5 mol) per subunit to apo-ferritin results in an e.p.r. signal at g' = 4.3 attributable to high spin ferric ions bound to the protein, resulting from 36% of the added ferrous ions. A ten-fold increase in the addition of ferrous ions causes a three fold reduction in the g' = 4.3 signal. At low temperatures an e.p.r. signal with g values of 1.94, 1.87 and 1.80 is found, which is indicative of an Fe(I1)-Fe(II1) dimer with S = l/2.48 Stoichiometry of metal binding of Mn(I1) and VO(1V) to apo-ferritin has been studied by e.p. r. spectroscopy. 49 Benkovic et al.50 have reviewed the mechanism of action of phenylalanine hydroxylase and have investigated the e.p.r. of copper bound to the enzyme. 5 0 The dioxygen-binding site of mononuclear non heme iron in putidamonooxin has been replaced by a nitrosyl group resulting in the formation of a stable Fe(III).NOcomplex characterized by 51 e.p.r. with g-values of 4 and 2. E . ~ . r , ~ and l Mossbauer studies52 of the ternary 'enzyme.substrate.NO' complex show that the iron is in the intermediate S=3/2 Fe(II1) state and is five coordinate. 3.2 Heme Iron Proteins.- Free radical formation was observed during oxidation of oxyhemoglobin by glyceraldehyde. 53 The locations of the various copper binding sites for horse and human hemoglobin were probed using nitroxide spin labels attached to 6-93 ~ y s t e i n e .Four ~ ~ binding sites were found and the Cu-NO distances were found to vary for human hemoglobin from -1.7nm in frozen

3: Metalloproteins

solution to-0.7

101

nm at 2 9 8 K. Variation of the e.p.r. properties as

a function of p H (4.8 to 7 . 8 ) of the nitrosyl derivative of hemoglobin from Ccrococlium dendriticum arc consistent with the dissociation of the proximal histidine from thc heme iron.55 A more systematic e.p.r. study of the nitrosyl Fc(II1) forms of human and Glycera dibranchiata hemoglobin, myoqlobin and somc model compounds has been reported.56 From e.p.r. studips of thc low affinity Cu(I1) binding site in human hemoglohin, it was concluded that the

Cu(I1)

site is less than 1 nm from the h e m e iron site and that proximal histidine particjpates in oxidation of heme iron and reduction of cupric ions. 57

the the

Powder and single crystal e.p.r. spectra for manganese and Q-band substituted myoqlohin wcre measured at S-. X-, frequencies from which the principal g , hyperfine and zero field splitting between the MS=?1/2 and MS=-t3/2 doublets were determined.58 By means of e.p.r. it was shown that the interaction between copper(I1) and met-myoglobin from G y s i a hrasiliana is p H dependent.59 At high pH, the e.p.r. data indicates coordination t o four nitrogens from four lysine residues. Copper(I1) is more strongly hound than manganese(I1). Comparison of the e.p.r. complexes indicates cobalt oxymyoglobin and model Co-O2

from that

restricted motion of oxygen in myoglobin is the result of hydrogen 60 bonding of the distal histidine to the terminal oxygen atom. Scholes et al. 61 carried out FMDOR on his ( imidazole )-ligated low spin ferric heme systems and identified, for the first time. isotropic hyperfine coupling from imidazole nitrogens ligated to MHz (heme) and the ferric ion. They found l 5 N courlings of -7.4 MHz ( imidazole 1. A protocatechuate 3,4--dioxygenasc was crystallized from Brevibacterium fuscum and found

-7.8

purified to have a

and new

subunit E . p . r . spectra due to high spin Fe(II1) with E/Dw1/3 and a g=9.67 resonance which is much narrower than previously observed ( a H = 1 8 9 . 5 M H z ) indicate a rigid structure in the vicinity of the iron atom E.p.r. characterization of the hinding of the transition state analogues of protocatechuate.

.

2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide with the enzyme indicate high spin Fe(II1) sites with negative zero field splitting parameters. 63 This confirmed that reduction of Fe(II1) is not responsible €or the apparent optical bleaching observed in the final dead end complexes. Broadening of the e.p.r. spectra of the enzyme after lyophilization and

102

Electron Spin Resonance

rehydration in 170 enriched water demonstrates water ligation to the active iron site.64 Binding of cyanide proceeds in two steps, firstly to high spin Fe (111) and then to low spin Fe (11) forms. Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes diol-type oxygenolytic cleavage of the substrate aromatic ring. The active Fe(I1) site binds to nitroxide to produce a S=3/2 e.p.r. active complex (g = 4 , 2) typical of spectra from a single Kramers doublet.65 170-labelled substrate in either the 3- or 4-hydroxyl group results in broadening of the e.p.r. spectrum, implying coordination of these groups to the iron atom. Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase both have Fe(I1) active sites binding t o enrichment shows nitroxide to give a S = 3/2 e.p.r. spectrum. ” 0 that water is bound t o Fe(I1) in the native enzyme.66 The native heme enzyme indoleamine 2,3-dioxygenaseI shows e.p.r. spectra due mainly t o high spin iron (gl= 6 , g , , = 2 ) with weak low spin iron (g = 2.86, 2 . 1 8 and 1.6) forms similar to the benzimidazole adduct of f e r r i - m y ~ g l o b i n . On ~ ~ substrate binding , different low spin heme e.p.r. signals (g = 2.53, 2.18, 1.86) are observed. Compound I of chloroperoxidase was prepared by freeze quenching the enzyme after rapid mixing with peracetic acid. Conventional e.p.r. and rapid passage dispersion mode spectra due to Compound I were obtained by subtraction of the native enzyme spectrum.68 The g-values (1.64, 1.73, 2.00) are consistent with the exchange coupling between an S = 1 ferryl (Fe(1V)) ion and an S = 1/2 porphyrin radical. Such a model accounts for all Compound I spectra studied so far.68 The pH dependence of the second order rate constant for the formation of bromoperoxidase Compound I demonstrates the presence of an ionizable group at the active site with a pKa of 5.3.69 ColtL@ounds I1 and I11 were examined using electronic absorption spectroscopy. E.p.r. spectra of the nitrosyl derivatives of human ferrous myeloperoxidase, human eosinophil ferrous peroxidase and bovine ferrous lactoperoxidase, indicate coupling to two nitrogens resulting from coordinated NO and histidine in the fifth ligand position of heme iron in these enzymes.” By way of contrast, the reduced form of the nitrosyl derivative of ferrous lactoperoxidase shows no evidence of an iron-histidine bond.” The different observations have been suggested as arising from the choice of reductant. 7 Q Mossbauer and e.p.r. measurements on Compound I of horse

103

3: Metalloproteins p e r ~ x i d a s e ~ a r~e

radish

consistent

with

those

€or

reported

c h l o r o p e r o x i d a s e . 68 Low t e m p e r a t u r e e . p . r .

spectra of l o w s p i n

and m.c.rl.

heme c a n d heme d i n n i t r i t e r e d u c t a s e aeruginosa, indicate axial histidine heme c a n d t w o a x i a l

histidine

c y a n i d e t o t h e heme g r o u p s i n

obtained and

for

heme

reductase

€or

ligands d. Binding

results

c

d i s p l a c e m e n t o f t h e m e t h i o n i n e l i g a n d i n heme

ferric

Pseudomonas

methionine

ligands nitrite

from

a

and

in

of the

histidine

s p e c t r a also r e v e a l an i n t e r a c t i o n between

l i g a n d i n heme d . E . p . r . 73 t h e s e chromophores.

Hydroxylamine o x i d o r e d u c t a s e from Nitrosomonas e u r o p e a e which c a t a l y z e s t h e o x i d a t i v e c o n v e r s i o n of N H 2 0 H t o NO2- c o n t a i n s c - t y p e heme g r o u p s a n d o n e P-460 o f t h e o x i d i z e d P-460

21-28

c e n t r e . Mgssbauer and e . p . r .

c e n t r e a r e i n d i c a t i v e of

high

spin

data

Fe(II1)

( g = 2 , 5 . 7 and 6 . 1 5 ) s i m i l a r t o m e t - m y ~ g l o b i n . ~ ~ H s y n t h a s e was

Ovine apo-prostaglandin

titrated

with

w h i c h bound a t t h e a c t i v e s i t e , a n d showed a h i g h s p i n 75 e p. r spectrum.

.

.

E.p.r.

which

g - m a t r i x was d e t e r m i n e d f o r t h e l a t t e r species f r o m s i n g l e at

ion

s t u d i e s o f y e a s t f e r r i c c y t o c h r o m e c p e r o x i d a s e showed

a m i x t u r e of h i g h and l o w s p i n F e ( I I 1 ) components, from

spectra

hemin

ferric

77K.

Powder

spectra

at

formed from t h e r e a c t i o n

of

hydrogen

X-

S-,

c o n j u n c t i o n w i t h s i n g l e c r y s t a l d a t a , show

that

peroxide

the

crystal

and

Q-band,

the

intermediate

with

the

in

enzyme

c o n s i s t s o f t w o d i s t i n c t r a d i c a l species. 76 Oxidized cytochrome c p e r o x i d a s e exhibited three e.p.r.

from (1) a

active centres:

indicating histidinyl-methionyl

Pseudomonas

low

stutzeri

spin

iron

c o o r d i n a t i o n ; ( 2 ) a low

spin

heme i n d i c a t i n g h i s t i d i n y l - h i s t i d i n y l

c o o r d i n a t i o n and ( 3 ) a

h i g h s p i n i r o n heme c o m p o n e n t . 7 7 T h e loss o f t h e h i g h s p i n component from t h e e.p.r.

spectrum

heme

of

the

half

iron minor

Fe(II1)

reduced

enzyme

i n d i c a t e s t h a t a s p i n s t a t e c h a n g e h a s o c c u r r e d . 77 r 7 8 E.p.r.

s p e c t r a of t h e n i t r o s y l d e r i v a t i v e

cytochrome c' i r o n - h i st i d i n e

of

f r o m A l c a l i g e n e s s p N C I B 1 1 0 1 5 showed t h a t t h e heme bond i s weak

and

is

cleaved

upon

report

g r o u p . 79 S c h e j t e r a n d E a t o n 8 '

coordination crystal

a

of

field

nitrosyl

correlations

between n e a r i n f r a r e d a b s o r p t i o n s and e.p.r. d a t a f o r a series o f low spin ferric complexes of cytochrome c and solubilized

myoglobin. Ferricytochrome

C,

c o m p l e x a t i o n w i t h 18-crown-6

ether, gives

rise

in

to

methanol

two

by

low

spin

F e ( I I 1 ) and one high s p i n F e ( I I 1 ) states, a l l of which d i f f e r

from

104

Electron Spin Resonance

what is observed in the native state.81 The electrochemistry and e.p.r, spectroscopy of a series of axial complexes of hemin and hemeoctapeptide from equine cytochrome c have been reported. 8 2 Cytochrome c-544, which functions in the ammonia oxidizing system of Nitrosomonas europeae, gives rise to e.p.r. and Mossbauer spectra due to 75% high spin ferric heme at pH 2 and 100% low spin ferric heme above pH 10. While the Mossbauer evidence is consistent with a 75% ; 25% l o w spin to high spin mixture, the e.p.r. spectra indicate an unusual g = 3.3 feature at X-band which becomes g = 3.0 83 at S-band. Heme-heme interactions are suggested. A method for the preparation of the cytochrome bd complex of cytochrome b-558 and cytochrome d from Escherichia coli has been described. Preliminary characterization by e.p.r. indicated the presence of two high spin heme Fe(II1) centres.84 Similar but more 85 extensive e.p.r. results have been reported by Kumar et al. The spectral intermediate complex formed upon reduction of halothane (CF3CHC1Br), by microsomal cytochrome P-450 (Soret band 470 nm) exhibited a characteristic low spin ferric heme e.p.r. spectrum (g = 2.71, 2.27 and 1.80). By comparison with spectral properties of model tetraphenylporphyrin complexes, the intermediate is believed to be [P-450]-[CF3CHC1-]. 86 The mechanism for the formation of the complexes between various nitrosobenzenes (NBD) and cytochrome P-450 have been investigated. E.p.r. spectra show the formation of a ferrous-heme-NBD complex in cytochrome P-450. 87 Thromboxane synthase from human platelets was purified to apparent homogeneity by chromatographic methods and found to contain one heme per polypeptide chain. Using optical and e.p.r. spectroscopy, a close analogy to the group of cytochrome P-450 proteins was established. The untreated, oxidized enzyme (Soret Band 424 nm) showed two slightly different low spin heme Bisthiolato-hemin model complexes €or cytochrome P-450 were formed from Fe(II1) protoporphyrin IX and characterized by e.p.r. and absorption spectra. 8 9 Superoxide generation by an iron tetra-phenylporphyrin-thiolate-oxygen system has been described and its significance in relation to the coordination site of cytochrome P-450 indicated. Optical, resonance Raman and e.p.r. spectroscopy show that the chloroplast cytochrome b-559 can occur as low spin ferric and ferrous hemes. Histidine nitrogens provide the fifth and sixth axial ligands. The different g values for the high and low potential forms are thought to arise from changes in the

3: Metalloproteins

105

orientation of the heme centresSg1 4 Iron Sulphur Proteins Iron sulphur proteins, with one exception, aconitase, are oxidoreductase enzymes which are paramagnetic in certain oxidation states. Using e.p.r. spectroscopy, the iron sulphur clusters in these enzymes can be used as probes to follow the progress of the enzyme reaction, substrate binding and electron transfer processes. E.p.r. spectroscopy can also provide information about the arrangement of the iron sulphur clusters and other centres within the enzymes. The e.p.r. of the various kinds of t2Fe-2Sl (2+'1+) , [ 3 ~ ~ - 3 / (3+,2+;1+,0) 4~] and [ 4 ~ ~ - (3+,2+;2+,1+) 4 ~ ] centres has been reviewed by Cammack et a1.92 4.1 t2Fe-2Sj (2+r1+) Cluster Containing Enzymes.- Methylococcus capsulatus posesses a multi-component methane monooxygenase which in vivo catalyzes the conversion of methane to methanol. Preliminary e.p.r. characterization of the native enzyme indicates the presence of iron sulphur and radical signals.93 The absorption and e.p.r. spectra of the flavin and [2Fe-2Sl redox centres in the NADH:acceptor reductase component of the soluble monooxygenase indicate that the flavin is a neutral semiquinone and that the iron sulphur centre is similar to that found for spinach ferredoxin. Mid-point redox potentials for these centres have been measured using e.p.r. spectroscopy. 94 Benzene dioxygenase from Pseudomonas putida comprises three components, a flavoprotein, an intermediate electron transfer ferredoxin with a [2Fe-2S] cluster and a terminal dioxygenase containing two [2Fe-2Sl clusters. Presence of these clusters was indicated by their e.p.r. properties. Redox potentials were also determined by e.p.r redox titrations at pH 7 . 0 . 9 5 Existence of a radical species and an iron sulphur cluster in membrane fragments of Heliobacterium chlorum has been confirmed by e.p.r. spectroscopy.96 E.p.r. evidence for new iron sulphur centres present in electron transport particles derived from Escherichia coli, grown aerobically on a range of non-fermentable carbon sources, has been reported. 9 7 The spectra are similar to the 'classical' ferredoxin e.p.r.. The oxidized and reduced forms of the Rieske iron sulphur protein from Thermus thermophilus have been characterized by optical, e.p.r., c.d., m.c.d. and Mossbauer spectroscopy. Evidence

106

Electron Spin Resonance

f o r two i d e n t i c a l [2Fe-2Sl

clL?stersI e a c h c o o r d i n a t e d

most

at

by

t w o c y s t e i n e r e s i d u e s was p r e s e n t e d . 9 8 ENDOR a n d ESEEM s p e c t r a f r o m t h i s enzyme i n d i c a t e d i r e c t c o o r d i n a t i o n o f n i t r o g e n o u s l i g a n d s t o t h e [2Fe-2S]

c l u s t e r s . B o t h s t r o n g a n d weak

nitrogen

are observed, t h e l a t t e r i n d i c a t i n g p o s s i b l e u n p a i r e d s p i n t o remote n i t r o g e n atoms o f ligands.”

Bonner

identification

and

a

of

Princeloo Rieske

coordinated

sulphur

of

the

imidazolate

e.p.r.

using

iron

interactions

coordination

report

protein

the

in

plant

mitochondria f o r t h e f i r s t t i m e . 4.2

[3Fe-3/$S] ( 3 + ’ 2 + ; 1 + r 0 )

C l u s t e r C o n t a i n i n g Enzymes.-

Fumarate r e d u c t a s e c a t a l y z e s t h e r e d u c t i o n of f u m a r a t e t o s u c c i n a t e i n t h e t e r m i n a l r e a c t i o n of t h e E s c h e r i c h i a c o l i e l e c t r o n

transfer were o b t a i n e d f r o m f u m a r a t e r e d u c t a s e membranes: t h a t d u e t o a h i g h p o t e n t i a l i r o n s u l p h u r p r o t e i n when o x i d i z e d a n d a f e r r e d o x i n [ 2 F e - 2 S ] (2+’1+) s i g n a l when c h a i n . Two t y p e s o f e . p . r .

spectra

reduced with dithionite.”’

In addition to the

e . p . r . s p e c t r a f r om a [4Fe-4S] e n h a n c e d s p i n r e l a x a t i o n of reduction,

can

be

m.c.d.

and e . p . r .

c l u s t e r have the [2Fe-2S11+

to

attributed

t e t r a n u c l e a r and b i n u c l e a r

[2Fe-2S11+

1+

an

interaction

c l u s t e r s . lo2

spectroscopy suggests

signal,

b e e n o b s e r v e d . The upon dithionite

Further

that

between

the

work

involving

high

potential

the

Fe-S c e n t r e is a t h r e e i r o n t y p e of i r o n s u l p h u r c l u ~ t e r . ~ ~ ~ , ~ Two new

characteristics

of

the

of

e.p.r

c o n t a i n i n g f e r r e d o x i n f r o m Thermus t h e r m o p h i l u s

the

are

Hagen e t a d o 5 F i r s t , t h e r e d u c e d s t a t e o f t h e 3 F e h a s t r a d i t i o n a l l y been c o n s i d e r e d t o be f o u n d t o e x h i b i t a aMs=+4 t r a n s i t i o n ,

e.p.r.

s u l p h u r c e n t r e s . T h i s s i g n a l is s i m i l a r t o Fe(I1)-EDTA complex and s u p p o r t s t h e

that

suggestion

e l e c t r o n i c s t a t e of t h e 3Fe c l u s t e r i s the e.p.r.

is

which

S=2.

seven-iron

described

by

which

centre,

silent,

has

been

unique

€or

iron

high

spin

a

of that

the

Secondly,

spectrum of t h e f u l l y reduced p r o t e i n a t 9

ground

changes and

15

in GHz

a r e c o n s i s t e n t w i t h a weak e l e c t r o n i c s p i n - s p i n i n t e r a c t i o n b e t w e e n t h e [4Fe-4S11+

( S = 1 / 2 ) and t h e r e d u c e d

e x p l a n a t i o n h a s been g i v e n €or t h e

3Fe

centre. A

observation

of

theoretical

these

signals

with constant effective g values. D e s u l f o v i b r i o g i g a s f e r r e d o x i n I1 ( F d 11) c o n t a i n s [3Fe-4Sl

c l u s t e r with

d i s a p p e a r s upon a

one

a

strong electron

e.p.r.

signal

reduction.

In

at the

D e s u l f o v i b r i o g i g a s c e l l e x t r a c t s , supplemented w i t h e.p.r.

s i g n a l t y p i c a l of a reduced

[4Fe-4SI1+

cluster

a

9=2.02

single which

presence

of

pyruvate,

an

is

obtained

3: Metalloproteins

107

for Fd 11. The cluster interconversion under physiological conditions is discussed in the context of some of the relevant metabolic pathways in this bacterium. Addition of Co(N03) to Fd I1 resulted in the formation of a heterobinuclear [Co-3Fe-4Sl cluster in 94% yield. E.p.r. and M6ssbauer studies indicate a S = 1 / 2 spin system.lo7 Interestingly the magnetic properties of a model heterobinuclear cluster [Co-3Fe-4S] 2- are similar to those found in Fd II.lo8 M.c.d. and e.p.r. studies of Ferredoxin I and I1 isolated from Desulfovibrio africanus indicate the presence of a single [4Fe-4Sl (’+’ 2 + ) cluster per protein subunit. 109 In contrast to the X-ray crystal structure of Azotobacter vinelandii ferredoxin I (Fd I) which shows a solvent accessible 0x0 3+ ligand to the planar [3Fe-3Sl centre, ESEEM studies of both H20 and 2 H 2 0 equilibrated samples show far less deuterium modulation than expected.’” As normally isolated, this enzyme contains a [4Fe-4S12+ and a [3Fe-3S13+ cluster. E.p.r. studies of the reduced reconstituted enzyme indicate the presence of two [4Fe-4S11+ clusters, suggesting that the isolated enzyme results from oxidative degradation during purification. 4.3 [ 4Fe-4S1 ( 3+ r 2 + ;2 + ,1+1

Cluster Containing Enzymes.- l7O ENDOR measurements on the reduced active [4Fe-4S] centre of beef heart aconitase, both in H2I60 and H 2 1 7 0 , in the presence of substrate and several inhibitors, have shown that the iron sulphur cluster participates in substrate binding and can simultaneously coordinate a hydroxyl from a substrate and water.‘l2 X-ray crystallographic evidence confirms the existence of a 3Fe-4S11+ cluster in the inactive form of aconitase from pig heart.’l3 Aconitase as isolated is inactive and contains a [3Fe-4S11+ cluster. On incubation at pH > 9.5, or treatment with 4-8 M urea, a purple coloured protein is derived which can be converted in good yield to the active [4Fe-4S11+ form by reduction in the presence of iron. E.p.r. and Mzssbauer studies of the purple form indicate the presence of a linear [3Fe-4Sl cluster which is similar to synthetic iron sulphur clusters. Absence of nitrogen and proton ENDOR signals of the ferrisiroheme and [ 4Fe-4Sl 2+ centres in sulf ite reductase isolated from Escherichia coli suggests that the siroheme is five coordinate. This rules out the coordination of histidine as a bridging ligand between these centres. In addition, 57Fe ENDOR confirms the results from Massbauer spectroscopy for a [4Fe-4Sl

108

Electron Spin Resonance

c l u s t e r .l15 M o s s b a u e r a n d e . p . r .

s p e c t r o s c o p y of t h e

reduced

presence

ferrisiroheme

in

i n d i c a t e a weak e x c h a n g e ( S = 2 ) and

the

a

contains

the

interaction

[4Fe-4SI1+

low

spin

(S=1/2)

ferric

of

between

high

c l u s t e r . The

heme

two

electron

guanidinium

(S=1/2)

sulfate

spin

ferrous

oxidized

a

and

enzyme

[4Fe-4S12+

The l a t t e r c l u s t e r i s r e d u c e d u p o n a d d i t i o n o f s u l p h i d e . l 1 6

centre.

The

e.p.r.

spectra

of

the

reduced

selenium

substituted

c l u s t e r s from t h r e e b a c t e r i a f e r r e d o x i n s c o n t a i n i n g t w o [ 4Fe-4SI of t h e C l o s t r i d i u m g e n u s , d i s p l a y low f i e l d s i g n a l s a t g = 5 . 1 7 , g = 1 0 . 1 1 and g = l 2 . 7 6 . The p o s i t i o n s ,

line

shapes

and

temperature

d e p e n dence o f t h e s e s i g n a l s h a v e a l l o w e d them t o b e a s s i g n e d t o t h e t h r e e e x c i t e d s t a t e s of a n S = 7 / 2

is

spin

multiplet,

the

fundamental

as unusual f e a t u r e s i n t h e l o w t e m p e r a t u r e ( T < 20 K ) Mgssbauer spectra. I n a d d i t i o n t o t h e S=7/2 state

of

which

observed

s p i n s t a t e i n t h e two [4Fe-4Se11+

Clostridial

ferredoxins,

e.p.r.

s p e c t r a from t h e c l a s s i c a l S=1/2 s t a t e a n d t h e S=3/2 s t a t e a r e a l s o o b s e r v e d ; The f u n d a m e n t a l

of

doublet

a t t r i b u t e d t o t h e broad s i g n a l i n t h e i n t e n s i t i e s of t h e

e.p.r.

signals

latter

the g=3-4

state to

corresponding

t h e f e r r e d o x i n is i s o l a t e d . reduced

Clostridial

which

proteins

by

c o n t a i n s o n l y o n e [4Fe-4Sl

spin

from

which

In contrast to Clostridial ferredoxins,

selenium-substituted

stearothermophilus,

be

relative

these

s t a t e s d e p e n d o n t h e p a r t i c u l a r s p e c i e s of C l o s t r i d i u m the

can

r e g i o n . The

ferredoxin

differs

its

primary

structure,

cluster, displays

f r o m a n S=1/2 s p i n s t a t e . T h u s ,

from

significantly

is

it

Bacillus from since

e.p.r.

only

concluded

the

it spectra

and that

the

high

m u l t i p l i c i t y s p i n s t a t e s a r i s e from a s p e c i f i c i n t e r a c t i o n

between

the

reduced

Clostridial

[4Fe-4S11+

ferredoxin

i n d i c a t i v e of a spin-spin sulphur

polypeptide

~ 1 u s t e r s . l ’U~n u~s u~a l~ e~. p . r .

cluster

centres

interaction

is

reduced

and FMN

when

9=2

and

triplet

state

upon

anisotropic

dipolar

and

or with

s u b s t r a t e . S p e c t r a l s i m u l a t i o n s were b a s e d both

di-

4

iron

electrons

model

assuming

and

the

either

two

by

the

at

signals

between

observed

are

trimethylamine dehydrogenase

chain

a

exchange

i n t e r a c t i o n s . ‘19 A c o m p a r i s o n of

chemical and

s y n t h e s i s of t h e two [ 4 F e - 4 S l

enzymic

(employing

c l u s t e r s i n Clostridium

rhodenase)

pasteuranium

f e r r e d o x i n , r e v e a l s t h a t t h e n a t u r e of t h e c l u s t e r t o b e i n s e r t e d i s d e t e r m i n e d by t h e a p o - p r o t e i n i t s e l f , and t h e r a t e l i m i t i n g s t e p

of

cluster

insertion

is

the

refolding

of

the

backbone. Rhodenase a p p e a r s t o p l a y a role i n t h e r e c o v e r y

protein of

the

3: Metalloproteins

109

native architecture of the reconstituted iron sulphur proteins. 120 5 Nickel-Iron-Sulphur and Related Iron-Sulphur Enzymes

5.1 Hydrogenases.- Hydrogenase catalyzes the reversible oxidation of molecular hydrogen to protons in the presence of suitable electron donors and acceptors. Two types of hydrogenase have been well characterized for Desulfovibrio species. The [Ni-Fe-S] hydrogenase is involved in hydrogen uptake, while the second type of hydrogenase [Fe-Sl contains only Fe-S clusters and is catalytically reversible. Mzssbauer and e.p.r. studies of Desulfovibrio gigas hydrogenase established the presence of a [3Fe-3/4S] cluster and two [4Fe-4S12+ clusters. A rhombic e.p.r. signal (g = 2.31, 2.23, 2.02) is observed. Assignment of the rhombic signal to Ni(II1) was established by the observation of nickel hyperfine coupling using 61Ni isotopically enriched enzyme .l2l This hydrogenase , as isolated, must undergo an activation process in order to express full activity. Characterization of the enzyme, its redox properties and activation profile in the presence of either hydrogen or chemical reductants enabled a working hypothesis for the mechanism of the [Ni-Fe-Sl hydrogenase to be formulated .122 Purified periplasmic hydrogenase from Desulfovibrio vulgaris has been reported to contain 11 ? 1 non-heme iron atoms and no nickel atoms. E.p.r. and Mb'ssbauer studies indicate that two [4Fe-4Sl clusters are In addition, a novel e.p.r. signal at 9-5 is assumed to be a AM^ = f 2 transition within an S = 2 multiplet. It is suggested that O2 sensitization parallels a spin 124 state transition of an iron sulphur cluster. E.p.r. at 4 , 9 and 34 GHz of intact hydrogenase isolated from Chromatium vinosum has revealed a weakly-coupled Ni ( I11 ) - [4Fe-4Sl 3+ pair. The distance between these centres is estimated to be l e s s than 1.2 nm. Inactivated enzyme gives e.p.r. signals from non-interacting S = 1/2 systems of Ni(II1) and a [3Fe-3/4S] cluster.12'E.p.r. redox titrations of this enzyme indicate the nickel ion can exist in the 3+, 2+, 1+ and possibly 0 redox states. A photodissociation reaction of the nickel(1) species occurs which is six times slower in 2H20 than in H20 indicating that a hydrogen atom is directly coordinated to the nickel ion.126 A current understanding of the e.p.r. properties of the [Ni-Fe-S] hydrogenase from this bacterium has been

I10

~ u m m a r i s e d . ’ ~In ~ the iight of this, the role

Electron Spin Resonance

of

nickel

and

the

Fe-S cluster in the function of the enzyme has been considered.128 Optical absorption, m.c.d. and e.p.r. spectroscopy have been used to study the paramagnetic Ni(II1) centre in [Ni-Fe-Sl hydrogenase from Methanobacterium thermoautotrophicum. It is concluded that Ni(II1) resides in a tetragonal site.129 A soluble hydrogenase from Methanobacterium barkeri contains a flavin component (either FMN or riboflavin), 8-10 iron atoms and 0.6 to 0.8 nickel atoms per subunit. The e.p.r. spectrum of the native enzyme shows a rhombic signal probably due to nickel with g values of 2.24, 2.20 and ~ 2 . 0 . Reduction of the native enzyme with either sodium dithionite or molecular hydrogen yields at least two types

of e.p.r. signals. Differences in the effect of temperature and microwave power on the e.p.r. spectra were used to assign these signals to two different iron sulphur centres. Centre I has g values at 2.04, 1.90 and 1.86 while centre I1 has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite, the latter signal disappears and is replaced by signals at g values 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K.I3O The intact NAD-linked hydrogenase from Nocardia opaca lb, containing 14.3-2 0.4 atoms of sulphide, 13.6 2 1 atoms of iron 3.8 kO.1 atoms of nickel and 1 molecule of FMN per enzyme molecule has [4Fe-4Sl and [2Fe-2S11+ e.p.r. spectra characteristic of clusters. The e.p.r. properties of the two types of subunit dimers prepared by preparative pol.yacrylamide gel electrophoresis were reported .l3I E.p.r. characterization of the iron sulphur centre in the [Ni-Fe-Sl hydrogenase from Clostridium pasteuranium has been reported. Iron sulphur core extrusion and e.p.r. spectroscopy of [Fe-S] hydrogenase from Clostridium pasteuranium indicated two distinct paramagnetic species. These were interpreted as arising from one [4Fe-4S] (1+r2+) and one [4Fe-4Sl (2+v3+) cluster per molecule. 133 M8ssbauer and 57Fe ENDOR spectra are consistent with the g=2.1 centre arising from the oxidation of a [4Fe-4Sl centre.134 An e.p.r spectrum (g=2.101, 2.052 and 2.005) detected at liquid helium temperatures due to oxidized hydrogenase from Megasphaera elsdenii, and previously assigned to a [4Fe-4S] 3+ cluster, is now thought to arise from a cysteine persulfide radical. 135

3: Metalloproteins

111

5.2 Other Nickel containing Enzymes.- Evidence for an iron-nickel-carbon complex formed in the reaction of carbon monoxide with the 57Fe enriched carbonmonoxide dehydrogenase from Clostridium thermoaceticum is provided by broadening of the e.p.r. spectrum.136 EXAFS studies of jack bean urease show that the nickel centre is similar to that of a model compound tNi ( L I Z ( L ' ) 1 (C104) where L is l-n-propyl-2-~-hydroxybenzylbenzimidazole and L ' is the 137 deprotonated form. No e.p.r data was reported.

6 Molybdenum Containing Enzymes 6.1 Molybdenum Iron Enzymes.- A useful and brief introduction to the biochemistry and e.p.r. of nitrogenase has been given.138 A more extensive review13' and a recent book140 provide a fuller background to its enzymology. Nitrogenase consists of two proteins each of which exhibit e.p.r. signals in the resting state and during enzyme turnover. The larger MoFe protein with 2 g atoms of Mo, approximately 32 g atoms of Fe and approximately 30 g atoms of S 2 - has e.p.r. signals arising from the S = 1 / 2 doublet of an S = 3 / 2 spin system. E.p.r. spectra from the iron molybdenum cofactor ( FeMoco) are reported. 138 ENDOR spectra of the resting state of nitrogenase MoFe proteins from Azotobacter vinelandii, Klebsiella pneumoniae and Clostridium pasteuranium have been reported141 and indicate that a single molybdenum is integrated into the MoFe spin system as Mo(1V). ENDOR hyperfine data is reported €or 'HI 57Fe, 9 5 , 9 7 ~ o and 3 3 S nuclei. Spin quantitation of the MoFe protein e.p.r. signal shows the presence of two S = 3 / 2 spin centres with a zero field splitting of 15 1 cm-l, determined from variation of the double integral of the spectrum as a function of temperat~re.'~~Single crystal EXAFS spectra of the MoFe protein of nitrogenase suggest the existence of trinuclear Fe-Mo-Fe clusters with Fe-Mo-Fe angles lying between S O 0 and 130°.143 Activation of the inactive MoFe protein (nifB-Kpl) of nitrogenase from nifB mutants of Klebsiella pneumoniae by addition 144

of FeMoco from active protein was confirmed by e.p.r.. The e.p.r. spectrum of the reduced Fe protein of nitrogenase was reinvestigated as a function of temperature, microwave power and microwave frequency and was found to arise from a magnetically isolated [4Fe-4S]( 2 + r 1 + ) cluster. The signal can be simulated

112

Electron Spin Resonance

a s s u m i n g S=1/2 g=1.94 e.p.r. Fe p r o t e i n

and

g-strain

broadening.145

In

a

exhibits

weak

signal

with

to

addition

s i g n a l ( 0 . 2 t o 0.5 s p i n s per mole),

the

the

nitrogenase

g ~ 5 , the

temperature

d e p endence of which i s c o n s i s t e n t w i t h S=3/ 2, a n e g a t i v e z e r o f i e l d 0.7 cm-l and 0.6-0.8 s p i n s per m o l . 1 4 6 s p l i t t i n g D=-5.0 2 T r a n s i t i o n s were o b s e r v e d f r o m b o t h s p i n d o u b l e t s .

I n d i c a t i o n s of a

s i m i l a r r e s u l t h a v e b e e n i n d e p e n d e n t l y o b t a i n e d .147 p r o t e i n of n i t r o g e n a s e h a s been e x p r e s s e d i n

The

t h e a b s e n c e o f t h e MoFe p r o t e i n a n d i t i s f o u n d t h a t 148 set of genes is r e q u i r e d t o e f f e c t t h e p r o c e s s . O r i e n t e d whole c e l l

layers

Azotobacter

of

a

g=3.6

signals

are

consistent

Fe

coli

in

restricted

vinelandii

Rhodospirillum rubrum were analyzed spectroscopy. Orientation dependent c h a r a c t e r i s t i c s and

active

Escherichia

and

e.p.r.

bY of

the

g=4.3

a

with

possible structural 149 a s s o c i a t i o n b e t w e e n t h e MoFe p r o t e i n a n d t h e membrane. Enzymes. - B r a y

6 . 2 M o n o n u c l e a r Oxomolybdenum

h a v e r e v i e w e d t h e methods by which e . p . r . transient

intermediates

techniques. In

of

conjunction

s u b s t i t u t i o n and e . p . r .

xanthine with

may oxidase

this,

and

be

George150

to

used

by

study

rapid

selective

freeze isotopic

computer simulations have contributed t o

a

m e c h a n i s t i c p r o p o s a l f o r t h e f u n c t i o n of t h i s enzyme. A d d i t i o n o f t h e s u b s t r a t e formamide t o x a n t h i n e o x i d a s e g i v e s

rise t o t h e Very Rapid a n d Rapid Type 1 M o ( V ) e . p . r . s i g n a l s . The l a t t e r s i g n a l , o b s e r v e d w i t h l7O e n r i c h e d w a t e r , indicates the presence of a single 0x0 ligand to the molybdenum i o n . 15' O b s e r v a t i o n o f n o n e x c h a n g e a b l e H' n M I = + l t r a n s i t i o n s i n t h e Very Rapid and

Desulfo

Inhibited

Mo(V)

e.p.r

signals

probably

or

o r i g i n a t e from carbon-bound

p r o t o n s of amino a c i d l i g a n d s t h e molybdenum c o f a c t o r (Moco).1 5 2

from

rise

I n h i b i t i o n of x a n t h i n e o x i d a s e by 8- br o m o x an th in e g i v e s

t o t h e R a p i d Mo(V) e . p . r . substrates153,

signals characteristic

of

other

w h i l e i n h i b i t i o n by a l d e h y d e s g i v e s r i s e

M o ( V ) i n h i b i t e d s i g n a l s . 154 B a n d s i n t h e

low

visible

to

purine stable

temperature

spectrum of t h e Desulfo Inhibited e.p.r. detectable active species are a s s i g n e d t o M o ( V ) c r y s t a l f i e l d t r a n s i t i o n s . 155 R e e x a m i n a t i o n of t h e e . p . r . s p e c t r a of the iron sulphur

m.c.d.

c e n t r e s i n x a n t h i n e o x i d a s e shows t h a t t h e

second

[2Fe-2S] (2+'1+)

c e n t r e h a s o n l y one g v a l u e above 2 , which i s c o n t r a r y

reports.156 M k s b a u e r

spectra

show

that

the

two

to

earlier

iron

sulphur

c e n t r e s are i n d i s t i n g u i s h a b l e i n t h e oxidized state, while

in

the

3: Metalloproteins

113

reduced state t h e f e r r o u s ion i n one c e n t r e has an unusually

large

q u a d r u p o l e moment.

e t_a l .157 r e p o r t t h e p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n Wagner o f x a n t h i n e d e h y d r o g e n a s e from C l o s t r i d i u m a c i d i u r i c i grown i n

the

presence of selenium. Despite t h e requirement f o r selenium

in

the

for

the

s y n t h e s i s o f t h e a c t i v e enzyme,

no

was

evidence

incorporation of selenium i n t o e i t h e r

the

found

or

Mo

interconversion

of

[ 2 F e - 2 S l (2+’1+)

centres. George e t a d 5 * have r e i n v e s t i g a t e d t h e

n i t r a t e r e d u c t a s e f r o m E s c h e r i c h i a c o l i b e t w e e n t h e l o w a n d h i g h pH Mo(V) e.p.r.

a c t i v e f o r m s . T h e i r r e s u l t s show t h a t t h e p r o t o n w h i c h

rise

c o n t r o l s t h e i n t e r c o n v e r s i o n is n o t t h a t which g i v e s superhyperf ine coupling i n nitrate

reductase

from

both

of

these

Pseudomonas

aeruginosa

exhibits

~ i g n a 1 s . l ~ T’ h e y a r e a h i g h p H

d i f f e r e n t Mo(V) e.p.r.

to

the

forms. P r e p a r a t i o n

of

three

species

and

l o w p H species. E . p . r . e v i d e n c e was a l s o f o u n d f o r a [ 3 F e - 4 S I 3 + c l u s t e r i n t h e o x i d i z e d nitrate

and

enzyme a n d a

nitrite

low

complexes

potential

of

the

[4Fe-4SI1+

cluster

in

the

reduced

enzyme. Low t e m p e r a t u r e e . p . r .

used t o determine t h e [4Fe-4Sl (2+r1+’

a n d i r o n s u l p h u r core

presence

centres per

of

one

molecule

3Fe

of

and

extrusion

were

or

four

three

nitrate

reductase

E s c h e r i c h i a c o l i .l6O Two d i s t i n c t i r o n s u l p h u r c l u s t e r s were

from found

i n formate dehydrogenase from C l o s t r i d i u m pasteuranium as i n d i c a t e d by t h e i r d i f f e r e n t e . p . r . Ra]agopalanl6’ oxomolybdenum

has

s p e c t r a and redox potentials.161 reviewed

e n z y m e s . Moco

has

properties

the been

Moco

of

to

shown

contain

from

a

m o l y b d o p t e r i n g r o u p which c o n t a i n s a n e s s e n t i a l p h o s p h a t e g r o u p f o r

its f u n c t i o n , b u t no p e p t i d e . P r e p a r a t i o n

of

Moco

from

xanthine

o x i d a s e u n d e r d e n a t u r a t i n g c o n d i t i o n s and s u b s e q u e n t o x i d a t i o n w i t h dimethylsulfoxide

gives

rise

to

e.p.r.

spectra

typical

of

m o n o n u c l e a r molybdenum ( V ) s p e c i e s w i t h a s i n g l e 0 x 0 g r o u p a n d f o u r t h i o l a t e l i g a n d s . lCi3 Chemical approaches t o t h e modelling

of

the

structure

and

r e a c t i v i t y o f m o n o n u c l e a r molybdenum c e n t r e s i n t h e

molybdoenzymes

h a v e b e e n d i s c u s s e d . 164’165

superhyperf i n e

Comparison o f t h e

p a r a m e t e r s f o r [Mo 170 ( S P h ) 4 ] - a n d

Cis-

”0

[Mo l7O 1 7 0 H

observed

in

the

e.p.r.

spectra

m o l y b d o e n z y m e s . 166 An e . p . r .

of

the

active

suggests

L]

t h a t a h y d r o x y l g r o u p c o o r d i n a t e d t o t h e M o ( V ) atom c i s t h e S 2 - or 02- l i g a n d s i s r e s p o n s i b l e f o r t h e TO

to either structure

forms

of

the

s p e c t r u m of model c o m p le x es e x h i b i t i n g

Electron Spin Resonance

i 14

t h i o l a t e a n d t h i o e t h e r l i n k a g e s h a s b e e n r e p o r t e d .167 7 Vanadium

A n a l y t i c a l and e . p . r .

measurements

have

been

reported

to

q u a n t i f y t h e n a t u r e o f s u l f a t e , vanadium and a c i d i t y i n v a n a d o c y t e s of A s c i d i a c e r a t o d e s . I t i s c l a i m e d t h a t t h e e . p . r . s i g n a l s a t room t e m p e r a t u r e and i n f r o z e n s o l u t i o n are c o n s i s t e n t w i t h aquated VO( I V ) .1b8

8 P a r a m a g n e t i c Metal S u b s t i t u t e d Enzymes

8 . 1 S u b s t i t u t e d Z i n c Enzymes.- A simple a n d e f f i c i e n t m e th o d f o r t h e p u r i f i c a t i o n (employing chromatafocussing as t h e l a s t s t e p )

o f two isomers o f Cu2Zn2 s u p e r o x i d e d i s m u t a s e w i t h i s o e l e c t r i c p o i n t s 5.2 and 4 . 9 h a s be e n d e s c r i b e d . 1 6 ’ C.d., e.p.r. and U.V. a b s o r p t i o n s p e c t r a f o r t h e s e c o n d isomer d i f f e r f r o m t h o s e o f the f i r s t isomer t r e a t e d w i t h h y d r o g e n p e r o x i d e . Zinc deprived bovine s u p e r o x i d e d i s m u t a s e and i t s c om pl e xe s w i t h a z i d e and t h i o c y a n a t e have been examined with e.p.r. and proton relaxation m e a s ~ r e r n e n t s .The ~ ~ ~ results indicate a more symmetrical e n v i r o n m e n t f o r t h e c o p p e r ( I 1 ) i o n s and t h a t a h i s t i d i n e o t h e r t h a n the bridging one is displaced upon the addition of a n i o n s . E q u a t i o n s f o r t h e m a g n e t i c f i e l d d e p e n d e n c e o f n u c l e a r T1-l enhancement due t o d i p o l a r c o u p l i n g w i t h an e l e c t r o n s p i n 1 / 2 , w i t h a n a n i s o t r o p i c g m a t r i x and a n i s o t r o p i c h y p e r f i n e m a t r i x , I=3/2 h a v e been d e v e l o p e d and s u c c e s s f u l l y a p p l i e d t o t h e s t u d y o f water -1 v a l u e s i n t h e copper e n z y m e s u p e r o x i d e d i s m u t a s e . 17’ p r o t o n T1 The c h e l a t i o n p r o p e r t i e s o f T r i s t o w a r d s a q u o C u ( I 1 ) i o n s a n d c o p p e r bound i n m e t a l l o e n z y m e s ( e . g . , superoxide dismutase) have b e e n e x a m i n e d by e . p . r . s p e c t r o s c o p y . 172 T h e s e r e s u l t s show t h a t T r i s i s a poor c h o i c e of b u f f e r f o r m e t a l l o e n z y m e s . B i s [ ( h e m e b l c o p p e r l p r o t e i n , a p r o t e i n o t h e r t h a n a Cu2Zn2 s u p e r o x i d e d i s m u t a s e , c o n t a i n i n g 20-50% of t h e t o t a l c e l l u l a r copper was i s o l a t e d f r o m b o t h b o v i n e a n d human e r y t h r o c y t e s . 1 7 3 T h e C u ( I 1 ) e . p . r . s p e c t r u m due t o t h i s complex i s s i m i l a r t o t h a t r e p o r t e d b y M c P h a i l a n d Goodman172 f o r t h e T r i s c o m p l e x of superoxide dismutase. Magnetic susceptibility measurements on the cobalt d e r i v a t i v e s o f s u p e r o x i d e d i s m u t a s e ( a p o 2 C 0 2 a n d Cu C o ) i n d i c a t e a 2 2 t e t r a h e d r a l geometry f o r t h e h i g h s p i n c o b a l t i o n and an

3: Metalloproteins

115

antiferromagnetic interaction (J = -16.5 cm-l) between the copper ( S = 1/21 and cobalt ( S = 3/21 ions. This interaction is propagated by the imidazolate bridge and is three times as large as the interaction in the Cu2Cu2 derivative, indicating that one of the three possible exchange pathways in the Cu2Cu2 derivative is more efficient.174 E.p.r. and absorption spectra of the Co2Zn2 metalloderivative of superoxide dismutase indicate a five coordinate geometry for the cobalt ion. 175 Binding of phosphate changes the coordination geometry to tetrahedral , while binding of cyanide causes a spin state change. Titration of the Co2C02 derivative with cyanide provides evidence for a magnetic interaction. Replacement of Mn(I1) with Cu(I1) in Bacillus stearothermophilus superoxide dismutase gives an e.p.r. signal which is almost identical to that of the Cu2ZnZ superoxide dismutase, indicating a tetrahedral arrangement of one carboxylate group and three imidazole ligands. 176 Radiationless energy transfer kinetics at subzero temperatures of carboxypeptidase A (CPA) have established identical reaction schemes for the hydrolysis of peptides and depsipeptides by the cobalt substituted and native zinc enzymes.177 E.p.r. spectra of the peptide and depsipeptide ES2 intermediates arising spin from transitions within a single Kramers doublet of a S = 3 / 2 system differ from each other and from the resting enzyme. In further studies, stabilization of these intermediates by equilibrium trapping has enabled their characterization using e.p.r, m.c.d. and absorption s p e c t r o ~ c o p y .E.p.r. ~~~ studies of the copper derivative of carboxypeptidase indicate a tetragonal geometry of ligands around the metal ion in contrast to the usual tetrahedral geometry for the native enzyme. This change reflects the decrease in activity towards peptide and depsipeptide substrates.179 The sign and magnitude of the splitting between the two lowest Kramers doublets ( A ) of high spin Co(I1) in a variety of structurally defined coordination complexes have been determined from the variation of T1 with temperature. The range of A values is found to be < 13 cm-l in tetracoordinate sites, 20 to 50 cm-l in pentacoordinate sites of trigonal-pyramidal or square-pyramidal geometry and > 50 cm-l for octahedral sites. It is shown on the basis of group theoretical arguments, and estimates of the zero field splitting derived by second order perturbation theory, that the observed range of valties correlates well with that predicted

116

Electron Spin Resonance

by theory.18' The zero field splitting for the Co(I1) ion is 8 . 3 cm-l in CoCPA, 51 cm-l in the CoCPA-L-benzylsuccinate inhibitor complex and 3 9 cm-l for the mixed anhydride reaction intermediate formed with 0-(trans-p-chlorocinnamoy1)L-0-phenyllactate. These values indicate a distorted tetrahedral complex for the native enzyme and pentacoordinate geometries for the inhibitor complex and 1 81 the reaction intermediate. Substitution of cobalt(I1) for zinc ions in the structural and catalytic sites of Bacillus cereus Phospholipase C enabled the spectroscopic characterization of these sites. E.p.r. studies indicate similar but not identical, distorted octahedral environments for both sites.182 Absorption, m.c.d. and e.p.r. spectra were employed to monitor the titration of rabbit liver apo-metallothionein-1 with cobalt. Addition of two equivalents of ColII) to the apo-enzyme gives rise to e.p.r. spectra typical of isolated tetrahedral tetrathiolate Co(I1) complexes. Further addition of cobalt indicates the existence of cluster formation. 183 A pH dependent binding study of the imidazole moiety of His 6 with Mn(I1) ions in angiotensin(I1) has been performed in order to show that this interaction is not important f o r the transport of Mn( 11) ions across lecithin bilayers. 184 the 8 . 2 Substituted Magnesium Enzymes.- Reed185 has reviewed e.p.r. properties of Mn(I1) substituted proteins with particular reference to metal binding sites and ligand interactions. Detection of l 7 O superhyperfine structure due to labellcd substrates has led to the characterization of enzyme-substrate complexes. Both Co(I1) and Mg(I1) are required by Ribulose 1,5-bisphosphate (RuBP) carboxylase for catalysis of C 0 2 fixation in photosynthetic organisms. Substitution of Mg(I1) by Mn(I1) has enabled the characterization of transition state analogues by e.p. r. spectroscopy. Gutteridge et a1.186 has reviewed the current status of the activation process and the active site of RuBP. E.p.r. examination of the ternary complex involving, Mn(I1) bound at the active site, C 0 2 and the transition state analogue carhoxyarabinitol bisphosphate (cabp) reveals an anisotropic environment for the metal ion.187 N.m.r. relaxation measurements in conjunction with t.he H 2 1 7 0 e.p.r. data are consistent with direct coordination of water to manganese. In addition, selectively labelling the C - 2 carboxyl group with 170 leads to a broadening of

3: Metalloproteins

t h e e.p.r.

117

s p e c t r u m i n d i c a t i n g t h a t c a b p b i n d s t o t h e metal i o n .

E.p.r. spectra of the Cu(I1) derivative of RuBP carboxylase/oxygenase i n d i c a t e t h e p r e s e n c e of one nitrogen ligand. A d d i t i o n of t h e s u b s t r a t e RuBP t o t h e c o p p e r enzyme y i e l d s t h r e e d i s t i n c t e . p . r . s i g n a l s , o n e of w h i c h i s o n l y t r a n s i e n t . Th e

9 and

5

matrices i n d i c a t e a n oxygen c o o r d i n a t i o n s p h e r e

for

these

c o m p l e x e s . 1 8 8 A d d i t i o n o f t h e s u b s t r a t e RuBP t o t h e c o p p e r enzyme i n t h e p r e s e n c e of 170 lead to t h e observation of l7O s u p e r h y p e r f i n e c o u p l i n g . Is’ F u r t h e r s t u d i e s w i t h 170 l a b e l l e d

cabp

and 3 - p h o s p h o g l y c e r i c a c i d i n d i c a t e t h a t t h e s e molecules are c o o r d i n a t e d t o t h e c o p p e r i o n s t h r o u g h o x y g e n atoms. E. p. r . s p e c t r o s c o p y o f t h e c o b a l t a c t i v a t e d RuBP h a s b e e n r e p o r t e d . l g l T h e

s p e c t r a i n d i c a t e t h a t t h e metal t a k e s p a r t

in

catalysis

and

the

r e a c t i o n i n v o l v e s inner-sphere s u b s t r a t e or i n t e r m e d i a t e complexes. Yeast i n o r g a n i c p y r o p h o s p h a t a s e b i n d s t w o M n ( I 1 ) i o n s p e r s u b u n i t i n t h e a b s e n c e o f p h o s p h a t e a n d t h r e e M n ( I 1 ) i o n s per s u b u n i t i n t h e p r e s e n c e of p h o s p h a t e . E . p . r . spectra a n a l y s e d i n terms o f a d i p o l a r r e l a x a t i o n m e c h a n i s m b e t w e e n t h e Mn-Mn d i s t a n c e p r o v i d e s a n estimate of

t h e Mn(I1) of 1.0 t o

ions 1.4

nm. A d d i t i o n of d i a m a g n e t i c s u b s t r a t e or i n t e r m e d i a t e a n a l o g u e s d e c r e a s e s t h e s e p a r a t i o n of t h e i o n s to 0.7 t o 0 . 9 .,.Ig2 E . p . r . h a s also been used t o p r o b e t h e c o o r d i n a t i o n s p h e r e of t h e copper d e r i v a t i v e o f t h i s e n z y m e . T h e a b s e n c e o f n i t r o g e n ESEEM t o g e t h e r w i t h t h e o b s e r v e d g a n d A v a l u e s is c o n s i s t e n t w i t h o x y g e n l i g a t i o n t o t h e c o p p e r . I n t e r a c t i o n s b e t w e e n t h e copper i o n s h a s b e e n i n t e r p r e t e d i n terms o f a t h r e e s i t e m o d e l , i n a g r e e m e n t

with

the

r e s u l t s f o r t h e Mn(I1) enzyme.lg3 C o o r d i n a t i o n of M n ( I 1 ) t o p h o s p h a t e g r o u p s i n s u b s t r a t e s

and

p r o d u c t s i n t h e c e n t r a l complexes of t h e c r e a t i n e k i n a s e r e a c t i o n m i x t u r e h a s b e e n i n v e s t i g a t e d by e . p . r w i t h r e g i o s p e c i f i c a l l y 170 l a b e l l e d s u b s t r a t e s . The e . p . r s p e c t r a o f t h e e q u i l i b r i u m m i x t u r e

is a superposition of t h e two c e n t r a l complexes.lg4 Inhomogeneous b r o a d e n i n g of t h e e.p.r. s i g n a l o f manganese a t t h e a c t i v e site i n 3-Phospoglycerate kinase, r e s u l t i n g from u n r e s o l v e d s u p e r h y p e r f i n e c o u p l i n g t o I 7 O i n c o r p o r a t e d i n t o ADP a t the

a a n d p p h o s p h a t e g r o u p s , i n d i c a t e s t h a t ADP i s bound

directly

t o t h e manganese i o n . l g 5 Yeast e n o l a s e b i n d s two c o b a l t i o n s i n s t r u c t u r a l s i t e s a n d a f u r t h e r p a i r i n c a t a l y t i c sites. A b s o r p t i o n and e . p . r . s t u d i e s of the structural sites indicate tetragonally distorted geometries, w h i l e t h e s p e c t r a of t h e c a t a l y t i c s i t e s i n d i c a t e a more r e g u l a r

118

Electron Spin Resonance

tetrahedral site. Addition of Mg(I1) or substrate to the enzyme with metal ions in the structural site causes dramatic changes to the metal environments. 196 Structure of the divalent metal ion activator site of S-Adenosylmethionine synthetase has been studied by Vanadyl(1V) e.p.r. Active enzyme preparations require an additional Mg(I1) or Ca(I1) ion. 31P and 170 e.p.r. data indicate the presence of two 1 97 bidentate phosphate ligands coordinated to the vanadyl ion. A distance of 1 to 1.2 nm between the two catalytically important manganese ions of adenylated and unadenylated glutamine synthetase was determined from e.p.r. data analysed by means of dipolar electron-electron relaxation theory.lg8 Atomic absorption and e.p.r. spectroscopy distinguished two non-exchangeable tight binding metal sites from a tight exchangeable metal binding site in beef heart mitochondria1 ATPase (F1).lg9 The latter site has been substituted with Mn(I1) and studied with e.p.r. at pH 6.8 and 8.0 and show that Mn(I1) and CrATP do not bind at the same site.199 Addition of guanosine triphosphate (GTP) to the manganese substituted elongation factor Tu(EF-Tu) from Bacillus stearothermophilus yields three distinct e.p.r. signals identified as EF-Tu.Mn.GTP, EF-Tu.Mn.GDP.Pi and EF-Tu.Mn.GDP. Use of l70-labelled GTP demonstrates an interaction of Mn( 11) with the P-phosphate in both EF-Tu.Mn.GDP.Pi and EF-Tu.Mn.GDP. 2oo 8.3 Extrinsic Metal Binding Site in Proteins.- Computer simulations of the e.p.r. spectra at S-band (2-4 GHz) at 77K of the tight binding complex of bovine serum albumin (BSA) at physiological pH indicate coordination of Cu(I1) to four-in-plane nitrogen atoms.201 Improved resolution of the S-band e.p.r. spectrum of BSA enabled the coordination sphere for this copper binding site to be assigned. Nitrogen superhyperfine coupling is evident in the e.p.r. spectra of Cu(I1) coordinated to collagen, CH3-collagen and DNP-collagen. * 0 2 The aerobic uptake of Mn(I1) in rat liver mitochondria yields an e.p.r. spectrum consisting of a six line signal corresponding to hydrated Mn(I1) and a single line arising from spin exchange.203 A simple e.p.r. method for resolving the binding modes (intercalation versus outside) of copper porphyrin and porphyrazine complexes to DNA has been reported.204 The results show increased intercalation over outside binding for these copper dyes to DNA as the buffer's ionic strength is increased from 0.1 to 1.0 M.

3: Metalloproteins

119

9 Mitochondria1 E l e c t r o n T r a n s p o r t Chain In

et al.

the

of

context

205 h a v e

outlined

mitochondria1 e l e c t r o n

lactic

congenital the

relationships

transfer

chain

acidosis within

1). By

(Figure

judicious choice of experimental conditions, e.p.r.

a l l components o f normal m i t o c h o n d r i a

can

be

of

to

s i g n a l s due

distinguished.

Thus

Complex I I

IFe-5: c-i

Succinate : ublquinone Oxidoreductase

Complex I

r

complete means

methods is f e a s i b l e .

d e t e c t i o n of c o n g e n i t a l d e f e c t s u s i n g e . p . r .

[

Moreadith

the

Complex Iv

1

NADH : Ubiqvinone

lF;;;!FTFJ

Oxidoreductase

Fatty Acy:

Ubiquinol: Cytochrome c Reductase

Cytochrome Oxidase

CoA

F i g u r e 1: S c h e m a t i c d i a g r a m o f t h e M i t o c h o n d r i a 1 E l e c t r o n

Transfer

Chain 9.1 m.c.d.,

Succinate

Dehydrogenase.-

LEFE and e.p.r.

Recent

studies206-208

i n s o l u b l e , homogeneous p r e p a r a t i o n s d e m o n s t r a t e d t h e t h r e e d i f f e r e n t i r o n s u l p h u r c l u s t e r s [ o n e e a c h of (S-1)

[4Fe-4S] (2+’1+)

using

of s u c c i n a t e d e h y d r o g e n a s e i n m e m b r a n e s a n d

(S-2)

presence of similar i r o n

presence

and [3Fe-3/4S] ( 3 + r 2 + ; 1 + ’ 0 )

sulphur

is

clusters

of

[ 2 F e - 2 S l (2+’1+) (S-3)].

reported

for

The the

b a c t e r i a l enzyme, f u m a r a t e r e d u c t a s e f r om E s c h e r i c h i a c o l i . 2 0 6 E.p.r.

spectroscopy

of

Bacillus

was enzyme, t o s t u d y s u b u n i t l o c a t i o n s and

dehydrogenase

defective

s u l p h u r c l u s t e r s . The

mutants

wild

type

subtilis

succinate

to characterize t h e b i o s y n t h e s i s of its iron

used

enzyme

contains

similar t o those of spectra from t h e [Fe-S]S-2

[Fe-S]S-l

[ F C - S ] ~ - ~c l u s t e r s

the

enzyme. E . p . r .

cluster

d e t e c t e d . However upon r e d u c t i o n w i t h d i t h i o n i t e

bovine greatly

and heart

were

not

enhanced

120

Electron Spin Resonance

c l u s t e r w a s observed, presumably s p i n r e l a x a t i o n o f t h e [Fe-S]S-l f r o m c o u p l i n g t o t h e [ F C ? - S ] ~ -c~e n t r e . 2 0 9 S i m i l a r r e s u l t s h a v e b e e n 210 o b t a i ned f o r s u c c i n a t e d e h y d r o g e n a s e f r o m E s c h e r i c h i a c o l i . The m a g n e t i c f i e l d d e p e n d e n c e o f t h e l i n e a r e l e c t r i c f i e l d e f f e c t i n e . p . r . s p e c t r o s c o p y r e v e a l s t h a t t h e [Fe-S]S-3 c e n t r e i n a i r o x i d i z e d p r e p a r a t i o n s o f c o m p l e x I1 i s a [3Fe-4Sl

r e d u c t i o n , t h e g=2.01 e.p.r.

c l u s t e r . Upon s i g n a l d i s a p p e a r s and a g = 1 . 9 4 s i g n a l

grows, c o n s i s t e n t w i t h t h e c o n v e r s i o n of t h e [3Fe-4Sl (3+'2+) c l u s t e r i n t o a n [4Fe-4Sl (2+'1+) c l u s t e r . LEFE m e a s u r e m e n t s o n c l u s t e r ( S - 1 ) of c o m p l e x I1 a r e c o n s i s t e n t w i t h a [ 2 F e - 2 S ] ( 2 + ' 1 + ) c l u s t e r . 211 9 . 2 Cytochrome 0 x i d a s e . -

An u n d e r s t a n d i n g o f t h e

mechanisms

of e l e c t r o n and p r o t o n t r a n s p o r t , coupled w i t h r e s p i r a t i o n

on

the

molecular l e v e l , r e q u i r e s d e t a i l e d information on t h e s p a t i a l o r g a n i z a t i o n of t h e r e d o x - a c t i v e c e n t r e s i n t h e i n n e r m i t o c h o n d r i a 1 membrane, Use o f s p i n - s p i n c o u p l i n g b e t w e e n d y s p r o s i u m c o m p l e x e s bound t o membrane s u r f a c e s , a n d e . p . r . a c t i v e c e n t r e s of c y t o c h r o m e has o x i d a s e h a s b e e n o n e a p p r o a c h t o t h i s p r o b l e m . 212 V;nngard2l3 reviewed t h e e.p.r.

s t u d i e s concerning t h e s t r u c t u r e

and

function

o f Pseudamonas c y t o c h r o m e c p e r o x i d a s e and b e e f h e a r t c y t o c h r o m e c o x i d a s e . A v a r i e t y o f h i g h a n d l o w s p i n heme F e ( I 1 ) e . p . r . signals were found.213 A h i s t i d i n e a u x o t r o p h o f Saccharomyces c e r e v i s i a e h a s b e e n u s e d t o m e t a b o l i c a l l y i n c o r p o r a t e [ l , 3-l5N1 h i s t i d i n e i n t o y e a s t c y t o c h r o m e c o x i d a s e . ENDOR o f c y t o c h r o m e a i n t h e [15N] h i s t i d i n e s u b s t i t u t e d enzyme r e v e a l s 1 5 N h y p e r f i n e c o u p l i n g f r o m h i s t i d i n e w i t h t h e l o w s p i n heme. T h e r e f o r e h i s t i d i n e i s a n a x i a l l i g a n d t o t h i s c y t o ~ h r o m e .E~. p~. r~. a t 1 5 K was u s e d t o p r o b e t h e magnetic i n t e r a c t i o n between 'visible' CuA(II) and ferric cytochrome a

in

the

carbon

monoxide

component

of

beef

heart

c y t o c h r o m e o x i d a s e . 215 P r o g r e s s i v e power saturation studies e n a b l e d T L t o b e m e a s u r e d a n d t h e o b s e r v e d v a r i a t i o n of T1 f o r CuA was d u e t o d i p o l a r r e l a x a t i o n b y c y t o c h r o m e a . A d i s t a n c e b e t w e e n C u A a n d c y t o c h r o m e a was e s t i m a t e d t o b e f r o m 0 . 8 t o 1 . 3 nm.215 I n t h e c o n t e x t of a d e t a i l e d s t u d y o f t h e r e l a x a t i o n o f CuA a n d hemes i n cytochrome c o x i d a s e , and o t h e r copper p r o t e i n s and complexes, B r u d v i g e t a 1 . 2 1 6 a l s o f i n d e v i d e n c e f o r r e l a x a t i o n o f CuA by c y t o c h r o m e a a n d e s t i m a t e t h e s e p a r a t i o n t o b e a t l e a s t 1 . 3 nm. Oxygen p u l s e d b o v i n e h e a r t c y t o c h r o m e c o x i d a s e h a s b e e n e x a m i n e d b y d u a l mode e . p . r . s p e c t r o s c o p y where t h e microwave m a g n e t i c f i e l d El i s a p p l i e d b o t h p e r p e n d i c u l a r a n d p a r a l l e l t o t h e

3: Metalloproteins

121

d . c . f i e l d B . I n t h i s way b o t h ' f o r b i d d e n ' t r a n s i t i o n s ( g = 1 0 , 4 . 5 ) and ' a l l o w e d ' t r a n s i t i o n s ( g 5 1 0 , 5 , 1 . 8 , 1 . 7 ) c a n be o b s e r v e d s e p a r a t e l y . On t h i s b a s i s , t h e s p i n H a m i l t o n i a n p a r a m e t e r s f o r a s t o i c h i o m e t r i c F e ( 1 V ) heme S = 2 s y s t e m a r e f o u n d t o b e 9=2,

I a I =O. 17cm? D=+2. l c m - l a n d 1 E 1-0.026cm-I .217 E.p. r . a n d o p t i c a l a n a l y s i s o f t h e r e d u c e d and r e o x i d i z e d cytochrome c o x i d a s e ( S o r e t b a n d a t 420 nm) a l s o s h o w s a 6 0 5 nm b a n d , a

blue

shifted

655

nm

band and a 9 = 5 e . p . r . s i g n a l . R e a c t i o n o f p e r o x i d e w i t h t h i s f o r m o f t h e enzyme c a u s e s t h e 9 = 5 e , p . r . s i g n a l t o d i s a p p e a r , i n d i c a t i n g

a peroxy intermediate is formed during enzyme that t u r n o v e r . 218 O x i d a t i o n o f p a r t i a l l y r e d u c e d c y t o c h r o m e o x i d a s e w i t h o x y g e n p r o d u c e s a n e . p . r . s i l e n t d i o x y g e n i n t e r m e d i a t e w h i c h was p r o p o s e d t o c o n t a i n a f e r r y 1 heme a j g r o u p . T h i s was b a s e d u p o n i t s r a p i d r e a c t i o n w i t h CO w h i c h p r o d u c e d l o w s p i n f e r r o u s heme a 3 a n d c ~ ~ ( Ie I. p), r . s i g n a l s . * " C o m p a r i s o n of a n e x c h a n g e c o u p l e d F e ( I I I ) - C u ( I I ) m o d e l w i t h a n Fe(1V) model f o r t h e O2 b i n d i n g s i t e i n o x i d i z e d cytochrome c o x i d a s e i s made by m e a n s o f e . p . r . s p e c t r a l s i m u l a t i o n s € o r t h e g = 1 2 f e a t u r e a t 9 GHz a n d g=9.3 s i g n a l a t

15

CHz.

It

is

concluded

t h a t t h e more c o r r e c t m o d e l i s t h e o n e i n w h ic h a n t i f e r r o m a g n e t i c s u p e r e x c h a n g e c o u p l i n g o c c u r s b e t w e e n Cug a n d heme a 3 . 2 2 0 E.p.r.

s p e c t r a of b o v i n e h e a r t

cytochrome

oxidase

titrated

species w i t h a z i d e y i e l d two d i s t i n c t l o w s p i n c y t o c h r o m e a 3 - a z i d e w i t h d i f f e r e n t r e d o x p o t e n t i a l s . The s i g n a l a p p e a r i n g a t t h e l o w e r p o t e n t i a l has g values of 2.88, 2.19 and 1 . 6 4 , w h ic h c a n b e a t t r i b u t e d t o a complex i n which cytochrome a is r e d u c e d . O x i d a t i o n

of t h i s s p e c i e s g i v e s r i s e t o t h e s e c o n d e . p . r . a c t i v e s p e c i e s w i t h 2.77, 2 . 1 8 a n d 1.74.221 C o m p l e x e s f o r m e d b e t w e e n c y t o c h r o m e c o x i d a s e a n d N3-, CNa n d S2- h a v e b e e n i n v e s t i g a t e d b y m . c . d . a n d e . p . r . . C y a n i d e f o r m s a l i n e a r b r i d g e b e t w e e n f e r r i c heme a 3 a n d Cu ( I f ) w i t h a n i r o n c o p p e r d i s t a n c e of 0 . 5 nm. I n c o n t r a s t , N3-, S'-, SH- d o n o t form b r i d g e s . 222 F u r t h e r s t u d i e s on cyanide i n h i b i t i o n of cytochrome c o x i d a s e h a v e been i n v e s t i g a t e d by r a p i d f r e e z e e . p . r methods l e a d i n g t o t h e s u g g e s t i o n o f a p o s s i b l e c y a n i d e b r i d g e . 2 2 3 Th e e f f e c t of s u l p h i d e o n r e s t i n g o x i d i z e d c y t o c h r o m e c o x i d a s e h a s b e e n r e e x a m i n e d by e . p . r . s p e c t r o s c o p y w h i c h p r o v i d e s e v i d e n c e f o r a low s p i n c y t o c h r o m e a 3 - t h i o l complex.224 E.p.r. s t u d i e s of c h e m i c a l l y m o d i f i e d b o v i n e h e a r t cytochrome c o x i d a s e w i t h sodium p-(hydroxymercuri)benzoate s u p p o r t a m o d e l i n w h i c h a s e c o n d a r y e l e c t r o n - t r a n s f e r p a t h w a y , o p e r a t i n g a t 20% o f t h e r a t e o f t h a t o f g values

122

Electron Spin Resonance

t h e n a t i v e enzyme, e x i s t s , b u t 225

which

does

not

the

low

azide

and

involve

p o t e n t i a l CuA c e n t r e .

A complex formed by

c

cytochrome

n i t r o x i d e h a s been s t u d i e d by e . p . r . p r e s e n c e o f a A M =+2 e . p . r .

oxidase

with

a t 9 . 2 a n d 35 GHz, s h o w i n g t h e

t r i p l e t s p e c t r u m and s t r o n g a n i s o t r o p i c

s i g n a l s d u e t o t h e i n t e r a c t i o n o f f e r r o u s c y t o c h r o m e a 3 , NO ( S = 1 / 2 ) a n d C u B ( I I ) ( S = 1 / 2 ) . When t h e p h o t o l y s e d s a m p l e i s warmed the e.p.r.

o f t h e f e r r o u s heme a3’+-N3--Cug(I)

to

o b s e r v e d . I n t h e t r i p l e t species i t i s s u g g e s t e d t h a t

azide

to CuB(II) whereas n i t r o x i d e is bridged e.p.r.

based

between

heme i r o n of c y t o c h r o m e a 3 . From

77K,

is

(S=1/2) c o m p l e x Cug(II)

simulations

binds

and

the on

a

d i p o l a r i n t e r a c t i o n , a c y t o c h r o m e a3 C u g ( I I ) . N O d i s t a n c e o f 0 . 3 3 nm 2 26 was d e t e r m i n e d . 9 . 3 B a c t e r i a l Cvtochrome 0 x i d a s e s . enzyme

was

activity

observed

by

ten-fold

A

pulsing

the

increase

in

caa3-terminal

c y t o c h r o m e c o x i d a s e i s o l a t e d f r o m t h e r m o p h i l i c b a c t e r i u m PS3. The e.p.r. s p e c t r u m of t h i s enzyme was s i m i l a r t o t h a t o f the m i t o c h o n d r i a l enzyme. The t r a n s i e n t g = 5 , 1 . 7 8 a n d 1 . 6 9 e . p . r . s i g n a l s o b s e r v e d by

pulsing

the

mitochondrial

enzyme

were

not

o b s e r v e d f o r t h e t h e r m o p h i l i c enzyme. 227 A

non-photosynthetic

mutant

of

(Ps-)

Rhodopseudomonas

c a p s u l a t a was a n a l y s e d f o r a d e f e c t i n t h e c y c l i c e l e c t r o n t r a n s f e r system. I t

showed

spectra

e.p.r.

from

all

components ( r e a c t i o n c e n t r e s , u b i q u i n o n e - 1 0 ,

c 2 , [2Fe-2SIRieske

of

actinic

s u l p h u r c e n t r e . 2 2 8 By m e a n s o f g=2.03

cytochrome

light. E.p.r.

d i s r u p t i o n of e l e c t r o n t r a n s f e r from s a t u r a t i o n of t h e

transfer b,cl

and

a n d a s e m i q u i n o n e c e n t r e ) . The m u t a n t f a i l e d

c a t a l y z e c y t o c h r o m e c1+c2 r e - r e d u c t i o n o r a f t e r a 1 0 ps f l a s h

electron

cytochromes

temperature

copper(I1)

evidence

to

quinol

b

the

e.p.r.

signal

membrane p r e p a r a t i o n s o f S y n e c h o c o c c u s 6 3 1 1 , i t i s

suggests

Rieske

dependence in

to

reduction

and

iron power

oxidized

concluded

that

t h e copper i s a f i r m l y bound c o n s t i t u e n t o f t h e t e r m i n a l o x i d a s e i n an environment similar, i f not identical, c y t o c h r o m e c o x i d a s e p r e p a r a t i o n s . 229

to

that

of

other

9 . 4 O t h e r M i t o c h o n d r i a 1 R e a c t i o n C e n t r e s . - Removal o f lipid from d e t e r g e n t - s o l u b i l i z e d s u c c i n a t e cytochrome c r e d u c t a s e is shown t o c a u s e c h a n g e s i n o p t i c a l cytochromes i n e.p.r.

the

inner

and

e.p.r. spectra of t h e b membrane. The r e s u l t i n g

mitochondrial

s p e c t r a a r e s i m i l a r t o t h o s e of p u r i f i e d b

cytochromes

and

123

3: Metalloproteins

were simulated using a 9-strain model €or low spin ferriheme complexes. 230 Binding of dibromothymoquinone (DPMIB) to the cytochrome b and Rieske iron sulphur centres in ubiquinol cytochrome c reductase (Complex 111, Figure l), has been shown by e.p.r. spectroscopy. In spite of this, DPMIB does not affect the first turnover of the fully oxidized Complex 111. 231 E.p.r. characteristics of the iron sulphur clusters of NADH-uhiquinone oxidoreductase (Figure 1) shows the presence of three [4Fe-4Sl and five or six [2Fe-2S] clusters which is in agreement with the iron content of the enzyme. A tentative scheme is proposed for the spatial organisation of these iron sulphur clusters in the enzyme and in the membrane.232 E.p.r. was used to characterize eight different iron sulphur clusters present in the human parasite, Trichomonas vaginalis, an anaerobic protozoan which lacks mitochondria. 233 10 Photosynthesis

Evans and Ford234 have summarised the main electronic processes photosynthetic reaction centres. Purple bacteria P 890 --- > B pheophytin -600 mV Photosystem I1 P 680 --- > pheophytin -600 mV Photosystem I P 700 --- > chlorophyll -1000 mV

--->

Q,

+

- 1 5 0 mV

--- >

u

QB 0 mV

--- >

- 4 3 0 mV

--- >

in

--->

Q, --- > -250 mV

--- >

Fe-SA+B -600 mV -500 mV In purple bacteria and Photosystem I1 (PSII), the electron acceptor centres appear to be two bound quinones which become reduced to semiquinones. The iron quinone centres have a characteristic g = 1.82 e.p.r. signal. Photosystem I (PSI) transfers electrons from P700 to two iron sulphur centres which function as the electron acceptors. An earlier article by the same authors and a colleague235 considers more generally the e.p.r. due to radical centres including the iron quinone centre. D i ~ m u k e shas ~ ~reviewed ~ the essential role played by manganese in oxygen evolution during A1

?

Fe-Sx -700 mV

QH 0 mV

Electron Spin Resonance

124 photosynthesis. 1 0 . 1 Photosystem I.- E . p . r .

evidence h a s been provided

s u g g e s t t h a t t h e low p o t e n t i a l ( - 7 0 0 m V ) e l e c t r o n a c c e p t o r

two

contains

iron

sulphur

which in

PSI

i s shown b y the s i g n a l is r e v e r s i b l y

centres.237 This

o b s e r v a t i o n t h a t t h e a m p l i t u d e of t h e e . p . r .

i n d u c e d by i l l u m i n a t i o n a t 7 . 5 K a n d i s n e v e r more t h a n 5 0 % o f

the

a m p l i t u d e of t h e s i g n a l when t h e i r o n s u l p h u r c e n t r e i s p r e - r e d u c e d by i l l u m i n a t i o n a t room t e m p e r a t u r e . E l e c t r o n t r a n s p o r t h a s been s t u d i e d i n PSI p a r t i c l e s p r e p a r e d w i t h d i g i t o n i n by e . p . r . K.238

In

the

presence

and f l a s h a b s o r p t i o n s p e c t r o s c o p y a t 10-30 of

ascorbate,

s e p a r a t i o n b e t w e e n P700 a n d Fe-SA

up

involving

to

a

maximum

of

is

an

two

P-700'

and

centre

charge

laser

flashes

thirds

c e n t r e s . When Fe-SA a n d Fe-SB a r e p r e r e d u c e d , relaxation processes

irreversible

produced

by of

the

both

slow

fast

are found to o c c u r . Recombination between Xo c c u r s t o 10-15%. A newly p u r i f i e d PSI

p a r t i c l e h a s b e e n d e s c r i b e d w h i c h h a s t h r e e a c t i v e Fe-S B and X .

reaction and

The a m i n o a c i d c o m p o s i t i o n o f

the

are

band r e v e a l s i m p o r t a n t f e a t u r e s which

8-kDa

c e n t r e s , A,

electrophoresis

common

with

those

of

s m a l l i r o n s u l p h u r p r o t e i n s o f t h e f e r r e d o x i n t y p e . 239 M o d i f i c a t i o n of c h l o r o p l a s t membranes w i t h d i a z o n i u m leads to a

loss

of

PSI

dependent

benzene

ferredoxin

s t u d i e s o f DABS-modif i e d m e m b r a n e s show

no

sulfonate

(DABS)

reduction. E.p.r.

inhibition

of

P-700'

f o r m a t i o n a t l o w t e m p e r a t u r e s , b u t p h o t o r e d u c t i o n o f b o t h Fe-SA a n d Fe-SB i s m a r k e d l y i n h i b i t e d . 2 4 0 B i n d i n g of s t i g m a t e l l i n ( a c h r o m o n e i n h i b i t o r a c t i n g

Qo c e n t r e

of

the

bcl

complex)

c y t ochrom e b and t h e [2Fe-2Sl and e . p . r .

to

the

p r o t e i n was

at

the

heme

b-566

domain

shown

using

absorption

of

spectroscopy.241

I t i s known t h a t c e r t a i n s p e c i e s o f h e t e r o c y s t o u s , N 2

cyanobacteria have t h e a b i l i t y photosynthesis to a bacterial-type t h e e l e c t r o n donor t o PSI.242 E.p.r.

e v i d e n c e shows

that

t r a n s f e r i n t h e n i t r o g e n a s e e n z y m e i n Nostoc muscorum i s u p o n c y t o c h r o m e b-599

w h i c h was d e t e c t e d o n l y i n

Na2S a l s o d e p l e t e d t h e ATP p o o l . T r e a t m e n t o f N . i n t h e l i g h t gave e . p . r .

fixing

to switch from oxygenetic photosynthesis, u s i n g Na2S a s electron dependent

cells.

vegetable

muscorum w i t h Na2S

due t o a chlorophyll r a d i c a l (g =

f e a t u r e s d u e t o r e d u c e d Fe-S c e n t r e s A a n d B ( g = 1 . 8 9 ,

2)

and

1.92,

1.94,

2 . 0 5 ) a n d some s i g n a l s d u e t o r e d u c e d Fe-SX c e n t r e ( g = 1 . 7 8 ,

1.88,

2.08).

3: Metalloproteins

125

10.2 Photosystem 11.- Light induced e.p.r. signals at 77K in oxygen evolving PSII membranes showed that the signal usually attributed to the iron semiquinone form of the primary Fe(I1)-QA acceptor at g = 1.82, was usually accompanied by a broad signal at 1.90. In some cases when the g = 1.82 signal was absent, the g = 1.90 feature was significantly increased. The latter signal is associated with a second form of the primary iron semiquinone acceptor of PSII and detailed reasons are given for this assignment. Both signals are affected by herbicides which block electron transfer between primary and secondaky quinone acceptors. Increasing pH favours the g = 1.90 signal while the g = 1.82 signal is enhanced at lower pH.243 It is normally found that the e.p.r. signal of the iron semiquinone electron acceptor of PSII in higher plant chloroplasts is difficult to observe. 244 If the preparation

is washed with formate to remove bound C02, the signal increases. Experiments are reported for the iron semiquinone complex of formate washed pea PSII particles.244 C 0 2 depletion is found to lead to an approximately ten-fold increase in the g = 1.82 signal due to the Fe(II)-QA complex in PSII-enriched thylakoid membrane fragments.245 The signal decreases to that of control samples upon reconstitution with HC03-. The Fe(II)-QA- and split pheophytine.p.r. signals from triazine-resistant Brassica napus were identical to those from triazine-susceptible samples showing that

-

the Fe(II)-QA complex does not undergo any conformational change. 245 An extensive e.p.r. study of the iron-semiquinone complex in photosynthetic bacterial cells and oriented chromophores of the bacterium Rhodopsuedomonas viridis has been reported. 246 The results show that the iron possesses a low symmetry ligand field and exists with a preferred orientation in the native reaction centre-membrane complex. It was found that the dominant crystal field axis was 64O away from the membrane normal. Analysis of the weak interactions between Fe (11) and Q1+ and QZ-, J=0.12 and 0.06 cm-l indicate that Q1 is not directly coordinated to the ferrous ion. 246 E.p.r. was employed to examine the effects of mild trypsinization of PSII particles at pH 6 . 0 , which almost completely blocks reoxidation of the primary plastoquinone acceptor, Fe(II)-QA-. The lack of any effect on the e.p.r. signal from Fe(II)-Q, was discussed in relation to the functional and 247 structural organization of the PSII acceptor site. A characteristic non heme high spin iron e.p.r. signal at 10

126

Electron Spin Resonance

K with g = 4.1 and width H= 183.6 MHz was found in dark-adapted, oxygen evolving PSII illuminated at 140 K then cooled prior to measurement. Formation of the g = 4.1 signal was inhibited by 400 pM NH20H in 0.8 M NaCl buffer.248 Other workers have also reported the g = 4.1 signal noting that it occurs upon formation of one of two intermediates in the photo-oxidation of the S1 state.249 Three

distinct electron pathways were observed. Below 100 K one molecule of cytochrome b-559 was photo-oxidized per reaction centre. The e.p. r. spectrum of cytochrome b-563 in spinach chloroplast membranes indicate the presence of low spin hemes with g=3.5. The orientation of the two heme planes is found to be perpendicular to the thylakoid membrane which may be important for electron transport coupled to proton translocation. 250 A multiline manganese e.p.r. signal is also reported and is believed to originate from the same site as the g = 4.1 Broadening of the manganese e.p.r. signal from the S 2 state of PSII, in the presence of H 2 1 7 0 indicates oxygen coordination to the manganese ion. 253 A wild type strain of Rhodopseudomonas sphacroides containing manganese instead of the expected iron exhibited a Mn(I1) e.p.r. signal in the dark attributed to manganese bound at the reaction - and Mn.QB - were centre. New e.p.r. signals attributed to Mn.QA also found. 254 It is found that exogenous reductants reduce and destroy the Mn-complex in PSII membranes depleted of the 1 7 and 2 3 kDa polypeptides.255 The e.p.r. results support the idea of a shield around the Mn complex comprised of the 33 kDa polypeptide, along with the 23 and 1 7 kDa protein and tightly bound Ca2+. Manganese involvement in photosynthetic water oxidation by higher plants was monitored by low temperature e.p.r. as an indicator of the S-state composition for manganese X-ray absorption edge measurements of a spinach PSII preparation. 256 Simulation of the hyperf ine structure is consistent with exchange coupled Mn(II1,IV) binuclear and Mn (111,111,111,IV) tetranuclear centres. McCain et al. 257 find that in plant leaves from fifty plant species, the Mn(I1) e.p.r. spectra are isotropic. In some species, including those that exhibit orientation-dependent H ' NMR spectra, the forbidden transitions are also orientation dependent, suggesting that chloroplasts of some plants are preferentially aligned with respect to the leaf surface. Power saturation and temperature dependence of the three e.p.r. signals generated by low temperature illumination of dark-adapted PSII mem.branes can be associated with the S 2 state of

127

3: Metalloproteins

the oxygen evolving complex (OEC) of photosynthesis. It is concluded that each e.p.r. spectrum originates from a thermally-excited state of one of the three configurations of the manganese complex in the active site. Antiferromagnetically coupled Mn(II)-Mn(III) or Mn(II1)-Mn(1V) dimers are proposed for the S 2 e.p.r. active species.258 During dark adaption, a change in the oxygen evolving complex of spinach PSI1 occurs, that affects both the structure of the Mn site, as shown by e.p.r. and the chemical properties of the OEC. This leads to a resting state which is incapable of oxygen consumption. 2 5 9 E.p.r. characterization of the iron-depleted reaction centre in the photosynthetic bacterium Rhodopseudononas spheroides and subsequent reconstitution with Fe(II), Mn(II), Co(II), Ni(II), Cu(I1) and Zn(II), demonstrate that a divalent metal ion is not required for rapid electron transfer from QA- to QB. However metal ions may play a role in the rate limiting step prior to electron growth transfer. 260 Separation of the photosynthetic membrane initiation site using sucrose gradient centrifugation has enabled the characterization by e.p.r. of light induced reaction centres and the Rieske iron-sulphur signal for the ubiquinol-cytochrome c2 oxidoreductase. 261 Aerobic addition of 2,3-dimercaptopropan-l-o1 (BAL) to spinach chloroplasts from Oscillatoria limnetica cells inhibits NADPH photo-reduction, which is consistent with damage to the Rieske iron sulphur centre. However, no change in the e.p.r. spectrum was noted after the addition of BAL. In addition, when BAL is added anaerobically to the chloroplasts, it donates electrons to the Rieske protein. 2 6 2 11 Statistical Model of E.p.r.

Line Broadening

There are three recent papers of particular significance in relation to the origin of line broadening in the e.p.r. of metalloproteins. Hagen et g .263 describe a statistical model to explain the origin of '9-strain', the dominant broadening mechanism in metalloproteins. The observed e.p.r. widths result from a distribution in the effective g-value as a function of (a) the point distribution function of the elements of a p matrix which does not, in general, share the same principal axes as s, and (b) 264 , the relation between CJ and p principal axes. In a second paper Hagen et al. develop a very efficient algorithm to simulate spectra

128

due

Electron Spin Resonance

to

iron

sulphur

centres

in

NADH:Q

QH2:ferricytochrome c oxidoreductase, controversies about the stoichiometries of

oxidoreductase

and

clarifying existing these centres. Fourier

transform filtering techniques are used. More recently, Heashen et al.265 have sought to improve the simulation of the e.p.r. spectrum from the 56Fe reconstituted [2Fe-2S1 cluster in fully deuterated ferredoxin from Synechococcus lividus and the L H Z O exchanged [2Fe-2S] ferredoxin from Pseudomonas putida. E.p.r. and Mossbauer data are fitted by a model based on a statistical distribution of crystal field parameters and the techniques are based on their earlier They conclude that the e.p.r. of metalloproteins is a reflection of a protein structure that distributes its spatial coordinates and accommodates different levels of rigidity, the more flexible parts being on the outside. They warn against use of ad-hoc simulation algorithms and fitting ’by eye’. Salerno266 also considers a distributed e.p. r. parameter model to account for spectra due to heme proteins. This is a more conventional approach and could be seen, in some sense, as a subset of the more exacting model of the Michigan group.263-265 12 Relaxation The theory of paramagnetic relaxation required for proteins now requires consideration of the idea of a fractal or fractional dimension (d ) Wagner et a1 267 present a geometrical model to interpret the anomalous TT2m dependence of the Raman spin lattice relaxation rate for Kramers ions in heme and iron sulphur proteins, an integer. A modified Debye where m is not necessarily relationship where the vibrational density of states is dependent on Urn-’ is consistent with the values of m determined from measurements on cytochrome c-551 and putidaredoxin where the results depend on changes in the solvent. Solvent effects were also noted. The apparent physical significance of m is revealed, in part, by correlation to the protein fractal geometry. From independent data on 7 0 proteins it is known that l30 MHz which are usually anyway resolved in the ESR spectrum. In

solid-state ENDOR

with

poorly

resolved ESR

spectra,

second order contributions are more relevant. The explicit formulae for an anisotropic system with low symmetry, however, are rather

Electron Spin Resonance

146

F o r axial g and A tensors and Bo parallel to g,, o r gl,

AE2 is given by AE2(k,mI + m +1) = T4PA,(mI+1) 2

(7)

I or

AE2(+,mI + mI+l) = Tp{(Ai

A:)2mI

+

(A,, + All 2}

+

Second order expressions respectively (p = (8pBgBo)-1). nuclear quadrupole interactions have also been reportede7.

for

the

3fhfs

is

For some paramagnetic species the assumption at EZ > > ‘hfs O r gQ is not fulfilled. The second order perturbation approach

then no

longer accurate

enough

for

the determination

of

the

magnetic parameters. In such situations, found for example in cobalt complexes with

a

large cobalt

hf s i o 4 and

in

cobalt

and

copper

complexes with nitrogen hf coupling constants comparable to the “N nuclear quadrupole

i n t e r a c t i ~ n s ~ ~, ’ the ~ ~ ’ matrix ‘~~

of

the

spin

Hamiltonian has to be diagonalized numerically. A method to obtain good

starting values

for the minimization procedure has

recently

been reportediob.

b) Nonequivalent nuclei: If two magnetically nonequivalent nuclei, I and K . are present in a spin system, the transition frequency of nucleus I is influenced by a cross-term (sometimes called indirect nuclear-nuclear interaction) between nucleus I and K e o ’ e 7 ’ 1 0 7. This cross-term produces

shifts or

splittings in

the ENDOR

spectrum.

Shifts are observed if the hfs of nucleus K is resolved in the ESR spectrum; i.e.. if ESR transitions with different mK can be used as observers. This is often the case in transition metal compounds with a large hfs of the central nucleus. In copper complexes, the shift of

nitrogen

ENDOR

transitions

caused

cross-term may amount to more than 1 MHz

by

the

.

108’109

nitrogen-metal

Splittings or line

broadening effects are observed, if several hf lines of nucleus K

4: ENDOR Methodology

147

are simultaneously saturated. Such splittings have been measured, for example, in proton ENDOR spectra of 7-irradiated glycineIo7. c) Magnetically equivalent nuclei:

a

center

of

symmetry:

inversion-related and

i.e.,

Many paramagnetic compounds have they

contain

nuclei

which

are

therefore magnetically equivalent in pairs.

For the description of such species a coupled nuclear spin base has to be used87 . g o 9 9 1 v 1 0 8 . The pronounced splitting of the four lines into doublets for a copper complex with two magnetically equivalent 14

N nuclei is shown in Fig. 3a. Similar second order effects have

been

observed

in

liquids

(Fig.

3b)89~'031"0*"'

and

in

solids90.91.98.112

b

a 22

q

24

w

I 52 MHt

v&

L8 I

Figure 3: Second-order splittings: a) Single crystal ENDOR of Cu/Ni-bis-salicylaldoxime. two magnetically equivalent nitrogen nucleilo8, b) Liquid phase ENDOR of a radical cation, two magnetically equivalent nitrogen nucleiIo3.

d) Woncrossing of energy levels: in

ENDOR

spectra

or

patternsl13'l14'l16

multiples

of

second-order

manifest in

in

transitions

unii6. Extensive perturbation

The effects of level noncrossing

itself

theory

model is

complicated at

higher-order

frequencies

calculations

usually

not

of

equal show

to that

sufficient

accuracy in the neighbourhood of noncrossing points. The additional splittings

depend

quadrupole

interactions and

tensorsiI4.

critically

on

the

magnitude

of

the degree of anisotropy

the

nuclear

of

the hfs

Electron Spin Resonance

148

Transition Probabilities. -

3.2

Nuclear transitions induced by an

rf

field B2(t)=B 2cosot applied along the x (p=l) or the y ( p = 2 ) direction in the laboratory frame are described by the coupling operator

Application of the operator transform yields for a single nucleus the following nuclear transition probability for z e r o t h - o r d e r base functions:

with

The

1(1 + 1) - ml(mI - 1) .

=

A2(I,mI)

first

term

in

(9) describes

the

interaction of

the

oscillating rf field with the electron spin and contributes only in a

first order

Expressions f o r given

by

spin base

to

the nuclear

transition probability.

first order transition probabilities W1 have been

several

authors8g'e3'117'11eand

are

found

to

be

in

excellent agreement with numerical ca!culztions'17. The change of the nuclear transition probability due to the electron

spin, usually

called

hyperfine

considered classically as originating from the modulation

may

be

of

the

magnetic field Be produced by the electron at the nucleus. For an isotropic hf interaction, the hyperfine enhancement factor is given by

For an anisotropic system the enhancement factor

E = may

easily

{W1/Wo}1'2 be

calculated

from

the

expressions

for

Wo

and

W1.

Pronounced hyperfine enhancements are expected for metal-ions with large hf couplings, and for ligand nuclei with small gyromagnetic ratios. For nitrogen hfs of 40-50 MHz, values of E

2:

20

are found at X-band frequencies. Furthermore, the enhancement may

4: ENDOR Methodology

149

also essentially depend on the d i r e c t i o n of the rf fieldI2'. Nuclear

transition probabilities are

also

influenced by

quadrupole interactions. F o r nitrogen nuclei with 3fhfSZI q , for examp 1 e , Wl is strongly orientation dependent (within a factor of

10) and near to zero for one of the two m -states6'.

In addition,

transitions with A m 1-2 become allowed69"06"2' . They turned out to be very useful for the assignment of the AmI=l lines to corresponding nuclei6g"0s. Sivns

3.3

of

Hyperfine

and

Quadrupole

Couplinp: Constants.

-

Different approaches for the determination of absolute and relative signs of the principal values of hfs and quadrupole tensors are in use. a) Signs

of

the hyperfine

According to (1). be found by a

least-squares f i t of

different orientations of B m

principal

values

of a

single nucleus:

the relative signs of the principal values Ai can

does not alter (1).

.

the ENDOR

data obtained from

Since a change of the signs of A and

only r e l a t i v e signs of Ai can be obtained.

The a b s o l u t e sign of the t s o t r o p t c part of protons may be determined from the dipolar interaction, since the largest principal value of the dipolar part of A is p o s i t i ~ e ~ ~ ' ~ ~ ' ' ~ ~ .

For nuclei with Ai>>vn. the first order frequencies c(m

)

are rather insensitive to the signs of Ai. The relative signs may then be found by including the large higher order contributions in the

fitting process'".

Since

the

sign of

the

largest coupling

constant is often known unambiguously from theoretical arguments, the absolute signs of all three principal values may be determined. It has also been shown that relative signs of hf components can be determined from powder ENDOR

spectra by shifting the Bo

position

from a parallel toward a perpendicular E S R peak'". b)

Relative

signs of

the hyperfine

splittings of

two nuclei:

In

single crystal studies the relative signs between hfs constants of different nuclei may sometimes be determined from second order terms between nuclei I and K. This has been demonstrated, for example, on

AsOZ- radicals in KHZA~04123. c) Relative signs of hyperfine and quadrupole coupling constants: In

an E S R

spectrum with resolved hfs

quadrupole

coupling

constants

may

the relative signs of easily

be

evaluated

hf

and

from

the

Electron Spin Resonance

150

dependence of the ENDOR intensities on the nuclear quantum number mI of the saturated ESR linei0g"24

.

For two magnetically equivalent

1=1 nuclei, the signs can be obtained from similar, though more complex, intensity patterns98"0a"26.

If

the

hfs

of

nucleus

a

is

resolved

not

in

the

ESR

spectrum, all nuclear spin states are simultaneously saturated and a sign

determination

possible.

In

determined

this

using

ENDOR

case

the

considering

contributions have

second

been

used

line

intensities

relative to

order

signs hf

is

may

no

longer

sometimes

contributions.

determine

the

sign of

be Such

the

"K

electric quadrupole moment using F centres in KCti26. d) Signs of hyperfine splittings in triplet states: According to (5) absolute sign determination of hf couplings is not difficult, if the signs of the principal values of the fine structure tensor are knowniz7'1z8 . If only one of the two ESR transition can be observed or if both ENDOR transitions are detected with equal intensity when one ESR line is monitored, different microwave frequencies have to be used to get the hf signsiz9. Sign determinations with the aid of more sophisticated ENDOR techniques will be discussed in sect. 5. 3.4

Evaluation of the Mapnetic Parameters.

3.4.1

Liquid Phase ENDOR. Data reduction from a well resolved and

symmetric liquid phase ENDOR spectrum is usually straight forward. The assignment of hf couplings to particular nuclei, however, may be a difficult task, since the intensities of the ENDOR line do not reflect the number

of

contributing nuclei. An

obvious method

to

check the assignment is by computer simulation of the ESR spectrum based on the measured ENDOR frequenciesi3'.

A more direct procedure

uses partially deuterated speciesi3'. 3.4.2

Single Crystal ENDOR.

The interpretation of single crystal

ENDOR spectra is commonly much more complex. Various reasons are responsible for this: - 1 )

The dipolar interactions are not averaged

out to zero. This results in a large number of transitions also from regions several hundreds pm apart from the paramagnetic center and in

a

strong

matrix

line

near

the

nuclear

Zeeman

frequency.

Furthermore, the ENDOR frequencies are described by a more complex formula

.

-2)

Quadrupole

interactions

and

various

higher-order

contributions produce shifts and splittings of the lines. - 3 )

The

4: ENDOR Methodology

151

ENDOR spectra

spectra show complicated orientation dependences. - 4 ) of various species may be overlapped.

Spectroscopic techniques capable of simplifying complicated ENDOR

spectra

are

discussed

in

5.

sect.

Another

approach

to

elucidate the magnetic parameters is based on a n extensive use of computer assistance.

2.6

2.,

7 .**.*.............

. ... :: ................................. . .

.

..

0

.

10

.*..

20

:

. .. .. . ... .. .

30

40

Ckgl./D*0

Figure 4 : Computer-aided ENDOR: Angular dependence of the ENDOR transitions of Ho -centers in KCe9. s,a

a) Computer

defect

aided ENDOR:

centers

may

Single crystal ENDOR

contain

hundreds

of

spectra of certain

linesg.

Studies

of

the

angular dependence of all these transitions have to be done in small angle increments ( < 2 ) '

and are very tedious. Such types of studies

can essentially be facilitated by using a fully computer controlled spectrometer for registration and data for the angular dependence of ENDOR

transitions

. An example collected and

treated by such an instrument is shown in Fig. 4 . b) Data analysis: In single crystal work the ENDOR frequencies are

usually measured for crystal rotations around three suitable but not necessarily orthogonal axes. The hyperfine and quadrupole parameters

Electron Spin Resonance

152

are

then

evaluated

by

simultaneous

least-squares

fitting

of

the

experimental datais3. Symmetry restrictions are generally not made Inon-coinciding algorithm which

A multiparameter coupling tensors). is particularly useful for this

fitting has

been published by MarquardtIa4. The resulting coupling parameters (second rank

tensors)

are

then characterized

by

three principal

values and the direction cosines of the principal axes either with respect to the g tensor principal axes or to suitable crystal axes. In

molecular

crystals

with

anisotropic

tensors,

g

the

former

representation is certainly the preferential one. A discussion of the interpretation of magnetic parameters

obtained

ENDOR

from

spectra

exceeds

the

scope

of

this

paper.

However, some brief comments with respect to hyperfine interactions should be made.

1)

Spin-only

The

contribution:

spin-only

electron-nuclear

dipole-dipole interaction tensor ADD is traceless and symmetric. In the ENDOR

literature, this hyperfine coupling has been treated at

several levels of approximation. For nuclei other than protons, the largest coupling generally z r i s e s frcm the one-center contribution (atomic orbital centered at the nucleus). For protons the dipolar coupling is determined by two- and three-center contributions. It has

been

shown

neglected

in

that

the

the

latter

computation

of

one

should

not,

in general, be

s:>isotr~pic proton

hf

coupling

constantsq7. If the proton Is scfficientZy apart from the orbitals containing the unpaired electron, ADD

may be approximated by

the

classical electron-nuclear point-dipole formula

where

pk

is

the spin density on the kth atom, rk

the distance

between the proton and the kth atom and nk the direction cosines of rk in the molecular frame.

In

transition metal

compounds

the

unpaired

electron

is

usually localized at the central ion and the ligand atoms in the first coordination sphere, s o

that summation over these nuclei

is

often sufficient for a determination of the proton positionsio8. The rough approximation that the spin density at the central ions, pM , is equal to unity allows one to get estimates of r M and nM and thus to assign

the hfs

tensors

to corresponding protons.

For

certain

lanthanide ions i t is reported that the accuracy of this method is

4: ENDOR Methodology

higher

than

153

that

of

an

x-ray

diffraction

analysi~'~~''~~'~~

Golding et a1.I3* mentioned, however, that even for f-electrons one has to be very careful in using the point-dipole model. The authors found

that

the

errors

of

the approach

are

only

negligible

for

spherical f7-ions.

2) A s y m m e t r i c

hfs

Compounds

tensors:

with

transition

lanthanide ions are often characterized by a

metal

and

substantial orbital

magnetic moment, reflected by strong deviations of the g-values from ge.

This

leads

to

an

anisotropic

as

well

as

to

an

isotropic

contribution to the electron-nuclear dipolar i n t e r a c t i ~ n ' ~ ~ ' ' ~ ~ ' ' ~ ' and to a s y m m e t r i c hfs tensors. The

phenomenon

of

asymmetric

hfs

tensors

was

first

discussed by M~Connell'~'. Later. Kne~buehl'~~''~' proved the existence of asymmetric g and A tensors in paramagnetic systems with low symmetry. In the meantime, asymmetric A tensors in ESR and ENDOR spectroscopy have

been

considered by

several a ~ t h o r ~ ' ~ ~ ' ~ ~

N.V

N

Since the term

+ Ag)C

C(gA

in

(1)

is linear in A. all nine matrix

elements of the hfs tensor A can, in principle, be determined by ENDOR. If in (13)

pk=O

A = aisoE +

T =

with

is assumed, the hfs tensor writes

, pn=l

gT

p Bp Ngn (3nMiM

.

- E)/ri

(15)

Equation ( 1 4 ) amounts to a description of the anisotropic coupling

originating

magnetic

moment

isotropic

part

from

p=pgS

of

gT

the

with which

interaction

the

proton

is

of

an

magnetic

usually

called

electron

spin

moment'42.

The

pseudo-contact

interaction'4g, is only zero for isotropic g tensors. Since in (14) the g tensor is known from ESR data, the only remaining parameters which describe

A are aiso r M and the direction cosines nM'

Equation ( 1 4 ) has been applied, for example, to determine the proton coordinates

in

Nd(II1)-doped

crystals'36,

and

to

lanthanum

localize the

nicotinate

charge compensator

dihydrate (proton)

in

Co(I1)-doped 3.4.3

Powder ENDOR. In most practical cases, the evaluation of the

magnetic parameters from ENDOR spectra of polycrystalline or frozen solution samples recorded with an arbitrary position of

the ESR

observer is a difficult task, since a great many of orientations

1 54

Electron Spin Resonance

contribute

to

the

signal.

In

two

limiting

cases,

however,

the

interpretation becomes considerably simplified:

1) In systems with a weak anisotropy or with a n effective electron cross-relaxation, the ESR transitions of all orientations are saturated simultaneously. The resulting powder-ENDOR spectrum is then simulated by integrating over the whole unit sphereg3.

2) For slow

strongly

cross-relaxation,

anisotropic

easily

paramagnetic

interpretable

compounds

with

crystal-like

single

ENDOR spectra are recorded for certain selected Bo field settings"' (sect. 5.1).

8

Figure 5: Powder-ENDOR: Correspondences between magnetic field values in an ESR spectrum and field orientations within the molecular frameis1.

If

Bo

is

not

orientations

of

spectrum

pronounced

by

the

oriented

magnetic

along

parameters

spectral

one

of

the

manifested

features

(turning

canonical

in

the

points),

ESR a

simulation of the polycrystalline ENDOR pattern requires integration over the subset of orientations selected by the particular observing field. Fig. 5 shows the field orientations which are simultaneously saturated for different B g tensorI6'.

settings in a system with an orthorhombic

If hf couplings are resolved in the ESR spectrum the

situation becomes even more complicated. Theoretical models for a comprehensive polycrystalline

ENDOR

.

a U t ~ o r s l S l ~ 1 6 2 ~ 1 6 3

importance

of

patterns

Kreilick

applying

the

have

and

been

published

analysis by

collaboratorsis3 emphasized

correct

formulae

for

the

of

several the

ENDOR

155

4: ENDOR Methodology

frequencies to obtain a proper description of the powder However, signals

an by

unambiguous using

this

interpretation type

of

of

model

spectra.

polycrystalline

calculations

may

ENDOR

only

be

expected for relatively simple systemsis4 with a restricted number of interacting nuclei. A very recent report on the interpretation of powder ENDOR spectra is based on incorrect assumptionsi66.

THEORETICAL APPROACHES, MECHANISMS AND CONDITIONS

4

Various types of mechanisms taking place in solid and liquid phase ENDOR are summarized based

elementary

011

in the book of Kevan and Kispert'. considerations

of

level

They are

populations,

rf

and

microwave pumping rates and relaxation path-ways. 4.1.

Theory

of

Liquid

treatment of ENDOR

Phase

-

ENDOR.

A

rigorous

in solutions has been published

theoretical

by

Freed and

co-workers in a series of fundamental papers'6b-ibo . The theory makes use of the density matrix method and allows a qucntitatiue description

of

experimentally

observed

phenomena

like

ENDOR

enhancements, ENDOR line shapes or coherence effects. The papers are rather pretentious; this might be the reason why in recent years the approach has been used by only a few research groups. In a relaxation

pioneering

theory

work,

to study

Plato

et

the optimum

a1.l6'

applied

ENDOR on different nuclei. The results are presented and graphical parameter

in analytical

form and are applicable over a wide range of input

values

(e.g.

temperature,

viscosity,

microwave

field strengths). The authors found that for organic optimum conditions are essentially determined by squared

Freed's

conditions for performing

dipolar

hfs

tensor,

TrA'.

and

the

K

and

rf

radicals, the

the trace of the

spin-rotational

and

Heisenberg exchange parameter. The published data also allow one to predict the detectability of interesting nuclei not yet observed in liquid phase ENDOR. Based on Plato's general theory, Moehl et al. 1 6 ' the

first

ENDOR-in-solution

experiment

on

a

designed

transition

metal

complex. The margin for successful solution ENDOR on this type of compound, anisotropy.

however,

seems

to

be

very

narrow

(small

g

tensors

I = 0 for the central nucleus, small ligand hfs),

so

that ENDOR signals can only be expected for especially well selected complexes.

Electron Spin Resonance

156

Freed's parameters

if

theory

the

ESR

has

also

lines are

been not

applied

to

evaluate

ENDOR

resolved163. Brustolon

and

co-worker~'~ adapted ~ the theory to oriented radicals and calculated the orientation dependence of ENDOR enhancements and linewidths for a-protons. The same group developed an elegant technique for the investigation of methyl dynamics in solids'66.

The method is based

on an analysis of the ENDOR enhancement using Freed's approach and is even applicable for rotation rates to high to affect

the ENDOR

linewidths. The

ENDOR

phenomenoLogica1

response

theories

based

can on

also changes

be

described

in

the

by

effective

electronic spin-lattice relaxation time. The approach was introduced by the inventor of steady-state ENDOR, Horst Seidel'66, and extended by Allendoerfer and Maki'67

to describe the spin dynamics in liquid

phase ENDOR. Shikatal6'

applied the method of spin population numbers to

describe the ENDOR intensities and placed emphasis on the effect of incomplete

hf

separation

(self-ELDOR)

which

decreases

the

ENDOR

enhancement. Applicability and limitations of the approach have been checked up with experimental results from galvinoxyl radicals under various conditionsi6'. Multilevel analogues

to

spin

electric

systems

have

also

networks'i'170'i71

.

In

been this

considered

as

approach

the

various spin-lattice relaxation rates are regarded as proportional to the conductances. The ENDOR enhancement corresponds then to the relative

change

of

the

input

conductance

describing

the

ESR

transition after short-circuiting the branches of the resonant NMR transition. Freed's sophisticated density matrix approach is certainly much more difficult to handle than the simplified treatments cited above; however, the latter one do not necessarily take into account

all of the phenomena observed in an ENDOR therefore be applied with caution. 4.2.

Negative

ENDOR.

-

Negative

ENDOR

spectrum

enhancements.

d e c r e a s e rather than an increase in intensity of the

is observed, have

first been

reported

by

and

Atherton

should

where

a

ESR absorption et a1.I7'

liquids and by Miyagawa et a1.44 in solids. Negative ENDOR

in

lines

detected with intense rf fields may be much stronger (more than a factor of 10) than the corresponding positive ENDOR signals. This

4: ENDOR Methodology

important

157

advantage

has been used

to

improve

the

sensitivity of

proton-ENDOR studies on irradiated single

It could be established experimentally that negative ENDOR is not just an instrumental artifactI7'.

Theoretical descriptions of

the negative ENDOR mechanism based either on Bloch equations or on the

density

matrix

formalism

have

been

given

by

several

author^'^^-'^'. Recently, Miyagawa and c ~ l l a b o r a t o r s ' ~ ~ ' reported '~~ on an interesting phenomenon closely related to negative ENDOR. They found that the sign of the ENDOR enhancement of irradiated crystalline or polycrystalline distribution interacting

samples

of

is

sensitive

chemically

radicals

are

to

the

identical characterized

interacting radicals by positive ENDOR electron spin echo techniques and ESR

inhomogenity of

radical by

species.

negative,

the

Weakly strongly

signals. Thus, apart

from

tomography, continuous wave

ENDOR seems to offer an alternative way to study the distribution of radicals in irradiated crystals.

4.3

ENDOR - Detected Nuclear Magnetic Resonance. -

ENDOR-detected

NMR or distant-ENDOR has proved to be a very sensitive technique to determine

the

particularly

structure

well

of

suited

molecular

to get

crystals.

precise

The

method

is

information on hydrogen

(deuterium) atom locations. In a distant-ENDOR experiment, the signals from nuclei of diamagnetic

molecules

are

recorded

via

unpaired

electrons

of

statistically embedded paramagnetic probes. These nuclei are s o far off

from

the

paramagnetic

species

that

the

hfs

interaction

is

smaller than the internuclear dipolar coupling. Since the unpaired electrons with

their

large magnetic moment act as detectors, the

sensitivity of ENDOR-detected NMR exceeds the one of wide-line NMR by some orders of magnitude. Detailed knowledge about the structure

of the radicals (usually generated by irradiation) or ions used as detector centers is not required. Since distant-ENDOR mechanisms are based on nuclear spin diffusion, the experiments have to be carried out at

low temperatures (