Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem [1 ed.] 9811515832, 9789811515835

Table of contents :
Supervisor’s Foreword
Parts of this thesis have been published in the following journal articles:
Acknowledgements
Contents
Abbreviations
1 General Introduction
1.1 Photosystem II
1.2 Fourier Transform Infrared Spectroscopy (FTIR)
1.3 Quantum Chemical Calculation
1.4 Purpose of This Study
References
2 Hydrogen Bond Structure of Redox Active Tyrosines in Photosystem II
2.1 Introduction
2.2 Materials and Methods
2.3 Results
2.4 Discussion
References
3 Proton Release Reaction of Tyrosine D in Photosystem II
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.4 Discussion
References
4 Vibrational Analysis of Water Network Around the Mn Cluster
4.1 Introduction
4.2 Materials and Methods
4.3 Results
4.4 Discussion
References
5 Vibrational Analysis of Carboxylate Ligands in the Water Oxidizing Center
5.1 Introduction
5.2 Materials and Methods
5.3 Results
5.4 Discussion
References
6 Protonation Structure of a Key Histidine in the Water Oxidizing Center
6.1 Introduction
6.2 Materials and Methods
6.3 Results
6.4 Discussion
References
7 General Conclusion
Curriculum Vitae

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Springer Theses Recognizing Outstanding Ph.D. Research

Shin Nakamura

Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II

Springer Theses Recognizing Outstanding Ph.D. Research

Aims and Scope The series “Springer Theses” brings together a selection of the very best Ph.D. theses from around the world and across the physical sciences. Nominated and endorsed by two recognized specialists, each published volume has been selected for its scientific excellence and the high impact of its contents for the pertinent field of research. For greater accessibility to non-specialists, the published versions include an extended introduction, as well as a foreword by the student’s supervisor explaining the special relevance of the work for the field. As a whole, the series will provide a valuable resource both for newcomers to the research fields described, and for other scientists seeking detailed background information on special questions. Finally, it provides an accredited documentation of the valuable contributions made by today’s younger generation of scientists.

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Shin Nakamura

Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II Doctoral Thesis accepted by Nagoya University, Nagoya, Japan

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Author Dr. Shin Nakamura Department of Biochemical Sciences “A. Rossi Fanelli” University of Rome “Sapienza” Rome, Italy

Supervisor Prof. Takumi Noguchi Division of Material Science (Physics) Graduate School of Science Nagoya University Nagoya, Japan

ISSN 2190-5053 ISSN 2190-5061 (electronic) Springer Theses ISBN 978-981-15-1583-5 ISBN 978-981-15-1584-2 (eBook) https://doi.org/10.1007/978-981-15-1584-2 © Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Supervisor’s Foreword

Photosynthetic water oxidation, which is performed in photosystem II (PSII) in plants and cyanobacteria, is one of the most essential enzymatic reactions in biological systems. Utilizing light energy, water oxidation provides electrons necessary for reduction in CO2 to synthesize sugars and produces molecular oxygen to form the oxygenic atmosphere, and thus, it supports the sustenance of the environment and life on the Earth. We now know the basic structure of the catalytic center of water oxidation, the so-called Mn cluster, thanks to the recent high-resolution X-ray crystallographic studies of PSII. However, the molecular mechanism of water oxidation, in which protons play the main role, is still the greatest mystery in photosynthesis research. The key to answer the question is to understand how protons are coupled with electron transfer, which controls the whole mechanism of water oxidation. In this thesis, Shin Nakamura tackled the elusive problem of the water oxidation mechanism using both experimental and theoretical approaches such as Fourier transform infrared measurements and quantum mechanics/molecular mechanics simulations. Using these methodologies, he first addressed the mechanism of asymmetric electron transfer on the electron-donor side of PSII, which is relevant to the high quantum yield of water oxidation. He also analyzed the structures and vibrations of water networks near the Mn cluster and proposed a new mechanism of proton transfer during water oxidation. Furthermore, he clarified the roles of amino acid residues interacting with the Mn cluster with respect to the control of the redox potential and the activation of water molecules. I believe that his work not only provides a significant contribution to research of the water oxidation mechanism but also offers an essential basis for the development of efficient photocatalysts of water oxidation, which is the bottleneck in artificial photosynthesis. Nagoya, Japan December 2019

Prof. Takumi Noguchi

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Parts of this thesis have been published in the following journal articles: (1) S. Nakamura, R. Nagao, R. Takahashi, and T. Noguchi (2014) Fourier transform infrared detection of a polarizable proton trapped between photooxidized tyrosine YZ and a coupled histidine in photosystem II: relevance to the proton transfer mechanism of water oxidation. Biochemistry 53, 3131–3144. (2) S. Nakamura and T. Noguchi (2015) Infrared detection of a proton released from tyrosine YD to the bulk upon its photo-oxidation in photosystem II, Biochemistry 54, 5045–5053 (3) S. Nakamura, K. Ota, Y. Shibuya, and T. Noguchi (2016) Role of a water network around the Mn4CaO5 cluster in photosynthetic water oxidation: A Fourier transform infrared spectroscopy and quantum mechanics/molecular mechanics calculation study, Biochemistry 55, 597–607 (4) S. Nakamura, and T. Noguchi (2016) Quantum mechanics/molecular mechanics simulation of the ligand vibrations of the water-oxidizing Mn4CaO5 cluster in photosystem II, Proc. Natl. Acad. Sci. U.S.A. 113, 12727–12732 (5) S. Nakamura, and T. Noguchi (2017) Infrared determination of the protonation state of a key histidine residue in the photosynthetic water oxidizing center, J. Am. Chem. Soc. 139, 9364–9375.

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Acknowledgements

I would like to express my gratitude to all of those who gave me the opportunity to complete this thesis. I am especially indebted to my supervisor, Prof. Takumi Noguchi, for providing me his knowledge, guidance, and technical supports. I learned the importance of objectivity and perseverance in science through the research activities with him. I deeply appreciate Dr. Ryo Nagao for significant technical advices and supports for sample preparations. I learned most of the biochemical techniques used in the present work from him. I also appreciate Dr. Ryouta Takahashi and Mr. Kai Ota for their technical supports in FTIR measurements and Mr. Ryota Ashizawa for his advice on computational techniques. Finally, I greatly thank Prof. Hiroyuki Mino, Prof. Yuki Kato, and all members in the laboratory for helpful discussion and kind supports. This research was supported by Japan Society for the Promotion of Science (JSPS) Fellows Grant-in-Aid 15J10320. QM/MM calculations were performed at the Research Center for Computational Science, Okazaki, Japan, and Information Technology Center, Nagoya University.

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Contents

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2 Hydrogen Bond Structure of Redox Active Tyrosines in Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3 Proton Release Reaction of 3.1 Introduction . . . . . . . . 3.2 Materials and Methods 3.3 Results . . . . . . . . . . . . 3.4 Discussion . . . . . . . . . References . . . . . . . . . . . . .

Tyrosine D in Photosystem II . . . . . . . .

1 General Introduction . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Fourier Transform Infrared Spectroscopy (FTIR) 1.3 Quantum Chemical Calculation . . . . . . . . . . . . . 1.4 Purpose of This Study . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 Vibrational Analysis of Water Network Around the Mn Cluster . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Materials and Methods . . . . . . . . . . . . . . . . . 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5 Vibrational Analysis of Carboxylate in the Water Oxidizing Center . . . . 5.1 Introduction . . . . . . . . . . . . . . . 5.2 Materials and Methods . . . . . . . 5.3 Results . . . . . . . . . . . . . . . . . . . 5.4 Discussion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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6 Protonation Structure of a Key Histidine in the Water Oxidizing Center . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . 6.2 Materials and Methods . . . . . . . . . . . 6.3 Results . . . . . . . . . . . . . . . . . . . . . . . 6.4 Discussion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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7 General Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Curriculum Vitae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Abbreviations

DFT DM FTIR Hepes MeIm Mes ONIOM P680 PDB PSII QA QB QM/MM WOC XFEL YD YZ

Density functional theory n-dodecyl-b-D-maltoside Fourier transform infrared 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Methylimidazole 2-(N-morpholino)ethanesulfonic acid Our own n-layered integrated molecular orbital and molecular mechanics The special pair chlorophyll of photosystem II Protein Data Bank Photosystem II Primary quinone electron acceptor Secondary quinone electron acceptor Quantum mechanics/molecular mechanics Water-oxidizing center X-ray free-electron laser Redox-active tyrosine on the D2 protein Redox-active tyrosine on the D1 protein

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Chapter 1

General Introduction

1.1 Photosystem II In plants and cyanobacteria, photosystem II (PSII) has a function of photosynthetic water oxidation [1–7]. PSII is a multisubunit protein complex embedded in thylakoid membranes (Fig. 1.1). Water oxidation splits water into molecular oxygen and protons. Virtually all of atmospheric oxygen, which is essential for the sustenance of life on Earth, is produced by this reaction. Released protons generate a proton gradient across the membranes, which is utilized to produce ATP, a form of chemical energy,

Fig. 1.1 Redox cofactors and electron transfer chain of PSII © Springer Nature Singapore Pte Ltd. 2020 S. Nakamura, Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II, Springer Theses, https://doi.org/10.1007/978-981-15-1584-2_1

1

2

1 General Introduction

Fig. 1.2 Proton-coupled electron transfer of a tyrosine side chain

at ATPase. Electrons obtained by water oxidation are utilized for reducing carbon dioxide to synthesize sugars together with ATP. Electron transfer and water oxidation reactions in PSII starts with light-induced charge separation at the excited state of reaction center chlorophylls (a coupled excited state of special pair chlorophyll, P680, and monomer chlorophyll, ChlD1 ). An electron is ejected to a pheophytin electron acceptor (Pheo) and then transferred to plastoquinone electron acceptors (QA and QB ) [8]. On the electron-donor side, the generated P680+ cation abstracts an electron from the water-oxidizing center (WOC) via a redox-active tyrosine YZ (D1-Y161). There is also another redox-active tyrosine, YD (D2-Y160), in PSII (Fig. 1.1). These two redox-active tyrosines, YZ and YD , are symmetrically located in PSII [9, 10]. It is known that these tyrosines give rise to proton-coupled electron transfer reactions (Fig. 1.2) [11–25]. Namely, when a tyrosine side chain is oxidized, its phenolic proton is released to form a neutral radical (Y•Z or Y•D ) due to the very low pK a value (pK a = −2.0) in the oxidized state [23, 26, 27]. However, the roles of these tyrosines are significantly different. YD functions as a secondly electron donor to P680+ , whereas YZ functions as a major electron donor to P680+ and then accepts an electron from the WOC. This asymmetry of electron transfer is crucial for the high quantum efficiency of water oxidation in PSII. However, the origin of this asymmetry has not been clarified. Recent X-ray crystallographic analysis has revealed the detailed structure of the WOC and the surrounding protein environment (Fig. 1.3a) [9, 10, 28–30]. The catalytic core is the Mn cluster, which is a cubane-like cluster formed with three Mn atoms (Mn1–Mn3), one Ca atom, and four oxygen atoms (O1–O3, and O5), combined with a dangling Mn atom (Mn4) connected by two oxygen atoms (O4 and O5). The Mn cluster is surrounded by amino acid ligands of six carboxylate groups [D1– D170, D1–E189, D1–E333, D1–D342, D1–A344 (C-terminus), and CP43–E354] and an imidazole group (D1–H332). In addition, four water ligands on Mn4 (W1 and W2) and Ca (W3 and W4) have been identified. A guanidine group (CP43–R357), an imidazole group (D1–H337), and water molecules are hydrogen-bonded with the Mn cluster. Moreover, several water molecules form a water cluster near the Mn cluster (Fig. 1.3b). However, the roles of these amino acid residues and water molecules for water oxidation remain unresolved. The water oxidation reaction proceeds through the cycle of five intermediates called the Si state (i = 0–4) (Fig. 1.4) [31, 32]. The S1 state is most stable in the

1.1 Photosystem II

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Fig. 1.3 Structures of a the Mn cluster and the surrounding amino acids and b the water cluster

Fig. 1.4 S-state cycle of WOC

dark, and the Si state (i = 0–3) advances to the next Si+1 state upon oxidation by Y•Z . The S4 state is the highest oxidation state and immediately relaxes to the S0 state releasing an oxygen molecule. In the S-state cycle, four protons are produced from two water molecules and released into the lumen. Proton release occurs in the S0 → S1 , S1 → S2 , S2 → S3 , and S3 → S0 transitions with the stoichiometry of 1: 0: 1: 2 [33–35]. This stoichiometry together with electron abstraction at each transition implies that an excessive positive charge is accumulated on the Mn cluster in the S2 and S3 states. In water oxidation, proton transfer is a crucial process. The recent high-resolution X-ray structure [9, 10, 29] suggested several candidates of proton transfer pathways from the WOC to the lumen. However, it has not been clarified yet which pathways are suitable for proton release. One candidate is the hydrogen bond network from the Mn cluster to the lumen via YZ (Fig. 1.5) [9]. Thus, it is possible that YZ plays a key role in not only electron transfer but also proton transfer in the water oxidation mechanism. In spite that the high-resolution X-ray crystallographic structure of the WOC has been revealed, the molecular mechanism of photosynthetic water oxidation is still largely unresolved. One of the main reasons for this is that the X-ray structures reported so far have not resolved hydrogen atoms [9, 10, 28–30]. The information

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1 General Introduction

Fig. 1.5 Hydrogen bond network from the Mn cluster to the lumen via YZ

of the structures and reactions of protons is crucial to reveal the process of water oxidation. Thus, it is necessary to investigate the protonation and hydrogen-bonded structures of water molecules and proteins in the WOC for full understanding of the water oxidation mechanism.

1.2 Fourier Transform Infrared Spectroscopy (FTIR) Infrared spectroscopy is widely used to investigate the structural information of molecules. The energy of infrared light corresponds to the excitation of molecular vibrations, which reflect the structures and interactions of molecules. Infrared absorption occurs when the dipole moments of the vibrations are changed upon excitation. The coordinates of normal modes, their vibrational frequencies, and the intensities of infrared absorption are very sensitive to subtle structural changes of the molecules. Thus, infrared spectroscopy is a powerful method to detect the changes in chemical bonds in reactions, in particular, changes in protonation structures and hydrogen-bond interactions. For instance, in an OH stretching vibration, a hydrogenbond interaction induces a downshift of the vibrational frequency by weakening the strength of the O–H bond. Infrared spectroscopy has been extensively used to obtain the structural information of proteins, because all of amino acids forming polypeptide chains and water molecules are infrared active. However, infrared absorption spectra of proteins mainly provide the bands of amide groups of polypeptide main chains. In addition, the bands of amino-acid side chains and water molecules significantly overlap each other to form relatively broad spectral features. Hence, specific information of the active site of a protein, which is necessary for detailed analysis of the enzymatic reaction, cannot be obtained only from its original infrared spectrum. To extract signals specific to the active site of a protein, the reaction-induced Fourier transform infrared (FTIR) difference technique has been developed [36]. In this method, by

1.2 Fourier Transform Infrared Spectroscopy (FTIR)

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Fig. 1.6 a The diagram of light-induced FTIR difference measurements and b an example of the different spectrum (blue line) between spectra before (red line) and after (black line) illumination

taking a difference between spectra measured before and after the reaction, a large absorption that is not involved in the reaction is canceled and only the infrared bands of the active site affected by the reaction are obtained (Fig. 1.6). With this method, the change of even a single chemical bond in a very large protein (e.g., >500 kDa) can be detected. In such a measurement, a very small absorption change (A < 10−4 ) must be detected and hence this technique requires an extremely high accuracy. FTIR spectroscopy realizes such a high accuracy in the wavenumber axis by using a Michelson interferometer. Infrared light from an IR source, which passes through a Michelson interferometer and a sample, is monitored at a mercury cadmium telluride (MCT) detector as a function of the position of a moving mirror (Fig. 1.6). Because this position of the moving mirror is monitored using a He–Ne laser, which has a wavenumber accuracy of more than 7 digits, the obtained spectra also have a high wavenumber accuracy. Thus, FTIR spectroscopy is a suitable method for detecting very small spectral changes in a large protein. The attenuated total reflectance (ATR) technique is also useful in the measurement of reaction-induced FTIR difference spectra (Fig. 1.7) [37, 38]. In contrast to a conventional transmission method, it detects infrared absorption utilizing the Fig. 1.7 Diagram of polarized ATR-FTIR measurement

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1 General Introduction

penetration of the evanescent wave of infrared light into the sample on the surface of the internal reflection element (IRE) [39]. Using the ATR method, the condition of the sample can be changed by exchanging buffers on the sample adsorbed on the IRE surface [40]. Furthermore, polarized FTIR measurement is readily applied to an oriented sample on the IRE surface, to estimate the directions of the dipole moments of molecular vibrations [41]. The reaction-induced FTIR different spectroscopy has been applied to various biological systems. As a trigger to initiate reactions in proteins, light illumination is the most convenient. This method, light-induced FTIR difference spectroscopy, has been used to investigate the reaction mechanism of many photo-sensitive proteins, such as photosynthetic proteins and bacteriorhodopsin [42–44]. In particular, the mechanism of water oxidation in PSII has been investigated using this technique, and the information of the structural and interaction changes in amino acid side chains, polypeptide main chains, and water molecules, and that of proton release and transition efficiencies during the S-state cycle have been obtained [45–47].

1.3 Quantum Chemical Calculation Quantum chemical calculations theoretically predict the most stable structures and the vibrational frequencies of chemical compounds [48]. In particular, density functional theory (DFT) is a highly versatile method for the analysis of various types of compounds from a small molecule to a biopolymer [49]. Quantum chemical calculations make it possible to simulate the protonation structure and the conformational changes during the reaction in a protein, which are difficult to obtain by X-ray crystallography. Thus, with the aid of vibrational analysis using quantum chemical calculations, detailed structural information can be obtained from experimental infrared spectra [50–52]. The QM/MM method is widely applied to the simulations of large systems. The calculation system is separated into two regions, MM and QM regions (Fig. 1.8) [53– 55]. QM calculations provide highly accumulate simulation, although calculation costs are very high. In contrast, the cost of MM calculations is low in spite of lower accuracy than the QM calculations. The QM/MM calculations combine these merits of the two calculations. Thus, QM/MM method is suitable for the simulation of an active-site structure in a large system such as WOC in a PSII protein complex.

1.4 Purpose of This Study Unanswered essential questions regarding the electron transfer and water oxidation mechanisms of PSII mentioned above are summarized as follows. (1) What is the reason for the asymmetry in electron transfer of YZ and YD in spite of their symmetric locations? The electron transfer rate of YZ is much faster than that of YD , causing a

1.4 Purpose of This Study

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Fig. 1.8 a Example of the region of the QM/MM system in PSII and b the MM and QM regions in QM/MM calculation

high quantum efficiency of water oxidation. (2) What is the role of a hydrogen-bond network formed by water molecules around the Mn cluster in proton transfer? Which pathway is used for proton exit during water oxidation? (3) What are the roles of amino acid residues near the Mn cluster in the water oxidation mechanism? To answer these questions, we used a combinatorial approach of experimental and computational methods, i.e., light-induced FTIR difference spectroscopy and QM/MM calculations. As for the question (1), proton-coupled electron transfer of a tyrosine side chain should be a key process to solve the problem, and hence the hydrogen-bonded structures of YZ and YD were investigated and compared (Chap. 2). To further examine the difference in proton transfer reactions, the quantities of protons released from YZ and YD upon their oxidation were estimated by monitoring protonation signals of buffer molecules using the isotope-edited FTIR method (Chap. 3). From these studies, it was found that YZ and YD indeed have significantly different hydrogen-bonded structures and proton release reactions, which explains the significant difference in the electron transfer rate. The question (2) was investigated using QM/MM calculations (Chap. 4). The OH stretching bands of water molecules in the FTIR difference spectrum of WOC were analyzed and the experimental spectrum was reproduced. We suggested that a delocalized water vibration plays an important role in rapid proton transfer by the Grotthuss mechanism. For the question (3), the role of carboxylate ligands to the Mn cluster was studied by normal-mode analysis using QM/MM calculations (Chap. 5). It was shown that the vibration of a key carboxylate ligand bridging the Mn and Ca ions regulates the reactivity of water ligands. Furthermore, polarized ATR-FTIR spectroscopy combined with the QM/MM analysis was used to investigate the role of a His side chain hydrogen-bonded with the Mn cluster (Chap. 6). It was proved that this His residue has a protonated cation form during the S-state cycle of water oxidation, playing an important role in keeping the redox potential of the Mn cluster high enough to oxidize water. In the last chapter (Chap. 7), the conclusion of this work is summarized.

8

1 General Introduction

References 1. Debus RJ (1992) The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102:269–352 2. Grundmeier A, Dau H (2012) Structural models of the manganese complex of photosystem II and mechanistic implications. Biochim Biophys Acta 1817:88–105 3. McEvoy JP, Brudvig GW (2006) Water-splitting chemistry of photosystem II. Chem Rev 106:4455–4483 4. Vinyard DJ, Ananyev GM, Dismukes GC (2013) Photosystem II: the reaction center of oxygenic photosynthesis. Annu Rev Biochem 82:577–606 5. Hillier W, Messinger J (2005) Mechanism of photosynthetic oxygen production. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water: plastoquinone oxidoreductase. Springer, Dordrecht, The Netherlands, pp 567–608 6. Renger G (2012) Photosynthetic water splitting: apparatus and mechanism. In: Eaton-Rye JJ, Tripathy BC, Sharkey TD (eds) Photosynthesis: plastid biology, energy conversion and carbon assimilation. Springer, Dordrecht, The Netherlands, pp 359–414 7. Messinger J, Noguchi T, Yano J (2011) Photosynthetic O2 evolution. In: Wydrzynski TJ, Hillier W (eds) Molecular solar fuels. Royal Society of Chemistry, Cambridge, U.K., pp 163–207 8. Renger G, Holzwarth AR (2005) Primary electron transfer. In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water: plastoquinone oxidoreductase. Springer, Dordrecht, The Netherlands, pp 139–175 9. Umena Y, Kawakami K, Shen JR, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473:55–60 10. Suga M, Akita F, Hirata K, Ueno G, Murakami H, Nakajima Y, Shimizu T, Yamashita K, Yamamoto M, Ago H, Shen JR (2015) Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature 517:99–103 11. Diner BA, Britt RD (2005) The redox-active tyrosines YZ and YD . In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water: plastoquinone oxidoreductase. Springer, Dordrecht, The Netherlands, pp 207–233 12. Renger G (2012) Mechanism of light induced water splitting in Photosystem II of oxygen evolving photosynthetic organisms. Biochim Biophys Acta 1817:1164–1176 13. Styring S, Sjöholm J, Mamedov F (2012) Two tyrosines that changed the world: interfacing the oxidizing power of photochemistry to water splitting in photosystem II. Biochim Biophys Acta 1817:76–87 14. Babcock GT, Barry BA, Debus RJ, Hoganson CW, Atamian M, McIntosh L, Sithole I, Yocum CF (1989) Water oxidation in photosystem II: from radical chemistry to multielectron chemistry. Biochemistry 28:9557–9565 15. Faller P, Debus RJ, Brettel K, Sugiura M, Rutherford AW, Boussac A (2001) Rapid formation of the stable tyrosyl radical in photosystem II. Proc Natl Acad Sci USA 98:14368–14373 16. Faller P, Rutherford AW, Debus RJ (2002) Tyrosine D oxidation at cryogenic temperature in photosystem II. Biochemistry 41:12914–12920 17. Faller P, Goussias C, Rutherford AW, Un S (2003) Resolving intermediates in biological protoncoupled electron transfer: a tyrosyl radical prior to proton movement. Proc Natl Acad Sci USA 100:8732–8735 18. Rutherford AW, Boussac A, Faller P (2004) The stable tyrosyl radical in photosystem II: why D? Biochim Biophys Acta 1655:222–230 19. Berthomieu C, Hienerwadel R (2005) Vibrational spectroscopy to study the properties of redoxactive tyrosines in photosystem II and other proteins. Biochim Biophys Acta 1707:51–66 20. Havelius KG, Styring S (2007) pH dependent competition between YZ and YD in photosystem II probed by illumination at 5 K. Biochemistry 46:7865–7874 21. Hammarström L, Styring S (2011) Proton-coupled electron transfer of tyrosines in Photosystem II and model systems for artificial photosynthesis: the role of a redox-active link between catalyst and photosensitizer. Energy Environ Sci 4:2379–2388

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22. Matsuoka H, Shen JR, Kawamori A, Nishiyama K, Ohba Y, Yamauchi S (2011) Proton-coupled electron-transfer processes in photosystem II probed by highly resolved g-anisotropy of redoxactive tyrosine YZ . J Am Chem Soc 133:4655–4660 23. Saito K, Shen JR, Ishida T, Ishikita H (2011) Short hydrogen bond between redox-active tyrosine YZ and D1-his190 in the photosystem II crystal structure. Biochemistry 50:9836–9844 24. Chatterjee R, Coates CS, Milikisiyants S, Lee CI, Wagner A, Poluektov OG, Lakshmi KV (2013) High-frequency electron nuclear double-resonance spectroscopy studies of the mechanism of proton-coupled electron transfer at the tyrosine-D residue of photosystem II. Biochemistry 52:4781–4790 25. Saito K, Rutherford AW, Ishikita H (2013) Mechanism of tyrosine D oxidation in Photosystem II. Proc Natl Acad Sci USA 110:7690–7695 26. Dixon WT, Murphy D (1976) Determination of the acidity constants of some phenol radical cations by means of electron spin resonance. J Chem Soc Faraday Trans 2(72):1221–1230 27. Rappaport F, Boussac A, Force DA, Peloquin J, Brynda M, Sugiura M, Un S, Britt RD, Diner BA (2009) Probing the coupling between proton and electron transfer in photosystem II core complexes containing a 3-fluorotyrosine. J Am Chem Soc 131:4425–4433 28. Suga M, Akita F, Sugahara M, Kubo M, Nakajima Y, Nakane T, Yamashita K, Umena Y, Nakabayashi M, Yamane T, Nakano T, Suzuki M, Masuda T, Inoue S, Kimura T, Nomura T, Yonekura S, Yu LJ, Sakamoto T, Motomura T, Chen JH, Kato Y, Noguchi T, Tono K, Joti Y, Kameshima T, Hatsui T, Nango E, Tanaka R, Naitow H, Matsuura Y, Yamashita A, Yamamoto M, Nureki O, Yabashi M, Ishikawa T, Iwata S, Shen JR (2017) Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543:131–135 29. Tanaka A, Fukushima Y, Kamiya N (2017) Two different structures of the oxygen-evolving complex in the same polypeptide frameworks of photosystem II. J Am Chem Soc 139:1718– 1721 30. Young ID, Ibrahim M, Chatterjee R, Gul S, Fuller F, Koroidov S, Brewster AS, Tran R, AlonsoMori R, Kroll T, Michels-Clark T, Laksmono H, Sierra RG, Stan CA, Hussein R, Zhang M, Douthit L, Kubin M, de Lichtenberg C, Long Vo P, Nilsson H, Cheah MH, Shevela D, Saracini C, Bean MA, Seuffert I, Sokaras D, Weng TC, Pastor E, Weninger C, Fransson T, Lassalle L, Brauer P, Aller P, Docker PT, Andi B, Orville AM, Glownia JM, Nelson S, Sikorski M, Zhu D, Hunter MS, Lane TJ, Aquila A, Koglin JE, Robinson J, Liang M, Boutet S, Lyubimov AY, Uervirojnangkoorn M, Moriarty NW, Liebschner D, Afonine PV, Waterman DG, Evans G, Wernet P, Dobbek H, Weis WI, Brunger AT, Zwart PH, Adams PD, Zouni A, Messinger J, Bergmann U, Sauter NK, Kern J, Yachandra VK, Yano J (2016) Structure of photosystem II and substrate binding at room temperature. Nature 540:453–457 31. Joliot P, Barbieri G, Chabaud R (1969) A new model of photochemical centers in system II. Photochem Photobiol 10:309–329 32. Kok B, Forbush B, McGloin M (1970) Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism. Photochem Photobiol 11:457–475 33. Fowler CF (1977) Proton evolution from photosystem II. Stoichiometry and mechanistic considerations. Biochim Biophys Acta 462:414–421 34. Schlodder E, Witt HT (1999) Stoichiometry of proton release from the catalytic center in photosynthetic water oxidation. Reexamination by a glass electrode study at Ph 5.5–7.2. J Biol Chem 274:30387–30392 35. Suzuki H, Sugiura M, Noguchi T (2009) Monitoring proton release during photosynthetic water oxidation in photosystem II by means of isotope-edited infrared spectroscopy. J Am Chem Soc 131:7849–7857 36. Mäntele W (1993) Reaction-induced infrared difference spectroscopy for the study of protein function and reaction mechanisms. Trends Biochem Sci 18:197–202 37. Harrick NJ (1960) Surface chemistry from spectral analysis of totally internally reflected radiation. J Phys Chem 64:1110–1114 38. Fahrenfort J (1961) Attenuated total reflection: a new principle for the production of useful infra-red reflection spectra of organic compounds. Spectrochim Acta 17:698

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39. Goormaghtigh E, Raussens V, Ruysschaert JM (1999) Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes. Biochim Biophys Acta 1422:105–185 40. Okubo T, Noguchi T (2007) Selective detection of the structural changes upon photoreactions of several redox cofactors in photosystem II by means of light-induced ATR-FTIR difference spectroscopy. Spectrochim Acta, Part A 66:863–868 41. Iizasa M, Suzuki H, Noguchi T (2010) Orientations of carboxylate groups coupled to the Mn cluster in the photosynthetic oxygen-evolving center as studied by polarized ATR-FTIR spectroscopy. Biochemistry 49:3074–3082 42. Zscherp C, Barth A (2001) Reaction-induced infrared difference spectroscopy for the study of protein reaction mechanisms. Biochemistry 40:1875–1883 43. Berthomieu C, Hienerwadel R (2009) Fourier transform infrared (FTIR) spectroscopy. Photosynth Res 101:157–170 44. Noguchi T, Berthomieu C (2005) Molecular analysis by vibrational spectroscopy. In: Wydrzynski T, Satoh K (eds) Photosystem II: the light-driven water: plastoquinone oxidoreductase. Springer, Dordrecht, The Netherlands 45. Chu HA (2013) Fourier transform infrared difference spectroscopy for studying the molecular mechanism of photosynthetic water oxidation. Front Plant Sci 4:146 46. Debus RJ (2015) FTIR studies of metal ligands, networks of hydrogen bonds, and water molecules near the active site Mn4 CaO5 cluster in Photosystem II. Biochim Biophys Acta 1847:19–34 47. Noguchi T (2015) Fourier transform infrared difference and time-resolved infrared detection of the electron and proton transfer dynamics in photosynthetic water oxidation. Biochim Biophys Acta 1847:35–45 48. Szabo A, Ostlund NS (1996) Modern quantum chemistry: introduction to advanced electronic structure theory. Courier Corporation 49. Parr RG (1980) Density functional theory of atoms and molecules. In: Fukui K, Pullman B (eds) Horizons of quantum chemistry. Springer, Dordrecht, The Netherlands, pp 5–15 50. Hasegawa K, Ono T, Noguchi T (2000) Vibrational spectra and ab initio DFT calculations of 4-methylimidazole and its different protonation forms: infrared and Raman markers of the protonation state of a histidine side chain. J Phys Chem B 104:4253–4265 51. O’Malley PJ (2002) Density functional calculations modelling tyrosine oxidation in oxygenic photosynthetic electron transfer. Biochim Biophys Acta 1553:212–217 52. Ashizawa R, Noguchi T (2014) Effects of hydrogen bonding interactions on the redox potential and molecular vibrations of plastoquinone as studied using density functional theory calculations. Phys Chem Chem Phys 16:11864–11876 53. Warshel A, Levitt M (1976) Theoretical studies of enzymic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. J Mol Biol 103:227– 249 54. Maseras F, Morokuma K (1995) IMOMM: a new integrated ab initio + molecular mechanics geometry optimization scheme of equilibrium structures and transition states. J Comput Chem 16:1170–1179 55. Vreven T, Byun KS, Komaromi I, Dapprich S, Montgomery JA, Morokuma K, Frisch MJ (2006) Combining quantum mechanics methods with molecular mechanics methods in ONIOM. J Chem Theory Comput 2:815–826

Chapter 2

Hydrogen Bond Structure of Redox Active Tyrosines in Photosystem II

2.1 Introduction During electron donation in PSII, redox-active tyrosine YZ (D1-Tyr161) is first oxidized by P680+ , and subsequently it abstracts an electron from the Mn4 CaO5 cluster. This process then continues to catalyze the oxidation of water through fiveintermediate cycles (Si state; i = 0–4), commonly called the S-state cycle [1–7]. On YZ oxidation, the phenolic proton is released because of its constantly decreasing pK a . Recent X-ray crystallographic studies of PSII showed that the oxygen atom of YZ is located within bonding distance of the hydrogen of the neighboring histidine residue, D1-His190 (Fig. 2.1a) [8–10]. Particularly, the recent X-ray structure revealed that a significantly short hydrogen bond exists between Nτ atom of D1-His190 and oxygen atom of YZ (2.46 Å) [10]. For the purpose of theoretical studies, this hydrogen bond can be reproduced and investigated by quantum mechanics/molecular mechanics (QM/MM) calculations, which show that the hydrogen bond has a single well potential due to its strong interactions [11]. Even though the phenolic proton in YZ shifts toward the Nτ site of D1-His190 on YZ oxidation via the well-established mechanism of rapid proton-coupled electron transfer (PCET) [11– 15] one remaining question is whether the positive charge stays on D1-His190 during Y•Z formation [15–19]. Numerous published reports support that the positive charge remains near Y•Z ; [15, 16, 20, 21] however, conclusive and irrefutable evidence is still necessary to draw a final conclusion. Recent X-ray crystallographic studies [10] have also revealed that a cluster of water molecules comprising several water molecules links YZ to the Mn4 CaO5 cluster (Fig. 2.1a). In particular, a water molecule within this cluster, namely W4, directly bridges the Ca atom in the Mn4 CaO5 cluster and the oxygen atom in YZ , whereas another water ligand, W3, interacts via W7 and YZ . One of the water ligands associated with Mn4, namely W2, interacts with several molecules in the water cluster (i.e., W3, W5, W6, and W7). X-ray structural studies also showed the existence of a hydrogen-bond network linking the Mn4 CaO5 cluster to the bulk surface via YZ , © Springer Nature Singapore Pte Ltd. 2020 S. Nakamura, Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II, Springer Theses, https://doi.org/10.1007/978-981-15-1584-2_2

11

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2 Hydrogen Bond Structure of Redox Active Tyrosines …

Fig. 2.1 Hydrogen-bonding structures of YZ (a) and YD (b) in the crystal structure of PS II (PDB code; 3ARC)

which may acts as an access point for water or proton exit pathways [10, 22–24]. Thus, YZ possibly functions as both a direct electron acceptor for the Mn4 CaO5 cluster and as a proton transfer mediator during water oxidation reactions. YD (D2-Tyr160), a redox-active tyrosine that is symmetrically related to YZ , functions as an electron donor for P680+ (Fig. 2.1b). The environment surrounding YD is quite similar to that found around YZ , where the YD oxygen is located within hydrogen bonding distance of a water molecule and the neighboring histidine residue, D2-His189 (Fig. 2.1b). In spite of this structural similarity, the electron transfer rate in YD is significantly slower than that in YZ , which results in relative stability of the Y•D radical even at room temperature [12, 15]. Hence, YD can only act as a peripheral electron donor for P680+ and cannot be directly related to the water oxidation process. Possible causes for the slow redox kinetics and the stability of Y•D radical include the presence of a proton transfer mechanism involving a mobile water molecule [25] and the lower redox potential of YD (700−800 mV) [26, 27] compared to that of YZ (900−1000 mV) [27, 28]. One crucial question still surrounding the function of YZ is the exact role it plays in water oxidation [14, 15, 17, 29, 30]. Is YZ key in proton transfer mechanisms during water oxidation, or does it only serve as an electron mediator between the Mn4 CaO5

2.1 Introduction

13

cluster and P680+ ? If the former case is true, then which PCET mechanism incorporates both the Mn4 CaO5 cluster and YZ , and which S-state transitions are actually employed for the proton transfer pathway via YZ ? To answer these questions, it is important to clarify whether the positive charge remains on D1-His190 during Y•Z formation or the proton is released from histidine during YZ oxidation. When D1His190 releases a proton into the bulk, the Y•Z is able to use the surrounding water cluster to abstract a proton from the substrate water molecule in a concerted manner with the help of YZ re-reduction; this is often referred to as the “Hydrogen Abstraction Model” [17]. Moreover, there is debate as to whether electrostatic interactions between a positive charge near Y•Z and the Mn4 CaO5 cluster trigger the release of protons from substrate water molecules [29–33]. In order to argue the possibility of proton transfer via a hydrogen-bond network located near YZ , it is also important to elucidate hydrogen bonding interactions and the protonation structures present in the YZ -His moiety. In this chapter, the hydrogen-bonded structure of YZ and the structural changes which take place during YZ oxidation are investigated using light-induced Fourier transform infrared (FTIR) difference spectroscopy and quantum chemical calculations. The Y•Z /YZ difference spectra with a higher frequency region, which provided invaluable vibrational information about the protons, was successfully acquired. In previous FTIR studies, similar spectra were reported in the 1000–1800 cm−1 region [34]. We analyzed these spectral region not only by focusing on the pH changes and isotopic substitutions but also by comparing them with a Y•D /YD difference spectra. To obtain structural information and to better interpret the experimental data that was collected, quantum chemical calculations were performed using density functional theory (DFT) and QM/MM methods for tyrosine and histidine pairs. On oxidation, the proton between D1-His190 and YZ was detected as a broad feature of NH vibration for this histidine residue, which meant the presence of a highly polarizable proton. Structural information about the hydrogen-bond network located near YZ -His, as well as the associated hydrogen bond and protonation structures were also studied. Based on these results, we discuss the role of YZ and D1-His190 in PCET reaction for water oxidation processes.

2.2 Materials and Methods Sample Preparation. Thermosynechococcus elongatus cells with a histidine-tagged CP47 protein were cultured in a BG-11 medium [35] containing 10 mM Hepes at pH 7.5 and bubbled with air that had been supplemented with 3% (v/v) CO2 gas. Global 15 N substitution of the PSII core complexes were performed using a BG-11 medium containing CoCl2 and Na15 NO3 (SI Science Co. Ltd.; 99.7 atom % 15 N) as a replacement for Co(NO3 )2 and unlabeled NaNO3 , respectively, in the cell culture. The PSII core complexes were extracted from the cells and purified using the method reported by Boussac et al. [36]. After washing the cells with a buffer containing 40 mM sodium phosphate at pH 7.0 and 1 mM EDTA, the cells were recovered through suspension in Buffer A, which contained 1 M betaine, 40 mM MES at pH

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2 Hydrogen Bond Structure of Redox Active Tyrosines …

6.5, 10 mM of MgCl2 , 10 mM CaCl2 , and 10% (w/v) glycerol. The cells were then disrupted via agitation with glass beads that had a diameter of 100 μm on a repeating cycle of 10 s “On” and 3 min “Off”; this process was conducted nineteen times in a solution of Buffer A supplemented with 1 mM benzamidine, 1 mM aminocaproic acid, 0.2% (w/v) bovine serum albumin, and 50 μg/mL DNase I on ice in the dark. The disrupted cells were diluted with an identical volume of a buffer containing 40 mM MES at pH 6.5, 10 mM MgCl2 , and 10 mM CaCl2 . After weak centrifugation at 3000 g for 5 min to remove any unbroken cells, the thylakoid membranes were centrifuged at 48,000 g for 20 min before being suspended in a solution of Buffer A and solubilized with 1% (w/v) DM at the concentration of 1.0 mg Chl/mL for 10 min on ice in the dark. The lysate was centrifuged at 48,000 g for 10 min and then the supernatant was loaded onto a nickel-affinity chromatograph equilibrated with Buffer A that had been supplemented with 100 mM NaCl, 20 mM imidazole, and 0.03% DM. PSII core complexes were eluted from the column using Buffer A supplemented 100 mM NaCl, 200 mM imidazole, and 0.03% DM. After washing the purified PSII core complexes with a solution of Buffer A containing 0.03% DM via ultrafiltration (Vivaspin 20, Sartorium Stedim, molecular mass cutoff of 100 kDa), the oxygen evolution activity was observed in the range of 2400–2700 μmol of O2 (mg Chl)−1 h−1 in the presence of 0.5 mM 2,6-dichloro-1,4-benzoquinone. Treatment with 10 mM NH2 OH for Mn depletion was performed for 30 min at room temperature with a solution of Buffer A containing 0.03% DM via ultrafiltration (Vivaspin 20). FTIR Measurements. To perform Y•Z /YZ measurements, the Mn-depleted PSII core complexes were suspended in a buffer containing 20 mM MES at pH 6.5 or 5.5 (or 20 mM Hepes for pH 7.5), 40 mM sucrose, 10 mM NaCl, 5 mM MgCl2 , and 0.06% DM before being concentrated to ~6 mg Chl/mL via ultrafiltration (Vivaspin 500, molecular mass cutoff of 100 kDa). A solution containing 1 μL of 100 mM potassium ferricyanide as the electron acceptor was added to 4 μL of the suspension. The mixture was lightly dried on a BaF2 plate (13 mm in diameter) under N2 gas. The dried sample was then hydrated via the addition of 0.8 μL of water on the BaF2 plate before being quickly sandwiched with another BaF2 plate. For D2 O substitutions, re-suspending the dried sample in 3 μL of D2 O and drying the suspension were repeated several times. The temperature was fixed at 250 K using a cryostat (Oxford DN1704) during Y•Z /YZ data collection. A Bruker IFS66/S spectrophotometer with an MCT detector (D313-L) was used for recording flash-induced FTIR spectra. For Y•Z /YZ measurements, single-beam spectra with 50 scans for a 25-s accumulation were recorded twice before flash illumination and once afterwards; this was performed using a Q-switched Nd: YAG laser (INDI-40-10; 532 nm; ~7 ns full width at half-maximum; ~7 mJ pulse−1 cm−2 ). The scheme was repeated 350–1000 times with a dark relaxation process for 225 s between each scheme using 1 to 4 samples. The resulting spectra were averaged to obtain the light-minus-dark and a dark-minus-dark (before illumination) difference spectra for a Y•Z /YZ difference spectrum and the noise level baseline, respectively. The measurements scheme for the Y•D /YD difference spectrum was described previously [37]. Mn-depleted PSII core complexes suspended in a buffer solution

2.2 Materials and Methods

15

containing 10 mM MES at pH 6.5, 5 mM NaCl, and 0.06% DM, and then concentrated to ~3 mg Chl/mL via ultrafiltration (Vivaspin 500). The solution containing Mn-depleted PSII complexes (5 μL) was mixed with 1 μL of 20 mM potassium ferrocyanide and 1 μL of 20 mM potassium ferricyanide before being dried on a BaF2 plate (25 mm × 25 mm) under N2 gas to convert into a film. The sample was sealed with another BaF2 plate and a silicone spacer (0.5 mm in thickness) with 2 μL of a 40% (v/v) glycerol solution without touching the sample. For D2 O substitutions, re-suspending the dried sample in D2 O and drying the suspension were repeated several times using a method similar to that employed for D2 O substitution when performing YZ measurements. The sample temperature was kept at 283 K by circulating cold water through a copper holder. Single-beam spectra with 100 scans for a 50-s accumulation were measured twice before and once after five flashes at 1 Hz using a Q-switched Nd: YAG laser. The measurement was repeated 54 times with a dark adaption process for 750 s. The resulting spectra were averaged and calculated to obtain a light-minus-dark difference spectrum as a Y•D /YD spectrum and a dark-minus-dark difference spectrum as the baseline. Quantum Chemical Calculations. Quantum chemical calculations of redoxactive tyrosine residues were carried out using the Gaussian09 package [38]. For the Tyr-His model simulations, geometry optimization and normal mode calculations were performed at the B3LYP/6-31++G(d, p) level. The ONIOM method [39] was used for QM/MM calculations based on the X-ray structure of PSII [10]. The atomic coordinates for YZ simulation, which included amino acid residues, water molecules, the Mn4 CaO5 cluster, and Cl− ions located within 20 Å of the Ca atom, were extracted from the recent high-resolution X-ray structure of PSII (PDB code: 3ARC) [10]. The generation of the hydrogen atoms was performed using TLEAP software before being optimized using an Amber force field [40]. The structures of water-oxidizing center (WOC), the Mn4 CaO5 cluster and its surrounding residues, were primarily optimized in the QM region, which was defined as containing Cl− ions, the Mn4 CaO5 cluster, amino acid ligands to Mn, Ca, and Cl− , D1-Asp61, D1-Tyr161 (YZ ), D1His190, W1−W7, HOH446, and HOH442, fixing other atoms as an MM region. The theoretical model for protonation and spin states reported by Luber et al. [41] was used to perform geometry optimization studies of the Mn4 CaO5 cluster. Geometry optimization and normal mode calculations for YZ were then performed within the QM region, which was defined as having YZ , D1-His190, D1-Asn298, Ca ion, D1Gln165, W3–W7, HOH387, HOH394, HOH398, HOH778, HOH923, and HOH1117 fixing other atoms as an MM region. In these simulations, LANL2DZ for metal ions, Ca and Mn ions, and 6-31++G(d, p) for other atoms were used for the QM calculations. The atomic coordinates for the YD simulation, which included amino acid residues and water molecules located within 20 Å of YD , were extracted from the X-ray structure of PSII (PDB code: 3ARC) [10]. The method for generation and optimization of the hydrogen atoms for YD simulation was same as the process used for YZ simulation. Geometric optimization and normal mode calculations for YD simulation were performed in the QM region, which was defined as D2-His189, D2-Arg294, D2-Arg180, and HOH1, thus fixing other atoms as an MM region at the ONIOM [B3LYP/631++G(d, p): Amber] level.

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2 Hydrogen Bond Structure of Redox Active Tyrosines …

Vibrational frequencies which had been determined using normal mode calculations in DFT and QM/MM simulations were scaled to fit the experimental CO stretching frequencies observed for Y•D (1504 cm−1 ) and Y•Z (1514 cm−1 ). The factors of scaling were 0.987 and 0.979 for YZ -His and YD -His models in the DFT method, respectively, and 0.973 and 0.968 for YD and YZ simulation in the QM/MM method, respectively. Vibrational frequencies of the 4-methylimidazole (4MeIm) molecule were also scaled by a factor of 0.980, which followed the previously mentioned normal mode calculations [42].

2.3 Results A light-induced Y•Z /YZ difference spectrum acquired at pH 6.5, which was measured using the Mn-depleted PSII core complexes taken from T. elongatus, is shown in Fig. 2.2 (red line) with a dark-minus-dark difference spectrum (black line) that is representative of the baseline. The large peaks observed at 2116 and 2038 cm−1 arose from CN stretching vibrations of ferricyanide and ferrocyanide, respectively. These peaks reflected the electron flow from the electron donor (YZ ) to the electron acceptor (ferricyanide). Notably, small peaks at the same positions (2116/2038 cm−1 ) with opposite signs in the dark-minus-dark spectrum were due to residual back reactions, which were estimated to be ~4% of the total number of reactions observed while

Fig. 2.2 Light-induced FTIR difference spectrum upon YZ oxidation of the PSII core complexes from T. elongatus (red line) and the corresponding dark-minus-dark difference spectrum representing noise levels (black line) in the region of 3000–1000 cm−1 . Adapted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

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Fig. 2.3 Light-induced FTIR difference spectra upon YZ oxidation in the 1800–1000 cm−1 region of a unlabeled and b global 15 N-labeled PSII core complexes, and of c PSII core complexes in D2 O solution, together with d a FTIR difference spectrum upon YD oxidation. Ticks without frequencies represent the identical peak position as shown in spectrum a. The FTIR difference spectrum upon YD oxidation was scaled by multiplied by 0.25 due to the larger intensity than that upon YZ oxidation. These spectra were recorded at pH (pD) 6.5. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

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2.3 Results

1200

1000

-1

Wavenumber (cm )

taking measurements for the two dark spectra preceding illumination. The Y•Z /YZ difference spectrum in the 1000–1800 cm−1 region is shown in Fig. 2.3a, which is expanded in Fig. 2.2, was very similar to the spectrum acquired using Mn-depleted PSII membranes derived from spinach and the PSII core complexes of Synechocystis sp. PCC 6803, which had been previously reported by Berthomieu et al. [34]. The positive peak seen at 1514 cm−1 originated from CO stretching vibrations of Y•Z [34, 43], whereas those for Y•D were observed at 1504 cm−1 (Fig. 2.3d) [37, 44]. The negative band at 1255 cm−1 and a small negative peak at 1279 cm−1 in the Y•Z /YZ difference spectrum were assigned to COH bending and CO stretching vibrations of the reduced YZ , respectively [34]. Because the latter peak was not observed during our experiments, we used the negative band assigned at 1256 cm−1 (Fig. 2.3) to overlap the COH bending and CO stretching vibration coupling with each other [45, 46]. In D2 O solution (Fig. 2.3c), a narrower band previously assigned to pure CO stretching vibrations that were decoupled from the COH bending vibrations was upshifted to 1262 cm−1 [46]. Berthomieu et al. [34] had assigned the peaks seen at 1705 and 1697 cm−1 to electrochromic shifts of P680’s keto CO band during YZ oxidation. Amide II vibrations (NH and CN bands) coupled to Amide I vibrations (CO bands of the backbone) observed at 1560, 1553, and 1544 cm−1 were downshifted to 1545, 1538, and 1530 cm−1 via global 15 N substitution, respectively (Fig. 2.3b). Global 15 N substitution revealed the occurrence of downshifting of a negative band from 1101 to 1095 cm−1 (expanded in Fig. 2.4), which was indicative of the presence of the CN

1101 1095

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1102

1105

Fig. 2.4 Histidine CN vibration region (1160–1040 cm−1 ) of the FTIR difference spectra upon YZ oxidation of 15 N-labeled (red lines) and unlabeled (black lines) PSII core complexes in a H2 O and b D2 O. c Unlabeledminus-15 N-labeled double-difference spectra in D2 O (green line) and H2 O (blue line). These spectra were recorded at pH (pD) 6.5. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

2 Hydrogen Bond Structure of Redox Active Tyrosines …

ΔA

18

1080

1040

-1

Wavenumber (cm )

band in the histidine residue [42, 47, 48] in this region. Indeed, a peak downshift from 1102 to 1091 cm−1 was observed in the 14 N-minus-15 N double-difference spectrum (Fig. 2.4, blue line). The dependence of a deuteration shift of the CN bands in the imidazole group during one of its protonation states was previously established [42]. The CN band region in the histidine residue of the Y•Z /YZ difference spectra in D2 O exhibited differential signals at 1105 and 1096 cm−1 in the double-difference spectra of an unlabeled-minus15 N (Fig. 2.4, green line). Thus, the CN band was only upshifted by 3–5 cm−1 during D2 O substitution. A positive band with broad features around 2800 cm−1 was observed in the highfrequency (2200–3000 cm−1 ) region of the Y•Z /YZ difference spectrum, which has not been reported. The broad feature began at ~2300 cm−1 and ended just above 3000 cm−1 (higher frequency regions above 3000 cm−1 were unobservable owing to absorption saturation caused by the presence of bulk water). During global 15 N substitution, the broad feature was, itself, downshifted by 31–19 cm−1 and smaller peaks were seen at 2902, 2882, 2856, 2659, and 2630 cm−1 (Fig. 2.5a). These peaks, which were observed in the 2500–3000 cm−1 region on the broad band, were similar − to those reported previously for the S2 /S1 , Q− A /QA , and QB /QB difference spectra [48–52] in which the peaks had been assigned Fermi resonance for the combinations and overtones of NH stretching vibrations in the histidine residue. Because the broad positive feature, itself, was downshifted by ~10 cm−1 during global 15 N substitution

-5

1x10

2630

-5

2659

2640

2836

2606

2902

2882 2857 2856

A: unlabeled 15 vs. N

1x10

ΔA

Fig. 2.5 High frequency region (2950–2150 cm−1 ) of the FTIR difference spectra upon YZ oxidation of globally 15 N-labeled (A, red line) and unlabeled (A, black line) PSII core complexes in H2 O and in B D2 O together with C a FTIR difference spectrum upon YD oxidation. The dotted lines are the corresponding dark-minus-dark difference spectra representing noise levels. The dashed line region in spectrum B is saturated with OD vibration bands of D2 O solution. The inset is strong OD stretching vibration region (2300–1750 cm−1 ) of spectrum B. These spectra were recorded at pH (pD) 6.5. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

19

2871

2.3 Results

2200

2000

1800

x 0.25

2800

2600

B: D2O

C: YD /YD

2400

2200

-1

Wavenumber (cm )

(Fig. 2.5a), it was highly likely that this broad positive feature, which was accompanied by Fermi resonance peaks, arose from NH vibrations in the histidine side chain. This theory was supported by the disappearance of the broad positive band at around 2800 cm−1 upon D2 O substitution (Fig. 2.5), and the appearance of another broad positive band at around 2100 cm−1 which overlaps with the ferricyanide (2116 cm−1 ) and ferrocyanide (2038 cm−1 ) peaks, as shown in Fig. 2.5 (inset). One remarkable observation is the absence of a similar broad feature in the Y•D /YD difference spectrum (Fig. 2.5c). A broad band at a higher frequency region (~3000 cm−1 ) was expected because of the gradual increase in intensity from 2600 cm−1 to 3000 cm−1 . However, the intensity of the broad band was lower in D2 O (Fig. 2.6, blue line), and the small peaks observed at 2809, 2768, 2747, and 2702 cm−1 were not shifted during global 15 N substitution, except for the minor peaks seen at 2620 and 2602 cm−1 (Fig. 2.6, red line). Thus, it is likely that the gradually increasing intensity of the background mainly arose from the hydrogenbond network surrounding YD rather than from the NH band of the histidine residue coupled with YD oxidation. In addition, the minor peaks noted may have arisen from numerous combinations of the fundamental vibrations and/or the overtones of proteins, but not from Fermi resonance of the histidine residue vibrations. Figure 2.7 shows the Y•Z /YZ difference spectra measured at different pH levels (a: pH 7.5 and c: pH 5.5) in comparison with those obtained at pH 6.5. A significant change, which was the absence of the broad feature starting at ~2300 cm−1 (except for

-4

1504 1519

2x10

1553 1538 1543 1530

1684

1697

1655

(a)

1172

1704

1251

1674

ΔA 1800

1600

1400

1200

1000

-1

Wavenumber (cm )

(b)

2x10

-5

Fig. 2.6 Light-induced FTIR difference spectra upon YD oxidation in (a) 1800–1000 and (b) 2900–2200 cm−1 regions of 15 N-labeled (red lines) and unlabeled (black lines) PSII core complexes in H2 O and of PSII core complexes in D2 O (blue lines). These spectra were normalized for the bands at 1504 cm−1 that arise from the CO stretching vibration of Y•D . The dashed line region in the spectrum (b; blue line) is saturated with OD vibration bands of D2 O solution. These spectra were recorded at pH (pD) 6.5. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

2 Hydrogen Bond Structure of Redox Active Tyrosines …

2809

ΔA

20

2747 2620 2592 2768 2702

2800

2602

2574

2600

2400

2200

-1

Wavenumber (cm )

the small peaks seen at slightly shifted frequencies), was observed at pH 5.5. Other than a change in its intensity, the broad feature observed in the same region remained unchanged between pH 6.5 and 7.5, and the peak associated with CO stretching in Y•Z was virtually unchanged in the pH range between 5.5 and 7.5. On the other hand, the negative peak linked with CO stretching and COH bending vibrations was upshifted to 1259 cm−1 at pH 5.5 from 1256 cm−1 at pH 6.5. This band seems to have broadened or been split at pH 7.5 (Fig. 2.7A). These changes were indicative of the pH dependence of the hydrogen-bond structures near the phenolic oxygen of YZ , particularly in the pH range between 5.5 and 7.5. Although CO stretching frequency in the tyrosine side chain reflected the differences in hydrogen bond interactions [46], overlap and coupling of COH bending vibration prevented the identification of the pure CO stretching frequency of reduced YZ in the Y•Z /YZ difference spectra. To observe the pure CO stretching band at different pH levels, the Y•Z /YZ difference spectra were measured in D2 O to remove any influence from the COH bending band arising from the CO stretching region (Fig. 2.8). CO stretching bands of YZ were observed at 1259, 1262, and 1263 cm−1 at pD 5.5, 6.5, and 7.5, respectively (Fig. 2.8C). Thus, the frequency had downshifted

1318

-5

2635

1x10

1103

1263 1242

2882 2856

2660

2902

ΔA

1109 1101

1256

1535 1515

a

2630 2882 2856

2659

b

2903 2851 2887

1112 1099

1259

c 1405

1572 1559 1546

1698 1679

(B) 2905

-5

5x10

1514

1638

1553

1560 1544

1666

1627

a

b

ΔA

1730

1704

(A)

21

1729 1697 1680 1652

2.3 Results

2639

c

2667 2618

2822

2800

2600

2400

2200

-1

Wavenumber (cm ) 1800

1600

1400

1200

1000

-1

Wavenumber (cm )

Fig. 2.7 pH dependence of the Y•Z /YZ spectra (A 1800−1000 cm−1 and B 2950−2150 cm−1 ) at a pH 7.5, b 6.5, and c 5.5. The structures in 2400–2350 cm−1 region are artifacts due to CO2 absorption. Ticks without frequencies represent the identical peak position as in spectrum a. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

by 4 cm−1 with a change from pD 7.5 to pD 5.5, whereas the frequency in pD 6.5 had downshifted only by 1 cm−1 . On the other hand, CO stretching band of the oxidized Y•Z (1513 cm−1 ) did not change between pD 5.5 and 7.5 (Fig. 2.8B), which was the result of H2 O in the system (Fig. 2.7A). These vibrational observations supported the view that the hydrogen-bonded structure in YZ was changed across the pH range 5.5 and 7.5 due to the weakening of the CO bond that occurred when the pH (pD) decreased from 7.5 to 5.5. Conversely, the Y•Z remained unchanged under these conditions. To verify the assignment of the broad positive band in the Y•Z /YZ difference spectrum, as well as the structural changes associated with the hydrogen-bond networks in YZ and YD , DFT and QM/MM calculations of the Tyr-His pairs were performed. To simplify the tyrosine and histidine side chains, p-cresol and 4-MeIm were used in DFT calculations, respectively (Fig. 2.9). The imidazole group formed four different protonation patterns because it had two protonation sites located on the Nτ and Nπ atoms in its ring. Due to the many conformations of the hydrogen-bond structures in the Tyr-His models, YZ and YD models that had been predicted based on the structural information of the surrounding amino acids residues observed in the X-ray structure were assumed and examined. In the X-ray structure, the Nπ atom of D1-His190 hydrogen bonded with YZ was located within the distance typically seen during the formation of a hydrogen bond (2.60 Å) from the amide oxygen of D1-Asn298 (Fig. 2.1a) [10]. This strongly suggested that the Nπ of D1-His190 was protonated and formed a hydrogen bond with D1-Asn298. Hence, in the case of YZ

22

2 Hydrogen Bond Structure of Redox Active Tyrosines …

Fig. 2.8 pD dependence of Y•Z /YZ spectra in D2 O (A 1800−1000 cm−1 , B the region of the νCO frequency of Y•Z , and C the region of the νCO frequency of YZ ) at a pD 7.5, b pD 6.5, and c pD 5.5. Ticks without frequencies represent the identical peak position as in spectrum a. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

Fig. 2.9 Optimized structures of Tyr-His model complexes: a Y•Z -HisH+ , b YZ -His, c YZ -HisH+ , d Y•D -His, and e YD -His. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

2.3 Results

23

oxidation, D1-His190 formed a protonated cation state (HisH+ ) to receive a proton from the phenolic oxygen of Y•Z at the Nτ atom. However, two possible hydrogenbonding patterns could occur in the reduced YZ model: (1) a neutral Nπ-H formed by D1-His190 hydrogen bonded with YZ which acted as a hydrogen-bond acceptor at higher pH levels (Fig. 2.9b), and (2) a cation HisH+ formed by D1-His190 hydrogen bonded with YZ , which acted as a hydrogen bond donor at lower pH levels (Fig. 2.9c). In contrast, the Nπ atom in the D2-His189 hydrogen bonded with YD was located within the typical distance required for the formation of hydrogen bonds (2.81 Å) from the Nε atom of D2-Arg294’s guanidinium residue (Fig. 2.1b). This meant that the Nπ atom of D2-His189 was deprotonated to form a hydrogen bond with D2-Arg294, which acted as a hydrogen bond acceptor [25]. Hence, it is highly likely that D2-His189 formed a neutral Nτ-H form and was a hydrogen bond donor to both YD and Y•D (Fig. 2.9d, e). For QM/MM calculations of YZ , the QM region comprised YZ , D1Gln165, D1-His190, D1-Asn298, the Ca ion of the Mn4 CaO5 cluster, and eleven water molecules (Fig. 2.10), whereas the other atoms extracted from X-ray structure were assigned to the MM region. Most notably, the optimized geometry determined for heavy atoms in the QM region in the reduced YZ model was in agreement with the 1.9 Å X-ray structure [10] (Fig. 2.10a–c). For QM/MM calculations of YD , the QM region consisted of YD , D2-His189, D2-Arg180, D2-Arg294, and a water molecule located between YD and D2-Arg180 (Fig. 2.10d, e). The Nτ-H stretching frequencies calculated for histidine residues that were coupled with a tyrosine, the distances between Nτ and the phenolic oxygen are shown in Table 2.1. For the QM/MM calculations conducted in light of the YZ model (Fig. 2.10b), an exceedingly short hydrogen bond distance was observed between YZ and D1-His190 (2.49 Å), despite the longer distances obtained in the DFT calculations (2.82 Å). The distance was in agreement with that established in the QM/MM calculations previously reported by Saito et al. (2.47 Å) [11] and that seen in the X-ray structure of 2.46 Å [10]. The YD model in the present study resulted in the relatively longer hydrogen bond distances between YD and His in both DFT (3.02 Å) and QM/MM (2.79 Å) calculations, which was also in agreement with those taken from X-ray structure studies (2.74 Å) [10]. Hence, these calculations adequately reproduced and represented the hydrogen-bonding interactions often seen in YZ -His and YD -His complexes. The Nτ-H frequency was calculated at 2810 cm−1 in the Y•Z -HisH+ (DFT) model as a localized mode (Fig. 2.11 and Table 2.1). In the Y•Z -HisH+ (QM/MM) model, the frequencies of the asymmetric and symmetric Nτ-H/Nπ-H vibration were calculated at 2748 and 2986 cm−1 , respectively. These frequencies were remarkably lower than the Nτ-H vibration frequencies obtained for the isolated neutral 4-MeIm and cationic 4-MeImH+ (3601–3546 cm−1 ). IR intensities were significantly higher than those seen in the isolated 4-MeIms (Table 2.1). The histidine residue in YZ -His (DFT) model is thought to be deprotonated at the site of the Nτ atom (Fig. 2.9b), which meant that there was no Nτ-H mode in the neutral YZ state. In contrast to the YZ -His (DFT) model, the Nτ-H frequency for the YZ -HisH+ (DFT) model was

24

2 Hydrogen Bond Structure of Redox Active Tyrosines …

Fig. 2.10 Optimized structures around YZ(D) -His moiety in the QM/MM calculation. a Y•Z -HisH+ ; b YZ -His; c heavy atoms around YZ -His moiety together with their positions in the X-ray structure [10] (green); d Y•D -His; e YD -His. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

calculated at 3027 cm−1 , which was also lower in value than that noted for the isolated molecules (Fig. 2.9c). On the other hand, the YD -His and Y•D -His models (both in DFT and QM/MM calculations) showed higher frequencies for Nτ-H vibrations (3534–3250 and 3417–3162 cm−1 , respectively) than those seen in the YZ models. The data obtained from these calculations were consistent with what was seen in the FTIR spectra (Table 2.1). The broad positive feature in high-frequency region in the Y•Z /YZ difference spectrum (Figs. 2.5 and 2.7) corresponded to the NτH (or NτH/NπH) vibrations established using higher IR intensities over a frequency range 2700–3000 cm−1 in the Y•Z -HisH+ models, and was notably absent in the YZ -His models. The calculated NτD frequency for deuterated Y•Z -HisD+ using DFT calculations (Table 2.1) also reproduced the significant downshift of the broad positive feature to ~2100 cm−1 in D2 O (Fig. 2.5b, inset) as well as the expected shift in 15 N substitution, which was consistent with a 15 N-induced downshift of ~10 cm−1 (Fig. 2.5a). Moreover, the calculated NτH frequency obtained at ~3000 cm−1 in the

2.3 Results

25

Table 2.1 Nτ-H frequencies (cm−1 ) of the YZ(D) -His moieties calculated by DFT and QM/MM calculations Calculation

Experimental

Method

Frequency (IR intensity)a

rO  Nτ (Å)b

Frequencyc

DFT

2810 (4569)

2.63

~2800

QM/MM

2986 (1390)/2748 (3554)d

2.63

Y•Z -HisD+e

DFT

2106 (2154)

–

YZ -His

DFT

–

2.82

Y•Z -HisH+

QM/MM

–

2.49

YZ -HisH+

DFT

3027 (2211)

2.75

Y•D -His

DFT

3417 (1141)

2.95

QM/MM

3162 (2083)

2.77

YD -His

DFT

3511 (658)

3.02

QM/MM

3250 (1490)

2.79

4-MeIm (NτH)

DFT

3601 (58)

4-MeImH+

DFT

3554 (72)/3546 (281)d

a Calculated

rO  Nτ (Å)b

~2100 2.46f

2.74f

IR intensity in kilometers per mole distance between Nτ of His and the phenolic oxygen of Tyr c Frequency observed in the present study d Coupled asymmetric and symmetric stretching modes of Nπ-H and Nτ-H e Deuteration of the exchangeable protons of Tyr-His f Distance from the X-ray structure [10] Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society b Hydrogen-bonding

Fig. 2.11 Directions of the displacement vectors of the Nτ-H vibration modes of the a Y•Z -HisH+ and b Y•D -His. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

26

2 Hydrogen Bond Structure of Redox Active Tyrosines …

Table 2.2 CN vibration frequencies (cm−1 ) of the YZ(D) -His moieties calculated by DFT and QM/MM calculations Deuterateda

Protonated

DFT QM/MM

Yz-His

Frequency (15 N shift)b

IR intensityc

Frequency (15 N shift)b

1121 (−7)

54

1144 (−6)

H/D shiftd IR intensityc 48

−1

1136 (−4)

7

16

1149 (−4)

114

3

7

17

Yz• -HisH+

1097 (−5)

3

Yz-His

1109 (−4)

94

Yz• -HisH+

1086 (−3)

18

1139 (−2)

a Deuteration

of all exchangeable protons b All nitrogen atoms are substituted with 15 N c Calculated IR intensity in kilometers per mole d Frequency shifts by H/D exchange Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

YZ -HisH+ model rationalized the marked absence of a broad positive feature at pH 5.5 (Fig. 2.7B, c). The NτH frequency seen in YZ -HisH+ may cancel the positive band of Y•Z -HisH+ . The NτH vibrations calculated using the Y•D -His models were also consistent with those obtained experimentally, also in the YZ case; there were no broad positive features in the Y•D /YD difference spectrum in the lower frequency region than 3000 cm−1 (Fig. 2.5c). CN stretching frequencies associated with histidine residues in YZ -His complexes are shown in Table 2.2. These frequencies, which were calculated to be approximately ~1100 cm−1 , were in agreement with the FTIR data obtained (Fig. 2.4). The relatively lower frequency of cationic HisH+ in Y•Z -HisH+ , when compared to its neutral His counterpart in YZ -His, was consistent with the previous studies focused on free histidine (or 4-MeIm) [42, 47, 48]. 15 N substitution showed downshifts in the CN stretching vibration frequencies by 2–5 and 4–7 cm−1 in Y•Z -HisH+ and YZ -His, respectively. During H/D substitution, the YZ -His model apparently allowed for a small shift in the range between −1 and 3 cm−1 , whereas the Y•Z -HisH+ model allowed for significant shifts between 16 and 17 cm−1 ; this corresponded with our previously reported data [42]. Also notable were the IR intensities seen for CN stretching vibrations of the neutral His in the reduced YZ -His model. These intensities were much higher than those seen for cationic HisH+ in the oxidized Y•Z -HisH+ model. The significant difference in the IR intensities obtained strongly suggests that the bands in CN stretching region in Y•Z /YZ difference spectrum mainly arose from the reduced YZ state in Fig. 2.4. The negative band originating from the neutral NπH form was indicative of transformation into a different form (i.e., the cationic HisH+ form) upon YZ oxidation. The slight upshift in the observable CN bands in Fig. 2.4C upon H/D substitution corresponded with a small effect commonly seen in the deuterated YZ -His model (Table 2.2). H/D shifts in the cationic HisH+ were not directly observed in the Y•Z /YZ difference spectra, which was an expected observation due to the small IR intensity estimated using the DFT and QM/MM calculations. Therefore, FTIR and computational data regarding CN vibrations strongly supported

2.3 Results

27

Fig. 2.12 Hydrogen bond rearrangement around YZ -His upon YZ oxidation estimated by QM/MM calculations. When YZ is oxidized, the phenolic proton of YZ is transferred to D1-His190, the hydrogen bond between W4 and YZ is broken. Subsequently, the proton of W4 turns toward WA to form a hydrogen bond, while WA moves to the Nτ atom of D1-His190. Adapted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

the notion that the protonation state of D1-His190 was changed from the neutral NπH to the cation HisH+ upon YZ oxidation. The optimized geometries of YZ in the QM/MM calculations allowed for substantial rearrangements in the hydrogen bond conformation between YZ and the Mn4 CaO5 cluster, which meant that both proton release from YZ to D1-His190 and the movements of water molecules were accommodated during YZ oxidation (Fig. 2.12). The movements of the water molecules were due to the double bond character of the CO bond in Y•Z , the frequency of which showed considerable upshift from 1262 to 1513 cm−1 (Fig. 2.8), thus causing the disruption of the three hydrogen bonds (W4) and the formation of another hydrogen bond between W4 and WA (Fig. 2.12). The movement of WA , which shortened the distance between Nτ atom of D1-His190 and WA from 4.57 to 3.28 Å, was induced by conformational changes in the hydrogen bond network surrounding W4. This rearrangement encouraged the formation of a new hydrogen bond network, which was linked to the luminal side [10] of W4 through WA , WB , WC , and WD .

2.4 Discussion Upon YZ oxidation, a phenolic proton is released from the Y•Z radical. [12–15, 53] In the present study, the proton of interest was directly detected as an NτH band of the neighboring histidine, D1-His190, which subsequently trapped this proton at the Nτ site of the imidazole ring. The broad positive band observed at approximately 2800 cm−1 in the Y•Z /YZ difference spectrum was analyzed in relation to NτH vibration (or the coupled vibration of NτH/NπH) which originates from the protonated

28

2 Hydrogen Bond Structure of Redox Active Tyrosines …

HisH+ form of D1-His190 (Figs. 2.2, 2.5 and 2.7). Quantum chemical calculations and isotopic substitutions provided evidence in support of this assignment. A significant downshift of the broad band from ~2800 to ~2100 cm−1 during the H/D exchange (Fig. 2.5b) indicated that the vibration involved an exchangeable proton. Moreover, the downshifts seen in the broad band with the superimposed small peaks noted upon global 15 N substitution (Fig. 2.5a) also indicated that NH vibration was coupled with other His vibrations by Fermi resonance. DFT and QM/MM calculations (Figs. 2.9a and 2.10), which showed NτH or the coupled NτH/NπH vibrations of the neighboring cationic HisH+ observed within the range 2700–3000 cm−1 (Table 2.1), well reproduced the experimental frequencies. In the reduced YZ structure (Fig. 2.9b), the absence of the protonated Nτ atom in the histidine residue was consistent with the appearance of a sole positive broad band; this was indicative of NτH vibration of the protonated HisH+ form in the Y•Z /YZ difference spectrum. Moreover, DFT calculations using the deuterated model (Y•Z -HisD+ ), which resulted in a NτD frequency at 2106 cm−1 (Table 2.1), reproduced the significant downshift to ~2100 cm−1 seen during H/D exchange (Fig. 2.5c, inset). The vibrational properties associated with CN stretching vibration in the histidine residue upon deuteration and 15 N substitution in DFT and QM/MM calculations (Fig. 2.4) were consistent with the findings linked with the protonation of Nτ atom of histidine upon YZ oxidation. The Y•Z /YZ spectra acquired under different pH conditions strongly supported the vibrational assignments mentioned above. In the reduced and protonated YZ -HisH+ model, which was considered for the lower pH form (Fig. 2.13) [13, 15, 21, 53], the DFT calculations provided evidence of a low NτH frequency at 3027 cm−1 . This was very close to the frequency observed at 2700–3000 cm−1 for the Y•Z -HisH+ models. Hence, the absence of the broad positive band at around ~2800 cm−1 could be explained by canceling the NτH band between the YZ -HisH+ and Y•Z -HisH+ structures. We ascertained that changes in the hydrogen-bond structure in reduced YZ under low pH conditions could be determined by detecting the frequency shift which occurred the pure CO stretching band in reduced YZ via H/D exchange from 1263 cm−1 (pD 7.5) to 1259 cm−1 (pD 5.5) (Fig. 2.8C). On the other hand, CO stretching frequency related to the oxidized Y•Z was virtually the same throughout Fig. 2.13 Changes of hydrogen bond structures of D1-His190 and YZ with photooxidation and pH. Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

2.4 Discussion

29

the pH (pD) range from 5.5 to 7.5 (Figs. 2.7A and 2.8B), which was consistent with the structural model of Y•Z (Y•Z -HisH+ ) throughout this pH (pD) range (Fig. 2.13c). Furthermore, a positive band at around 1720 cm−1 which could be attributed to CO stretching vibrations of protonated COOH groups and negative bands seen at 1572 and 1405 cm−1 , which arose from carboxylate asymmetric and symmetric stretching vibrations, respectively, were all observed at pH 5.5; this was indicative of protonation of some specific or non-specific carboxylate groups in the proteins during YZ oxidation. This was experimental evidence of the structural models seen in Fig. 2.13 in which a proton was released from YZ −His at pH 5.5, and also indicated that the pK a of D1-His190 was around 6 in the samples taken from T. elongatus. This pK a value was, however, lower than in previous reports (∼7) [13, 15, 53] and its consistency with that of the pK a values acquired for D1-His190, which was strongly dependent on the metal ion content and PSII preparations [21, 53]. The Y•D /YD difference spectra did not show any broad features around ~2800 cm−1 similar to those seen in the Y•Z /YZ difference spectrum at pH 6.5 (Fig. 2.5c). DFT and QM/MM results of both YD -His (Figs. 2.9e and 2.10) and Y•D -His (Figs. 2.9d and 2.10) models, which had a neutral histidine residue (NτH form), contained much higher frequencies for Nτ-H vibrations between 3550 and 3150 cm−1 (Table 2.1). This rationalized the absence of the broad band in the Y•D /YD difference spectrum, which in turn supported our previous assignment of the broad band to the NτH band in the Y•Z /YZ difference spectrum described above. All FTIR and quantum chemical calculation results indicated the presence of a protonated D1-His190 (HisH+ form) coupled with Y•Z . The relatively low NτH frequency of HisH+ was indicative of the formation of a strong hydrogen bond with Y•Z . This hydrogen bond probably originated from the so-called “charge-assisted” hydrogen bond [54–56] because similar hydrogen bond interactions with a neutral histidine residue in the Y•D -His model provided evidence of longer O-Nτ distances (2.77–2.96 Å) and higher NτH frequencies (3417–3162 cm−1 ) than those found in the Y•Z -HisH+ model. The higher CO stretching frequency of Y•Z (1514 cm−1 ), when compared with that seen in Y•D (1504 cm−1 ) (Fig. 2.3), also pointed to much stronger hydrogen bonds in Y•Z than those seen in Y•D [34]. Theoretical studies have shown that the upshift in the CO stretching frequency of Tyr• was due to its strong hydrogen bonds [57, 58]. Retaining a positive charge on D1-His190 in the oxidized Y•Z state was consistent with the oxidizing capabilities of YZ at ultra-low temperatures [15], the electrochromic shift in Chl absorption which occurred upon YZ oxidation [18], the absence of a proton release [21], and the low gx value seen in high-field electron paramagnetic resonance (EPR) spectrum [16]. Nτ-H stretching band in the Y•Z /YZ difference spectrum was shown to be extremely broad at around ~2800 cm−1 (Figs. 2.2 and 2.5a). This broad feature in the IR spectra was suggestive of a strong hydrogen bond with large proton polarizability, which played a key role in proton transfer [59–63] The width of the Nτ-H stretching band in oxidized Y•Z indicated that the proton between Y•Z and HisH+ became highly polarizable via strong hydrogen bonding during YZ oxidation, namely, this proton easily moved in response to changes in the electrostatic field surrounding it. The slightly broader width of the CO stretching band seen at 1514 cm−1 in the Y•Z /YZ

30

2 Hydrogen Bond Structure of Redox Active Tyrosines …

difference spectrum, when compared to the band observed at 1504 cm−1 in the Y•D /YD difference spectrum (Fig. 2.3) (which was previously observed by Berthomieu et al. [34]) also supported the notion of a highly polarizable proton on Y•Z . The Mn-depleted PSII sample was used for conducting FTIR measurement to obtain the Y•Z /YZ difference spectra. Mn depletion was predicted to induce changes in the properties associated with YZ [14, 53]. In particular, it is thought that the hydrogen bond network surrounding the Ca ion of the Mn4 CaO5 cluster was broken upon removal of the Mn4 CaO5 cluster. However, according to the QM/MM calculations, this hydrogen bond network (i.e., the amino acid residues and the water molecule around YZ ) in the QM region had a relatively low N–H frequency (2748 cm−1 ) which was close to the frequency taken through DFT calculations without the hydrogen bond network (2810 cm−1 ) (Table 2.1). Experimentally, it was shown through high-field EPR studies that the orientation of YZ did not change after Mn depletion. These EPR studies concluded that the hydrogen-bonding pattern noted for Y•Z was not drastically influenced by removing the Mn4 CaO5 cluster [16]. Within the alkaline pH range, the kinetic properties of the Mn-depleted PSII were quite similar to that of the intact PSII, which was indicative of the same PCET reaction mechanism of YZ [13]. Thus, hydrogen-bonding interactions of Y•Z acquired via Mn-depleted PSII remained the same as those observed in Mn-intact PSII. The role of a positive charge on the Y•Z -HisH+ moiety during water oxidation is thought to facilitate the release of a proton from the substrate water, particularly in the S2 and S3 states, because these states have an excess positive charge on the Mn4 CaO5 cluster [15, 29, 31, 64]. In contrast, protonation at the Nτ site of D1-His190 without any proton release, even from Nπ site itself, was not consistent with the previously reported “hydrogen abstraction model” [17] in which a phenolic proton on YZ was released to the luminal side upon YZ oxidation and a hydrogen atom was subsequently abstracted from substrate water by the radical Y•Z . Many experimental [15, 16, 18, 19] and theoretical [65] studies have been published which contradict this model. The current study adds further experimental evidence in opposition to the model. However, there is a possibility of direct involvement of YZ in the proton transfer from the substrate water molecule without any proton release upon YZ oxidation; namely, the YZ -His moiety may be part of a proton transfer pathway from the substrate water molecule. Based on information taken from X-ray studies of PSII [8–10] candidates for the proton transfer pathways from the Mn4 CaO5 cluster to the bulk have been proposed [10, 22–24, 66] the most probable of which was expected to be from the network associated with D1-Asp61 via Cl-1 [32, 33, 66–73]. The hydrogen bond network via YZ to the luminal side also has been proposed as another candidate for the proton transfer pathway [10, 24]. The presence of the water cluster, which consisted of several water molecules located between YZ and the Ca ion of the Mn4 CaO5 cluster, could facilitate transfer protons from the water ligand (i.e., W2, W3, and W4) and the oxo-bridge (O5) to the phenolic oxygen of YZ via the Grotthuss mechanism [74, 75] (Fig. 2.14). The YZ -His moiety, which connected the water cluster with the channel via D1-Asn298 to the luminal side, functioned as the “gate” for this proton transfer process. Our QM/MM calculations showed that the structural changes took place

2.4 Discussion

31

Fig. 2.14 Proposed reaction mechanism for the proton release from the substrate water via the Y•Z -HisH+ moiety. The polarizable proton observed in the present study is marked with a green circle. Hopping a proton to WA triggers a rapid proton release from the substrate water to YZ through the water network by the Grotthuss mechanism (red arrows). Reprinted with permission from Nakamura et al. [78]. Copyright (2014) American Chemical Society

when the hydrogen bonds between YZ and W4 were broken and a new hydrogen bond was subsequently formed between W4 and another water (WA ) upon YZ oxidation, with proton transfer occurring from YZ to D1-His190 (Fig. 2.12). Through these structural changes, WA moved toward D1-His190 and encouraged shortening of the distance between the oxygen of WA and Nτ of His190 from 4.57 to 3.28 Å. Because the proton between YZ and D1-His190 was highly polarized, as mentioned above, it moved in response to changes encountered in the charge distribution, which was mainly formed through the interactions between the Mn4 CaO5 cluster and the fluctuations occurring in the different water and protein environments. This meant that there was a chance for “proton hopping” to take place from the proton donor, D1-His190, to the proton acceptor, WA , in this particular case. This process acted as the rate-limiting step, and triggered changes in the equilibrium of the proton transfer reaction between Y•Z and water ligands to the Y•Z . Simultaneously, the proton linked to WA is released to the hydrogen bond network surrounding D1-Asn298 (Figs. 2.12 and 2.14), hence, a proton was finally released from the substrate water molecule to the luminal side. Another possibility for proton hopping occurred from W7 to W4, where the transfer distance was simply 3.84 Å in the Y•Z state. W4 also connected the hydrogen bond network to the luminal side via WA –WD (Figs. 2.12 and 2.14). The crucial event, which took place in this novel proton transfer mechanism was the rearrangement of the hydrogen bond network via water movement. The importance of these “movable water molecules” through proton transfer pathways has been previously reported [24, 33, 63]. This proton transfer is prioritized before the electron transfer process from the Mn4 CaO5 cluster to the oxidized Y•Z . Such a “proton-first” PCET mechanism takes place in the S2 and S3 states owing to the excessive positive charge on the Mn4 CaO5 cluster, which decreases the redox potential for electron transfer [29, 31, 76, 77].

32

2 Hydrogen Bond Structure of Redox Active Tyrosines …

Exactly which hydrogen bond network is most suitable for the proton transfer process in each S-state may depend heavily on the structural relation that existed between the substrate water molecule and the hydrogen bond network near the Mn4 CaO5 cluster. In the S3 → S0 transition, the hydrogen bond network via Cl-1 and D1-Asp61 is used for proton release [32, 33, 66–73], which has been supported by mutagenetic studies at D2-Lys317 [69, 70] and D1-Asp61 [71–73] In contrast to the S3 → S0 transition, another network found around YZ was suggested to be active in the S2 → S3 transition [29]. The results of our study strongly indicated that the Coulomb repulsion between the positive charges on the Mn4 CaO5 cluster in the S2 state and those on the Y•Z -HisH+ moiety facilitate the proton release process by initiating the release of the polarizable proton from Y•Z -HisH+ . The protons associated with W2, W3, W4, and O5 can be released to the lumen by utilizing the proton transfer mechanisms described above (Fig. 2.14). “Proton release” involving the Y•Z -HisH+ moiety may also occur in a concerted PCET reaction, which is similar to the previously proposed “hydrogen abstraction model.” [17] However, the difference between that model and our proposed mechanism lies in the fact that the proton transfer leads to the protonated D1-His190, which acts as a triggering reaction and determines the reaction rate of the entire PCET process; generating the deprotonation form of Y•Z -His becomes, thus, unnecessary. “Proton rocking” between D1-His190 and YZ , without any further release of protons, greatly encourages the decrease in the energy barrier required for YZ reactions. The strong hydrogen bond, which existed between D1-His190 and YZ in both the reduced and oxidized forms (as shown in theoretical studies conducted by Saito et al. [11] and this group, respectively) particularly contributes to increases in the rates of reaction for YZ . On the other hand, the absence of a broad band in 76°, >53°, and >39° for D1-His190 Nτ, D1-His332 Nτ, and D1-His337 Nπ, respectively). Redox potentials of the S1 → S2 transition in various protonation structures. Table 6.5 shows the redox potential (E m ) of the S1 → S2 transition calculated using the four different protonation states upon Mn4-oxidation. For an estimation of E m , D2-Lys317 and two Cl− ions were added to the QM region to embed all charged ions and amino acids within 8 Å of the Mn4 CaO5 cluster (Fig. 6.2). The E m value of W2/H337 = H2 O/HisH+ model, total charge of which was 0, was calculated to be + 0.87 V. This was close to the value obtained for water oxidation (+0.88 V at pH 6.0). On the other hand, other models showed relatively low values (i.e., −3.28, −0.92, and −1.49 V for W2/H337 = OH− /His0 , H2 O/His0 , and OH− /HisH+ , respectively). This meant that the deprotonation of W2 triggered a decreasing in the redox potential of about 2.16 V, irrespective of protonation on the D1-His337 residue, whereas the deprotonation of D1-His337 promoted a decreasing in the redox potential of about 1.79 V, irrespective of W2 protonation.

6.4 Discussion In this chapter, polarized ATR-FTIR difference spectra were acquired for each Sstate transition using PSII membranes oriented on the ATR crystal and the results were reported. During S1 → S2 transition, relatively high intensities in the negative and positive bands were observed at ~2600 and ~2900 cm−1 , respectively, in the

−0.848 (+0.007)

−0.723 (+0.006)

−0.821 (+0.026)

−0.765 (+0.039)

−0.743 (+0.011)

1.038

0.937

1.130

−0.855

−0.729

−0.847

−0.805

−0.754

Mn3

Mn4

Ca

O1

O2

O3

O4

O5

1.116 (+0.179)

1.02 (−0.018)

1.100 (−0.010)

−0.731 (+0.023)

−0.800 (+0.005)

−0.822 (+0.025)

−0.713 (+0.017)

−0.815 (+0.040)

1.139 (+0.009)

1.049 (+0.112)

0.994 (−0.044)

1.109 (−0.001)

1.123 (+0.248)

−0.767

−0.805

−0.819

−0.746

−0.864

1.121

0.900

1.058

1.093

0.878

−0.747 (+0.021)

−0.769 (+0.036)

−0.803 (+0.016)

−0.729 (+0.017)

−0.856 (+0.007)

1.143 (+0.022)

1.107 (+0.207)

1.021(−0.037)

1.096 (+0.003)

0.926 (+0.048)

−0.738 (+0.029)

−0.806 (−0.001)

−0.791 (+0.028)

−0.721 (+0.025)

−0.824 (+0.040)

1.133 (+0.012)

1.035 (+0.134)

0.995 (−0.063)

1.096 (+0.003)

1.126 (+0.247)

−0.760

−0.797

−0.854

−0.726

−0.852

1.119

0.885

1.012

1.102

0.865

−0.760 (0.000)

−0.754 (+0.044)

−0.827 (+0.027)

−0.727 (−0.001)

−0.851 (0.000)

1.136 (+0.017)

1.048 (+0.163)

0.998 (−0.015)

1.094 (−0.008)

0.926 (+0.061)

in parentheses indicate deviations from the corresponding values in the S1 state Reprinted with permission from Nakamura. and Noguchi [61]. Copyright (2017) American Chemical Society

a Figures

1.15 (+0.020)

1.110

Mn2

0.927 (+0.052)

0.875

Mn1

S1

Mn4-oxi ()a

Mn1-oxi ()a

S2

S1

Mn4-oxi ()a

Mn1-oxi ()a

S2

Mn4-oxi ()a

S2

S1

Atom

W2/H337 = H2 O/His0

W2/H337 = H2 O/HisH+

−0.733 (+0.027)

−0.773 (+0.024)

−0.827 (+0.028)

−0.711 (+0.015)

−0.813 (+0.038)

1.130 (+0.011)

0.971 (+0.086)

0.957 (−0.055)

1.105 (+0.004)

1.125 (+0.260)

Mn1 oxi ()a

W2/H337 = OH− /HisH+

Table 6.4 Calculated Mulliken charges of atoms in the Mn cluster in each protonation model

−0.768

−0.804

−0.818

−0.741

−0.860

1.112

0.855

1.035

1.079

0.86

S1

−0.762 (+0.007)

−0.765 (+0.039)

−0.803 (+0.015)

−0.737 (+0.004)

−0.858 (+0.002)

1.128 (+0.016)

1.033 (+0.179)

1.013 (−0.022)

1.085 (+0.005)

0.922 (+0.062)

Mn4 oxi ()a

S2

W2/H337 = OH− /His0

−0.739 (+0.029)

−0.789 (+0.015)

−0.793 (+0.025)

−0.721 (+0.021)

−0.822 (+0.038)

1.123 (+0.011)

0.962 (+0.108)

0.964 (−0.071)

1.087 (+0.007)

1.129 (+0.269)

Mn1 oxi ()a

112 6 Protonation Structure of a Key Histidine …

6.4 Discussion Table 6.5 Redox potentials (E m ) of WOC upon the S1 → S2 transitiona estimated by QM/MM calculations

113 W2/H337b H2

O/HisH+

Total Charge of Sc1

E m (V)

0

0.87

H2 O/His0

−1

−0.92

OH− /HisH+

−1

−1.49

OH− /His0

−2

−3.28

a Mn4-oxidized

S2 state was used for calculation of E m is in its neutral NτH (His0 ) or cationic (HisH+ ) form c QM region to estimate E m values contains all charged species located within 8 Å from the Mn4 CaO5 cluster Reprinted with permission from Nakamura and Noguchi [61]. Copyright (2017) American Chemical Society b D1-H337

parallel-polarized spectrum (Fig. 6.7a), whereas countersignals were observed across the same frequency region during S3 → S0 transition (Fig. 6.7b). Perpendicularpolarized spectra could only detect weak intensities which had significantly large dichroic ratios (3.9 < R, Table 6.1). Moreover, several small peaks were observed on the broad features between 2400 and 3000 cm−1 during S1 → S2 transitions, and these were also the opposite was observed during S3 → S0 transitions (Fig. 6.7a, b). These small peaks in the S2 /S1 difference spectrum were though to arise from Fermi resonance of the NH vibrations of histidine residues coupled with combinations of other vibrations of a imidazole group and overtones, as confirmed via global 15 N-labeling of PSII membranes taken from spinach samples (Fig. 6.7c). Thus, the presence of NH bands in a histidine residue in the 2400–3000 cm−1 region, at least, in the S2 /S1 and S0 /S3 difference spectra was strongly supported by evidence of these small peaks linked to Fermi resonance. Furthermore, the disappearance of these Fermi resonance peaks in the presence of D2 ,O [52, 53] accompanied by a broad feature in the spectrum, was consistent with the existence of a NH band in this region. The QM region in our QM/MM system included three histidine side chains around the Mn4 CaO5 cluster (namely, D1-His190, D1-His332, and D1-His337). Indeed, frequency calculations performed for this system showed the presence of an NτH vibration of D1-His337 in which the NτH group was hydrogen-bonded with an oxygen atom (O3) of the Mn4 CaO5 cluster, as seen within the 3000–2000 cm−1 region; this was most notable in cases where the D1-His337 residue formed a protonated cation (Table 6.2; Fig. 6.9A, a). NτH frequencies calculated for protonated cationic D1-His337, in particular those linked with the W2 = H2 O model, were in agreement with the positions of the broad features in the parallel-polarized S2 /S1 spectrum. On the other hand, NτH frequencies in the W2 = OH− model were significantly lower than those obtained from the positions of the broad features on the spectra (Fig. 6.9A, a). In contrast to NτH frequencies from the protonated D1-His337 residue, NτH frequencies of neutral D1-His337 and NπH frequencies of the protonated D1-His337 residue were significantly higher (between 3500–3200 cm−1 ) for the S2 and S1 states (Table 6.2; Fig. 6.9A, a and b). Other nearby histidine residues, namely D1-His190 and D1-His332, also had frequencies higher than 3000 cm−1 linked to their respective NH vibrations (Table 6.2; Fig. 6.9A, c and d).

114

6 Protonation Structure of a Key Histidine …

Table 6.6 Hydrogen bond distances at the imidazole NH of His residues (H337, H332, and H190) around the Mn cluster in the S1 state Model QM/MM

Experimenta

Hydrogen bond distance (Å)

W2

H337b

H337 NτH

H337 NπH

H332 NπH

H190 NπH

H2 O

HisH+

2.60

2.75

2.71

2.72

H2 O

His0

2.78

–

2.72

2.75

OH−

HisH+

2.4

2.78

2.74

2.77

OH−

His0

2.75

–

2.77

2.78

–

–

2.63

2.69

2.78

2.77

a Averaged

distance of two monomers, A and B, in the crystal structure (PDB code: 4UB6) is in its neutral NτH (His0 ) or cationic (HisH+ ) form Reprinted with permission from Nakamura and Noguchi [61]. Copyright (2017) American Chemical Society

b D1-H337

Low NτH vibration frequencies for protonated D1-His337 indicated the formation of a strong hydrogen bond between NτH and O3. Subsequent calculations revealed that the Nτ–O3 bond distance for the protonated D1-His337 in the S1 state was 2.54 and 2.60 Å for W2 = OH− and H2 O, respectively, which were considerably shorter than the N–O distances of other nearby histidine residues and the Nπ atom of D1-His337 (2.71–2.78 Å) (Table 6.6). The Nτ–O3 distance in the S1 state was calculated using the W2/H337 = H2 O/HisH+ model (2.60 Å) and it was found to reproduce well the values obtained (2.63 Å) from the most recent XFEL structure [5] (Table 6.3). Other hydrogen bonds linked with D1-His190, D1-His332, and D1His337 in the XFEL structure were longer (2.69–2.78 Å), which was consistent with our data (Table 6.6). Our calculations also indicated a tendency that a lower NτH frequency of the protonated D1-His337 is correlated with a more negative charge of O3 in the Mn4 CaO5 cluster (Fig. 6.9B). Hence, more the negatively charged O3 atom in the W2/H337 = OH− /HisH+ model promoted a downshift in the NτH frequency from the value seen in the W2/H337 = H2 O/HisH+ model (Fig. 6.9). Moreover, when an electron was extracted from the Mn4 CaO5 cluster during the S1 → S2 transition, O3 only lost a portion of its negative charge with an accompanying frequency upshift in the NτH vibration of D1-His337 (Fig. 6.9B). Therefore, the presence of oppositely signed signals in the 2200–3000 cm−1 region in the S0 /S3 difference spectra meant that the O3 charge decreased during S1 → S2 transition and reverted to the same charge as that seen in the S1 state during S3 → S0 transition, whereas S0 → S1 and S2 → S3 transitions triggered no significant change in the charge attached to the O3 atom (Fig. 6.7). This was evidenced by small intensities in the 2200–3000 cm−1 region of the S1 /S0 and S3 /S2 difference spectra. The large experimental dichroic ratio noted at ~2600 cm−1 (R = 10.0) in the polarized S2 /S1 difference spectra (Table 6.1; Fig. 6.7a) was an indication that the angle of the transition moment for this vibration with respect to the membrane normal was relatively small (19°). The dichroic ratios (R) calculated for the NτH stretching

6.4 Discussion

115

vibration of D1-His337 (13–26°) faithfully reproduced the values obtained under experimental observation (Table 6.2). Other dichroic ratios for the NH vibrations of nearby histidine residues (namely, D1-His190, D1-His332, and Nπ of D1-His337) were relatively small (Table 6.2), and in reproducing these dichroic ratio, the assignment of the broad feature linked to the NτH vibration of D1-His337 was confirmed. Slightly small dichroic ratios as well as the presence of large angles in the broad features at ~2900 and ~2600 cm−1 in the S0 /S3 spectra and at ~2900 cm−1 in the S2 /S1 spectra (R = 3.9–6.4, 28–39°) might be caused by overlap interference from other vibrations, namely those associated with the OH vibrations of water molecules that were capable of strong hydrogen bond interactions [52, 53]. From these experimental observations and theoretical simulations, we concluded that the broad features at ~2900 and ~2600 cm−1 in the experimental S2 /S1 and S0 /S3 spectra originated from or were involved significant contributions from the NτH vibrations for protonated cationic D1-His337. This notion, moreover, indicated that D1-His337 maintained its protonated cation during the S-state cycle, even at high pH levels. This was clearly seen in the S2 /S1 spectra at between 2200–3000 cm−1 under pH conditions ranging from 4 to 9; the lack of change observed under these conditions [54] meant that the protonation of D1-His337 and its hydrogen bond interaction were practically identical. Note also that the reason for this lack of influence on the protonation was not due to the isolation of D1-His337 from the bulk region because experiments conducted in D2 O showed that the proton of the Nτ site was exchangeable. This meant that the protonated D1-His337 was energetically stabilized through a strong, charge-assisted hydrogen bond with the O3 atom in the Mn4 CaO5 cluster. Our previous studies showed another characteristic band associated with the histidine residue at 1114 cm−1 in the S2 /S1 spectrum arose from C5N1 vibrations of the neutral NπH form of D1-His332 [51]. FTIR studies by Kimura et al. [43] further revealed that this C5N1 band appeared in both the S1 /S0 and S3 /S2 difference spectra, but not in the S0 /S3 difference spectrum. In contrast to the broad features seen at 2400–3000 cm−1 in the S0 /S3 difference spectrum, in which opposite signs to those noted in S2 /S1 difference spectrum, this observation confirmed that the signals at 2400–3000 cm−1 and 1114 cm−1 were not of the same origin. Thus, we revised the minor peak assignments that originated from Fermi resonance in 2400– 3000 cm−1 of the S2 /S1 difference spectrum from the NπH vibrations of D1-His332 [51] to the NτH vibrations of D1-His337. Still, the question of why there was no C5N1 vibration signal for D1-His337 at ~1110 cm−1 remained. Current QM/MM simulations showed that the C5N1 stretching vibration of protonated D1-His337 was much smaller in intensity than that of D1-His332 in the S2 and S1 states (Table 6.7), and the C5N1 stretching vibration of neutral D1-His337 had the same low level of intensity as that seen for D1-His332 (Table 6.7). Deuteration experiments [51] on the assignable C5N1 band linked to the neutral NτH form of a histidine side chain at about 1110 cm−1 strongly supported the notion that D1-His337 formed a protonated cation under these conditions.

116

6 Protonation Structure of a Key Histidine …

Table 6.7 C5N1 stretching vibrations of imidazole groups in His side chains in WOC calculated by QM/MM calculations Model

S2 (Mn4-oxidized)

S2 (Mn1-oxidized)

W2

H337a

His (Structure)b

Frequency (cm−1 )c

Intensity (km/mol)

Frequency (cm−1 )c

Intensity (km/mol)

Frequency (cm−1 )c

H2 O

HisH+

H337 (HisH+ )

1172

0.1

1153

4

1172

2

H332 (NπH)

1146

101

1142

126

1128

82

H190 (NπH)

1137

13

1135

13

1135

61

H337 (NτH)

1152

22

1146

37

1148

30

H332 (NπH)

1147

98

1143

109

1128

69

H190 (NπH)

1146

199

1136

10

1135

74

H337 (HisH+ )

1174

6

1167

0.2

1176

1

H332 (NπH)

1148

68

1145

210

1129

28

H190 (NπH)

1149

90

1145

129

1138

78

H337 (NτH)

1155

11

1150

28

1152

23

H332 (NπH)

1149

75

1146

92

1129

29

H190 (NπH)

1150

67

1148

129

1148

118

H2 O

OH−

OH−

S1

His0

HisH+

His0

Intensity (km/mol)

a D1-H337

is in its neutral NτH (His0 ) or cationic (HsH+ ) form state of the imidazole group of each His side chain indicates in parentheses. NτH: neutral NτH form; NπH: neutral NπH form; HisH+ : protonated cation form c Frequencies were not scaled Reprinted with permission from Nakamura and Noguchi [61]. Copyright (2017) American Chemical Society b Protonation

Concluding that D1-His337 remained protonated during the S-state cycle might encourage the idea that this histidine residue was not involved in the proton transfer process during water oxidation, a notion was at odds with the theoretical studies that accurately predicted the deprotonation of D1-His337 during S2 → S3 transition [20]. Our notion was consistent with the evidence from the X-ray structure [5] because the hydrogen-bonded structure was shown to indirectly link the Nπ atom of D1-His337 and the bulk surface, which in turn, provided a high energy barrier for proton transfer reactions from D1-His337 to the bulk. Recent X-ray structure of PSII showed the various N–O distances observed between O3 and D1-His337 (2.46 and 2.75 Å) in the two monomers of the crystal. The authors believe that this difference arose from the presence of different protonation forms (i.e., protonated and deprotonated) for D1-His337 [4]. Therefore, if two different protonation states existed in the S1 state, the electron transfer rate in the S1 → S2 transition should be heterogeneous, which has not been observed through time-resolved spectroscopic studies [55–58]. Thus,

6.4 Discussion

117

it was expected that variations in the N–O distance between O3 and D1-His337 specifically occurred within the PSII crystal. Further X-ray crystallographic studies done in combination with FTIR spectroscopy and using PSII crystals [7] are necessary to address the origin of these varied N–O distances between O3 and D1-His337. Despite the apparent lack of involvement of D1-His337 in the proton transfer process, its positive charge was shown to affect the redox potential (E m ) of the Mn4 CaO5 cluster, a step which is crucial for electron transfer processes between Y•Z and the Mn4 CaO5 cluster. To determine E m values, QM/MM calculations were performed with the larger QM region and incorporating nearby charge species from the Mn4 CaO5 cluster (Fig. 6.2). Only the W2/H337 = H2 O/HisH+ model had positive E m value (+0.87 V), whereas calculations using the other models resulted in negative E m values (Table 6.5). The E m value calculated in the W2/H337 = H2 O/HisH+ model (+0.87 V) was close to that for water oxidation (+0.88 V at pH 6.0) and also lower than those observed for both P680 (∼ 1.2 V) [59, 60] and YZ (0.9–1.0 V) [9, 10]. Thus, this E m value was consistent with the principles of a simple electron transfer from the Mn4 CaO5 cluster to Y•Z during S1 → S2 transition. Our estimation of this E m value was also consistent with previous theoretical results reported by Amin et al. [20] (between +0.70 and +0.93 V for the S1 → S2 transition), which had been determined using Multi-conformation Continuum Electrostatic calculations. This agreement between the E m values for the W2/H337 = H2 O/HisH+ model also supported the notion that a protonated cationic from of D1-His337 was generated during oxidation. Based on our E m calculations, it can be said that the H2 O form of W2 was an ideal protonation state for electron transfer from the Mn4 CaO5 cluster to Y•Z rather than its OH− counterpart, at least, during the S1 and S2 states. This assignment of the protonation state of W2 was supported by the similarities in the results obtained for the Nτ–O3 distance and the NτH frequency calculations with the experimental polarized ATR-FTIR spectra and the XFEL structure [5] and our polarized ATR-FTIR spectra (Fig. 6.7), respectively, in the W2/H337 = H2 O/HisH+ model, which were much closer in line with each other than they were with the values estimated from the W2/H337 = OH− /HisH+ model (Table 6.3; Fig. 6.9A). Moreover, our previous QM/MM results regarding symmetric COO− vibrations (Chap. 5) were also in agreement with the S2 /S1 spectrum in the W2 = H2 O model rather than with the spectrum obtained using the W2 = OH− model [44] for the neutral D1-His337 residue. However, because a similar spectrum was acquired for the model with the protonated D1-His337 (Fig. 6.10), the protonation state of W2/H337 = H2 O/HisH+ in the S1 state was found to be consistent with previous theoretical studies performed using QM/MM [12] and MCCE [20] calculations. In summary, the charge species distribution located near the Mn4 CaO5 cluster was found to be very important with regards to its redox potential (E m ). The total charge in the S1 state of the entire QM region in the W2/H337 = H2 O/HisH+ model was 0, whereas other protonation models had overall negative charges of −1 and − 2 in the W2/H337 = H2 O/His0 and OH− /HisH+ and in the W2/H337 = OH− /His0 models, respectively (Fig. 6.2). The W2/H337 = H2 O/HisH+ model was most in line with the results obtained, and the overall charge on the Mn4 CaO5 cluster with its directly attached ligands (i.e., four neutral water molecules, one neutral imidazole

6 Protonation Structure of a Key Histidine …

IR intensity

118

1450

1400

1350

1300

1250

-1

Wavenumber (cm ) Fig. 6.10 S2 /S1 difference spectra calculated for the symmetric COO− stretching vibration acquired through QM/MM simulations: The protonated (red line) and neutral (black line) forms of D1-H337 together with the experimental data (green line). The spectra were scaled with the factor of 0.965 and 0.963 for protonated and neutral D1-H337, respectively. W2 was assumed to be neutral water (H2 O), ad the S2 spectra was averaged with the calculated spectra of Mn1- and Mn4-oxidized S2 states. Reprinted with permission from Nakamura and Noguchi [61]. Copyright (2017) American Chemical Society

group, and six COO− groups) was determined to be 0. Furthermore, it was shown that the nearby charge species canceled their charges [i.e., three positive charged residues (D1-His337, D2-Lys317, and CP43-Arg357) and three negative charged residues and ions (D1-Asp61 and two Cl− )]. The effect of the protonation state of D1-His337 on the E m of the Mn4 CaO5 cluster was estimated to be +1.79 V, whereas that of W2 protonation (i.e., between H2 O and OH− ) is +2.16 V (Table 6.5). This significant effect of the protonated D1-His337 (+1.79 V) was reasonable, considering the direct interaction that existed between this histidine residue and the Mn4 CaO5 cluster through hydrogen bonds. This highlighted the crucial role played by this histidine residue in adjusting the E m values of the Mn4 CaO5 cluster during water oxidation.

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Chapter 7

General Conclusion

Regarding the electron transfer and water oxidation mechanisms of PSII, many problems had been unresolved such as (1) the origin of the asymmetric electron transfer of two redox-active tyrosines, YZ and YD , which is crucial for the high quantum efficiency of water oxidation, (2) hydrogen bond networks used in proton release during water oxidation, (3) the roles of key amino acid residues around the Mn cluster in the water oxidation mechanism. To address these issues, in this study, we investigated the structures and reactions of these tyrosines and the WOC, especially focusing on their hydrogen-bond interactions and protonation structures, using the combinatorial approach of infrared spectroscopy and quantum chemical calculations. To clarify the molecular mechanism of the proton-coupled electron transfer reaction of YZ and YD , we first analyzed their hydrogen-bonded structures (Chap. 2). It was shown from FTIR analysis that a proton of YZ was transferred to the neighboring D1-His190 upon photooxidation, forming a strong hydrogen bond with a high proton polarizability, whereas such a strong hydrogen bond was absent in oxidized YD . The QM/MM calculations revealed the rearrangement of the hydrogen bond network of water molecules around YZ upon oxidation. These results suggested that YZ has a key role in proton release during water oxidation, and a novel proton release mechanism through Y•Z was proposed. Furthermore, we detected protons released to the bulk upon YZ and YD oxidation using the isotope-edited FTIR technique (Chap. 3). It was shown that a proton is released into the bulk upon YD oxidation, whereas no proton release was observed upon YZ oxidation. This indicated that long-distance proton transfer takes place upon YD oxidation in contrast to the YZ case, in which a proton was only shifted to the His through a hydrogen bond. A high activation energy originating from this long-distance proton transfer was concluded to be the reason for the redox reaction of YD much slower than that of YZ , causing the asymmetric electron transfer between YZ and YD . This difference was proposed to arise from amino acid residues, D1-N298 and D2-R294, hydrogen-bonded to the Nπ sites of the His residues coupled to YZ and YD , respectively.

© Springer Nature Singapore Pte Ltd. 2020 S. Nakamura, Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II, Springer Theses, https://doi.org/10.1007/978-981-15-1584-2_7

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In Chap. 4, to investigate the role of the hydrogen-bond network around the Mn cluster, we performed normal mode analysis of the OH stretching vibrations of water molecules by QM/MM calculations. The calculated OH stretching vibrations well reproduced the experimental FTIR spectrum, and among them, a vibrational mode delocalized over several water molecules located between YZ and the Mn cluster was found. It was suggested that this vibrational mode contributes to a rapid proton transfer from the Mn cluster to YZ according to the Grotthuss mechanism. This result supported the proton transfer via YZ during water oxidation proposed in Chap. 2. Because this mechanism needs an excessive positive charge on the Mn cluster as a driving force, this proton transfer was suggested to occur in the S2 → S3 or S3 → S0 transition. Furthermore, we investigated the roles of amino acid residues interacting with the Mn cluster in water oxidation (Chaps. 5 and 6). First, to clarify the role of the carboxylate ligands to the Mn cluster, we performed normal mode analysis of the COO− vibrations of these carboxylate groups by QM/MM calculations (Chap. 5). The COO− vibrations reproduced the experimental FTIR spectrum. The large downshift of the vibrational frequency and the changes in the CO lengths of D1-D170 in the S1 → S2 transition indicated the shift of a positive charge from Mn4 to Ca via the π conjugation of this carboxylate group. Together with the concomitant changes in the OH lengths of the water ligands to Ca, we concluded that D1-D170 bridging Ca and Mn has a crucial role in controlling the reactivity of the water ligands to Ca. Next, to clarify the role of histidine residues in WOC, we performed polarized ATR-FTIR measurements and QM/MM calculations (Chap. 6). In the polarized FTIR difference spectra upon the S-state transitions, bands with large dichroic ratios were observed in the high-frequency region in the S1 → S2 and S3 → S0 transitions. The QM/MM analysis showed that these bands arise from the NτH stretching vibration of the protonated cation form of D1-H337, which is hydrogen-bonded with the Mn cluster. From these results together with energy calculations, we concluded that D1-H337 stays in a protonated cation throughout the S-state cycle and this histidine regulates the redox potential of the Mn cluster to oxidize water molecules. All of these results in the present study showed that the hydrogen-bonded structure is important to characterize the proton transfer reactions and the protonation structures of water molecules and amino acid residues. Moreover, it was shown that molecular vibrations are suitable for monitoring slight structural changes in PSII proteins, which cannot be detected by other spectroscopic methods. Such information of hydrogen bond interactions and structural changes in the catalytic site is crucial to clarify the molecular mechanism of water oxidation. Therefore, the methodology and results in the present study will make a significant contribution to the future researches for full understanding of the mechanism of photosynthetic water oxidation.

Curriculum Vitae

Shin Nakamura Contact Details Address Department of biochemical sciences “A. Rossi Fanelli”, University of Rome “Sapienza”, P.le Aldo Moro 5, 00185, Rome, Italy E-mail [email protected] Current Position 2018–present Postdoctoral fellow, Sapienza University of Rome, Rome, Italy. Education 2015–2018 Doctor of Science, Nagoya University, Nagoya, Japan 2013–2015 Master of Science, Nagoya University, Nagoya, Japan 2009–2013 Bachelor of Education, Aichi University of Education, Aichi, Japan. Fellowships 2018–2020 Overseas Research Fellowships from Japan Society for Promotion of Science (JSPS) 2015–2018 Research Fellowship for Young Scientists (DC1) from JSPS. Research Interests The scientific activity covers computational and experimental biophysics, and is aimed at understanding the molecular mechanisms of proteins. © Springer Nature Singapore Pte Ltd. 2020 S. Nakamura, Molecular Mechanisms of Proton-coupled Electron Transfer and Water Oxidation in Photosystem II, Springer Theses, https://doi.org/10.1007/978-981-15-1584-2

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Curriculum Vitae

Awards and Honors Young Talents Award 2014 International Conference Photosynthesis Research for Sustainability, Russia.