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Studies on the Water Oxidising

Complex and Redox-Active Tyrosines

of Photosystem II

A thesis submitted for the degree of

Doctor of Philosophy in Chemistry of

The Australian National University

by

Wooi Yee Chuah

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The work in this thesis is my own except where otherwise noted, and was performed wholly

within my candidature for the degree of Doctor of Philosophy in Chemistry.

Wooi Yee Chuah

July 2015

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Abstract

This thesis presents a two-part study of Photosystem II. The first, is a spectroscopic study

pri-marily based on a novel low temperature FTIR system. The second is a series of computational

studies on carboxylate-ligated transition metal complexes, culminating in a direct study of the

vibrational frequencies associated with the first-shell carboxylate ligands of the Water Oxidising

Complex. The findings of this thesis are summarised in the following:

Low temperature (10 K) illuminations on ZnSe cryostat windows induces transient and

stable FTIR difference signals. The kinetic properties of these signals are highly

reminis-cent of the TyrZ “split signals” observed in EPR studies.

The FTIR analogue to the visible illumination-induced, transient S1 TyrZ “split signal” was observed. Some characteristic features of the QA–/QAsignal was observed, but

fea-tures of the donor(s) could not be extracted from the data.

The 10 K and 80 K TyrD◦/TyrD difference signals were obtained using multiple signal

subtractions. Small spectral differences, possibly caused by electrostatic effects, were

found between these signals. The possibility of TyrD◦ forming an imidazolium interme-diate at 10 K was found to be unlikely, however.

Deprotonation was found to suppress carboxylate ligand vibrational frequency shifts

asso-ciated with manganese-centred oxidation. This appears to be a general feature of

redox-active transition metals, as the same effect was also observed in vanadium, chromium,

iron and cobalt complexes.

Charge conservation via simultaneous anionic ligand substitution with metal centre

oxi-dation also suppresses carboxylate ligand frequency shifts. Solvent polarisation also has

a similar effect. It was concluded that carboxylate ligand frequency shifts are

primar-ily driven by electrostatic effects, i.e. the increased polarisation between the carboxylate

ligand and metal centre, as the metal centre undergoes oxidation.

Water or hydroxyl ligand deprotonation is most likely the reason for the sparsity of

fre-quency shifts observed in mutagenic studies of the PSII Water Oxidising Complex (WOC).

Frequency shift suppression via deprotonation appears to be most effective in clusters that

are neutrally charged, possess a mean Mn oxidation of 3 in the S1 state, and have water

and hydroxyl ligands in the O5 and W2 positions, respectively. Further, it is shown that

Mn2 is likely to be oxidised in the S1/S2transition, on the basis of the IR frequency shifts

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Over the course of the last 4 years, I have met so many wonderful people, who have each in

some way or another, contributed to the completion of this thesis. It is inevitable that I will

have omitted some of these names here, and I apologise to those that I have; all of you have my

gratitude for helping me along this journey.

Elmars Krausz, my supervisor, has been my friend and mentor for the last five years. I would

like to thank him for giving me the opportunity to join in the grand endeavour that is the study

of Photosystem II, and free use of the group’s facilities that have taken some twenty-odd years

to prepare. His vigour and enthusiasm for science is truly remarkable, and I hope I might match

him in that aspect for the rest of my career.

It is very likely that I would be considering different career options at this point, had it not been

for the assistance of Terry Frankcombe, my advisor in all matters computational. Thank you for

instructing me in the dark arts of scientific writing, and for introducing me to LaTeX, which I

have used for the writing of this thesis and all but one of my papers.

Ron Pace’s ideas have formed the basis of all the computational work in this thesis. I hope the

well he draws his inspiration from never runs dry.

Warwick Hillier was my go-to person for anything to do with FTIR spectroscopy. May he rest

in peace.

Paul Smith has taught me nearly everything I know about the practical aspects of biochemistry

and EPR spectroscopy. I cannot thank him enough for his patience.

The RSC workshop staff have been especially nice to me, and were absolutely essential for the

completion of this work. I would like to extend a special thank you to Link Williams, Hendrik

Maat and Trevor Wilson for bailing me out of trouble so many times that I have lost count,

and Adam Zupac for always looking out for me. Keith Jackson, despite being officially retired,

always manages to produce some magical gadget just when it is needed.

My friends from RSBS, Alonso Zavaleta and Michael Cheah, have been incredibly generous

with me, whether it was in giving their time (like those painful hours spent in the cold room

preparing spinach PSII), or in sharing their stock of chemicals with me. Likewise, my lab

mates, Jeremy Hall and Jennifer Morton, have been excellent friends.

I also thank Professor Jian-Ren Shen for theT.Vulcanussamples he has graciously given to our

group, which have been enormously useful in my project.

Hoang Trang, my girlfriend, took care of me when I was sick, then took me skydiving when I

was better – well, perhaps not in that exact order, but I suspect that it might be close. The last

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three-and-a-half years have been immeasurably improved by her love and companionship, that

I hope I might be worthy of.

Finally, I thank my family, to whom I owe a debt of gratitude too great to simply express in

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Declaration of Authorship ii

Abstract iii

Acknowledgements iv

Contents vi

Abbreviations xi

1 An Introduction to Oxygenic Photosynthesis 1

1.1 An Abridged History . . . 1

1.2 How does Oxygenic Photosynthesis Work? . . . 2

1.3 Photosystem II . . . 4

1.3.1 Overview of Photosystem II . . . 4

1.3.2 The Water Oxidising Complex . . . 6

1.3.2.1 Ambiguities in the Geometry and Electronic State of the Wa-ter Oxidising Complex . . . 9

1.4 The Aims of this Thesis . . . 10

1.4.1 Studying Tyrosine Oxidation at Cryogenic Temperatures using FTIR . . 11

1.4.1.1 TyrD . . . 11

1.4.1.2 TyrZ . . . 13

1.4.2 Interpreting WOC FTIR Data through Model Complex Calculations . . 14

2 Materials and Methods 27 2.1 PSII Samples . . . 27

2.1.1 Isolation of PSII Membranes from Spinach . . . 27

2.2 Infrared Spectroscopy . . . 30

2.2.1 Principles of Infrared Spectroscopy . . . 30

2.2.2 The Effect of Environmental Interactions on IR Absorptions . . . 32

2.2.2.1 Hydrogen Bonding . . . 32

2.2.2.2 Electrostatic Effects . . . 33

2.2.3 IR Spectroscopy in PSII - Difference Spectroscopy . . . 33

2.2.4 Infrared Instrumentation . . . 34

2.2.4.1 Dispersive Instruments . . . 34

2.2.4.2 Interferometric Instruments . . . 34

2.2.5 Experimental Setup . . . 37

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2.2.5.1 Optimising the Signal-to-Noise Ratio of FTIR Measurements 39

2.2.5.2 Theoretical Sensitivity of FTIR Difference Spectroscopy . . 42

2.2.6 FTIR Sample Preparation . . . 43

2.3 Electron Paramagnetic Resonance . . . 44

2.3.1 A Single Unpaired Electron in a Magnetic Field . . . 47

2.3.2 An Unpaired Electron in a Molecule - Spin-Orbit Coupling and theg -factor . . . 48

2.3.3 A Quantum Mechanical Formulation of EPR - The Spin Hamiltonian . 50 2.3.3.1 Electron Zeeman Interaction . . . 50

2.3.3.2 Zero-Field Interaction . . . 51

2.3.3.3 Hyperfine Interactions . . . 55

2.3.3.4 Electron Spin-Spin Interactions . . . 56

2.4 Computational Quantum Chemistry . . . 57

2.4.1 The Hartree-Fock Self-Consistent Field Method . . . 58

2.4.1.1 Simplifying the Electronic Schr¨odinger Equation . . . 58

2.4.1.2 The Hartree-Fock Approach . . . 60

2.4.1.3 The Roothaan-Hall Equations . . . 60

2.4.1.4 Selection of Basis Sets and Electron Correlation . . . 62

2.4.2 Post-Hartree-Fock Methods . . . 62

2.4.2.1 Møller-Plesset Perturbation Theory . . . 63

2.4.2.2 Coupled-Cluster Theory . . . 64

2.4.3 Density Functional Theory . . . 65

2.4.4 Overview of Computational Methodology Used in Thesis . . . 67

3 Cryogenic FTIR study of Tyrosine Oxidation in PSII 73 3.1 Introduction . . . 73

3.2 The Redox-Active Tyrosines of PSII, TyrD and TyrZ . . . 73

3.3 Materials and Methods . . . 76

3.4 Results . . . 78

3.4.1 An Unexpected Result: The Light-induced ZnSe FTIR Difference Signal 78 3.4.2 TyrD Oxidation at Cryogenic Temperatures . . . 81

3.4.3 TyrZ Oxidation at Liquid Helium Temperatures . . . 85

3.5 Discussion . . . 92

3.5.1 The Light-Induced ZnSe FTIR Signal . . . 92

3.5.2 Cryogenic TyrD◦FTIR Signals . . . 93

3.5.3 TyrZ Oxidation at Liquid Helium Temperatures . . . 96

4 The Effect of Deprotonation in Manganese-Carboxylate Complexes 105 4.1 Introduction . . . 105

4.2 Methods . . . 107

4.3 Results . . . 110

4.4 Discussion . . . 115

5 Deprotonation of water ligands in V, Cr, Mn, Fe and Co Complexes Reduces Oxidation-driven Carboxylate Ligand Frequency Shifts 125 5.1 Introduction . . . 125

5.2 Methods . . . 127

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5.4 Discussions and Conclusions . . . 142

6 Vibrational Frequency Calculations on Models of the Water-Oxidising Complex 149 6.1 Introduction . . . 149

6.2 Methods . . . 152

6.3 Results . . . 154

6.4 Discussion and Conclusion . . . 163

7 Conclusions 177 7.1 Cryogenic FTIR Study on the Redox-Active Tyrosines of PSII . . . 177

7.2 The Effect of Deprotonation on Metal Oxidation-Induced Carboxylate Frequency Shifts . . . 180

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Car; carotene

ChI; chlorophyll

ChIZD1; ChIacofactor in the PSII reaction centre bound at the periphery of the D1 protein ChIZD2; ChIacofactor in the PSII reaction centre bound at the periphery of the D2 protein Cyt; cytochrome

Cyt b559; the cytochrome b559subunit

Cyt c550; the cytochrome c550subunit

EPR; Electron Paramagnetic Resonance

FTIR; Fourier Transform Infrared

HOMO; Highest Occupied Molecular Orbital

LUMO; Lowest Unoccupied Molecular Orbital

LHCII; major light harvesting complex of PSII

NIR; Near-infrared

P680; primary electron donor of PSII

Pheo; pheophytin

PheoD1; Pheo cofactor in the PSII reaction centre bound to the D1 protein

PheoD2; Pheo cofactor in the PSII reaction centre bound to the D2 protein

PSI; photosystem I

PSII; photosystem II

QA; the tightly bound plastoquinone acceptor of the PSII reaction centre

QB; the loosely bound plastoquinone acceptor of the PSII reaction centre

TyrZ; redox active tyrosine residue of the D1 protein of the PSII reaction centre

TyrD; redox active tyrosine residue of the D2 protein of the PSII reaction centre

WOC; Water Oxidising Complex

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An Introduction to Oxygenic

Photosynthesis

1.1

An Abridged History

Oxygenic photosynthesis is a biochemical process in which energy from the Sun is used to fix

CO2from the atmosphere and transform it into sugars. Water is used a as source of electrons and

protons, while oxygen is released as a byproduct. Sugars derived from the process serve as the

primary source of chemical energy for the majority of life on Earth, while the oxygen byproduct

in turn enables aerobic respiration, a highly efficient method of metabolising the sugars derived

from photosynthesis.1,2

Oxygenic photosynthesis evolved approximately 3 billion years ago in cyanobacteria, when the

Earth’s atmosphere was thought to be mildly reducing and rich in methane (CH4), a potent

greenhouse gas.3–6When oxygenic photosynthesis first evolved, most of the oxygen produced

by the process would be quickly removed by reduced minerals, such as iron deposits on the

planet’s surface. However, as cyanobacteria proliferated, oxygen sinks on the surface of the

planet were eventually overwhelmed, leading to the rapid accumulation of free oxygen in the

atmosphere.

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This rapid accumulation is known as the Great Oxygenation Event, but also known to some as

the Oxygen Catastrophe. Although it would still be approximately 2 billion years before oxygen

levels approached the present atmospheric level, the increased level of oxygenation rendered the

atmosphere toxic to most organisms of the period, including methanogenic bacteria that

main-tained CH4 levels in the atmosphere.7 The elimination of these bacteria, combined with free

oxygen reacting with CH4 to produce CO2 (which is less effective than CH4 as a greenhouse

gas), caused a rapid decrease in global temperatures. This decrease in temperature was so

dras-tic that the entire surface became covered in ice, and the planet was plunged into a Snowball

Earth episode that lasted 300-400 million years.8–10This resulted in one of the most significant

extinction events in Earth’s history.

The availability of free oxygen in the atmosphere, allowed its usage as an electron acceptor

for aerobic respiration, which greatly outstripped its anaerobic predecessor, fermentation, in

efficiency. As the oceans warmed and organisms evolved the ability to respire aerobically, life

diversified rapidly and eventually evolved into multicellular forms.11UV radiation was absorbed

by atmospheric oxygen to generate the ozone layer, which shields life from the most deleterious

effects of the Sun’s radiation. Life has now colonised almost every imaginable environment and

niche on the planet; oxygenic photosynthesis had come full circle, and now serves as the driving

force for the vast majority of life on Earth.

1.2

How does Oxygenic Photosynthesis Work?

All organisms that perform oxygenic photosynthesis today use the same catalyst and cellular

blueprint, which is remarkable considering the innumerable niches and adaptations that life has

encountered on this planet over the last 3 billion years. It is thought that oxygenic

photosyn-thesis evolved only once, in cyanobacteria; algae and plant cells acquired the ability to perform

oxygenic photosynthesis by engulfing cyanobacteria, which eventually became incorporated in

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Oxygenic photosynthesis involves light-driven processes and light-independent, dark processes.

In the light-driven processes, water is broken down into molecular oxygen and protons. The

latter are used to assemble high energy compounds such as ADP (adenosine 5’-triphosphate)

and NADPH (nicotinamide adenine dinucleotide phosphate, reduced form). The dark processes

involve the fixation of CO2into carbohydrates, using the energetic compounds derived from the

light-driven processes. In this thesis, we focus only on aspects of the light-driven processes.

The light-driven processes of oxygenic photosynthesis rely on 4 enzymes in particular:

Photo-systems I and II, Cytochrome b6f and ATP Synthase, which are contained within the bi-lipid

layer of the thylakoid membrane, a closed membrane vesicle located inside cyanobacteria and

chloroplasts. The interior region is known as the lumen, while the exterior region is known as

the stroma (see Figure 1.1).

FIGURE1.1: A schematic representation of the light-dependent processes in photosynthesis; the blue and red arrows in the figure describe the flow of electrons and protons, respectively. This figure was adapted

from the book,Plant Physiology, by L. Taiz and E. Zeiger.

Light energy from the sun is captured by antenna complexes, which transfers excitation energy

to the P680 pigment system in Photosystem II (PSII). P680 then undergoes charge separation,

releasing an electron. This electron is ultimately donated to cytochrome b6f, via a

membrane-bound plastoquinone (PQ) pool. The P700 pigment system in Photosystem I (PS I) too,

under-goes light-induced charge separation, and transfers an electron to NADP to form NADPH. The

electron from PSII eventually reaches PS I via cytochrome b6f and plastocyanin (PC) to reduce

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PSII ultimately obtains its electrons from water, a seemingly unlikely source given that water

is very chemically stable, with a Gibbs free energy of -237 KJ mol−1. In spite of this stability, the Water Oxidising Complex (WOC) in combination with the oxidising potential of P680 is

able to split water into its constituent parts: protons, water and electrons. The electrons from

water reduce P680+back to P680, while protons are accumulated in the lumen, thus generating

a proton gradient across the thylakoid membrane. ATP synthase utilises this proton gradient

to convert ADP to ATP, and the enzyme pumps excess protons out of the lumen and into the

stroma.

1.3

Photosystem II

1.3.1 Overview of Photosystem II

PSII exists in vivo in thylakoid membranes as a homodimer, with each monomer containing at

least 20 protein subunits and nearly 60 organic and inorganic cofactors, including 35 Chl a, 11

carotenoids, two pheophytins and two plastoquinones.13 The primary protein subunits of the

monomer include the membrane spanning polypeptides CP47 (56 kDa), CP43 (52 kDa), D2 (39

kDa) and D1 (38 kDa), and the extrinsic polypeptide PsbO (26.8 kDa, also known as the

Mn-stabilising protein); the total molecular mass of each monomer unit is 350 kDa, thus giving PSII

a total molecular mass of 700 kDa.2,13–17All of the cofactors participating in electron-transfer

and water-splitting are associated with the pseudo-symmetrical dimer composed of the D1 and

D2 proteins, also known as the PSII core complex.

Most of the features associated with the PSII core complex are conserved amongst all

organ-isms performing oxygenic photosynthesis, as inter-organism differences in PSII structure lie

primarily in the light harvesting antennas and also some of the extrinsic, lumenal side

pro-tein subunits.18 The main light-harvesting antenna of higher-plant PSII consists of

membrane-intrinsic trimers of LHCII protein complexes, whereas a light-harvesting system consisting of

membrane-bound phycobilisomes that do not contain chlorophylls is found in cyanobacteria and

red algae.19–21In green algae and higher plant PSII, the lumenal side protein subunits are PsbO,

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hand are PsbO, and CyanoP and CyanoQ, which are homologous to PsbP and PsbQ. In

addi-tion, cyanobacterial PSII also contains PsbU (12 kDa) and PsbV on the lumenal side, with PsbV

being a redox-active heme group that is also known as cyt c550. The precise role of cyt c550 is

still somewhat uncertain, although it is known that it enhances PSII activity by promoting the

binding of PsbO, PsbU, Ca2+and Cl–.22–28

FIGURE 1.2: A diagrammatic representation of the PSII Core Complex. The flow of electrons within PSII is indicated by small red arrows.

As previously mentioned, P680 accepts excitation energy from the antenna complexes and

un-dergoes charge separation; this forms a Pheo◦−P680◦+ charge pair. The separated charges are then stabilised by electron transfer from Pheo◦− to QA, and the rapid reduction of P680◦+ by TyrZ, one of the two redox-active tyrosine residues in PSII. This is then followed by

proton-coupled electron transfer from QA– to QB, and electron donation by the WOC to TyrZ◦.QB converts into a plastoquinol after two such charge separation events, and exchanges into a

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to the lumen.

The WOC can be damaged using a high light flux under physiological conditions, or artificially

removed via chemical treatments. In such cases, it is no longer available as an electron donor,

so secondary electron donors such as TyrD (the second redox-active tyrosine, situated in D2),

the carotenoids (CarD1and CarD2) chlorophylls (ChlZD1and ChlZD2) and cytochrome b559(Cyt

b559) act as electron donors to P680+ instead. The rapid reduction of the P680+ radical is

important, as it has the highest known oxidative potential in nature (∼1.2V) and would cause

damage to the enzyme otherwise.29

1.3.2 The Water Oxidising Complex

The WOC is embedded within a pocket formed by residues of D1 and CP43 in the lumenal

sur-face of thylakoid membrane.17The catalytic centre of the WOC is an organometallic Mn4CaO5

cluster, composed of a distorted cubane core made up of three Mn ions (Mn1 – Mn3), four

oxy-gen atoms (O1 – O3 and O5) and a Ca2+ion, with a fourth “dangling” Mn ion (Mn4) connected

to the core by twoµ-oxo-bridges, O4 and O5. The recently-obtained 1.95 ˚A resolution X-ray diffraction (XRD) structure of the WOC is shown in Figure 1.3.30 All oxo groups and metal

centres of the WOC becomes resolved, and the femtosecond X-ray free electron laser (XFEL)

technique used in obtaining this structure also precludes the possibility of X-ray-induced

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FIGURE 1.3: The 1.95 ˚A X-ray diffraction (XRD) structure of the WOC, Protein Data Bank (PDB) ID 4UB8.30The atoms are coloured Mn: purple, Ca: green, C: dark grey, O: red, N: blue. The carboxylate

ligands were contracted to acetates, while all oxygen atoms that do not directly bind to metal centres were omitted for clarity.

The first-shell amino acid cofactors to the Mn and Ca ions of the WOC are six carboxylates

(D1-D170, CP43-E354, D1-E333, D1-E189, D1-D342, D1-A344) and one histidine residue

(D1-His332), while four terminal waters bind to Mn4 (W1, W2) and the Ca2+ ion (W3, W4).

TyrZ (not shown) lies within the second shell of ligands to the WOC and weakly hydrogen bonds

one of the Ca-ligating waters (W4). Many of the remaining second-shell residues (and beyond)

participate in extended hydrogen bond networks; it is known that there are at least three such

networks that extend from the WOC into the lumen, and are thus likely to serve as water and/or

proton channels.32,33In some proposals, TyrZ is also thought to participate in a proton egress

pathway from the WOC.34,35

The water oxidation reaction itself is a 4-electron process, in which two water molecules are

simultaneously oxidised and split into molecular oxygen and protons. To couple the

single-electron processes of PSII to the 4-single-electron water-splitting reaction, the WOC undergoes

step-wise oxidation, and cycles through 5 different oxidation states dubbed S0, S1, S2, S3 and S4,

i.e. S-States, where the subscript denotes the oxidative equivalents stored by the WOC. When

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of dioxygen occurs in a single concerted step, and the WOC undergoes a 4-electron reduction

to revert to the S0 state. To maintain the redox potential of the Mn4CaO5 cluster throughout

the S-state cycle, such that it does not prevent subsequent oxidation by TyrZ◦, proton release to prevent an accumulation of charge is necessary. Photothermal beam deflection (PBD) and

Fourier-transform infrared (FTIR) spectroscopy experiments strongly suggest that throughout

the S-state cycle, electron transfer and proton removal occur in strictly alternating steps, with

the S1 to S2 transition being the only step not accompanied by proton release.36,37The S-state

cycle including electron and proton removal steps from the WOC is shown in Figure 1.4.

FIGURE 1.4: The extended S-state cycle from the work by Klauss and coworkers.36 A classical Kok

model is shown in the inner circle, while the extended model that includes S-state intermediate states as well as electron and proton removal kinetics is shown on the outer circle.

The water splitting reaction in the WOC utilises an oxidative potential very close to the

ther-modynamic limit required to oxidise water.38This feature also avoids the generation of reactive

intermediates such as hydrogen peroxide. While a broad understanding of the WOC’s function

has been attained, the underlying mechanism by which water is split remains unresolved, as

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1.3.2.1 Ambiguities in the Geometry and Electronic State of the Water Oxidising Com-plex

In dark-adapted PSII, the S1state is the natural resting state of the WOC. Thus, the 1.95 ˚A XRD

structure shown in Figure 1.3 was assumed to correspond to the S1state.30,31However, some of

the Mn-Mn distances in the XRD structure, particularly the Mn3–Mn4 vector, are longer than

those indicated by extended X-ray absorption fine structure (EXAFS) experiments on samples

poised in the S1 state.39–42 To explain this discrepancy, Askerkaet al. have suggested that the

XRD structure represents a mixture of S0and S1, whereas Petrieet al.proposed that the WOC

undergoes a tautomerisation that is able to explain both sets of observed Mn-Mn distances.40,41

This apparent contradiction also fuels a long-running debate on the two plausible electronic state

configurations for the WOC, that is, whether the WOC has a mean Mn oxidation state of 3.5 or

3 in the S1state – these are the so-called “high” and “low” oxidation paradigms.2,43–46

A related point of contention is the protonation state of O5. This oxygen occupies a central

location within the Mn4CaO5cluster, and exhibits abnormally long Mn-O distances in the XRD

structure; these elongated bonds are likely, the direct cause of the discrepancy between XRD

and EXAFS data. While electron spin echo envelope modulation (ESEEM) and electron

nu-clear double resonance (ENDOR) data suggest that O5 is fully deprotonated in S1 (indirectly

via studies on the S2 multiline – the lack of deprotonation in the S1 to S2 transition implies

that O5 is already deprotonated in S1),42,47computational models consistent with EXAFS data

that incorporate O5 as a fully-deprotonated oxo-bridge in the S1 state are generally unable to

reproduce the Mn-O distances observed in the XRD structure.41,48–50As previously mentioned,

it has been suggested that the XRD structure is a superposition of S0 and S1; following that,

it was also proposed that O5 is a hydroxide in S0 that undergoes deprotonation in the S0 to S1

transition. This would explain the long O5 bonding distances observed in the XRD structure.

The opposing viewpoint is that O5 isnotfully deprotonated in S1. Sugaet al.have proposed that

O5 is a hydroxide in S1, while Petrieet al. argue that O5 could either be a water or hydroxide,

depending on the tautomeric state of the WOC.30,41,51 This particular viewpoint is supported

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S1 transition is unlikely, because O4 has a “pre-organised” proton transfer chain that makes it

the more likely candidate for deprotonation.42If that is indeed the case, then any superposition

of S0 and S1 in the XRD structure may not cause a significant elongation of O5 bond lengths

relative to the “true” S1bonding distances.

The electronic state of the WOC and the protonation state of O5 are but two of the many issues

that must be resolved for an accurate portrayal of the water splitting mechanism within the

WOC. In this thesis, these issues are indirectly touched upon by IR frequency calculations on

model complexes based on the WOC. We have found that the results of these calculations were

dependent on the electronic state of the WOC, as well as the protonation state of O5.

1.4

The Aims of this Thesis

Spectroscopic and computational approaches were applied in investigations of the redox-active

tyrosines (TyrZ and TyrD) and the WOC, respectively. We sought to study the redox reactions

of TyrZ and TyrD using a new FTIR-based approach at cryogenic temperatures, to provide

com-plementary data to current studies that are primarily based on EPR spectroscopy. As a second

component, using computational chemistry, we calculated the vibrational frequencies and

en-ergies of various model Mn complexes based on the WOC, with and without deprotonation of

water/hydroxo ligands, to help explain key aspects of the FTIR data in the literature. This

com-putational study was also extended to examine the effects of simultaneous deprotonation with

oxidation in metal complexes based on other first-row transition metals.

1.4.1 Studying Tyrosine Oxidation at Cryogenic Temperatures using FTIR

PSII contains two redox active tyrosines called TyrZ and TyrD, that occupy homologous

posi-tions in D1 and D2, respectively. TyrZ and TyrD share a number of structural similarities, as

they are equally situated relative to the two central chlorophylls, PD1and PD2and form hydro-gen bonding pairs with a histidine residue (TyrZ with D1-His190 and TyrD with D2-His189, in

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on the S-state of the WOC, while the TyrD◦ radical is stable for hours.53This difference in ki-netics is necessary for the role that each tyrosine plays in PSII, as the fast redox kiki-netics of TyrZ

are necessary to enable rapid electron transfer between P680+and the WOC, while it has been

proposed that the stable TyrD◦radical may increase the potential of P680+through electrostatic effects.54,55

Given that tyrosines function as electron transfer intermediates in a wide range of biological

enzymes, it would be of great utility to learn how the protein environment in PSII modulates the

kinetic properties of TyrD and TyrZ; such knowledge may one day be applied in the design of

artificial photosynthetic systems.

1.4.1.1 TyrD

The paramagnetic signal arising from the TyrD◦ state was one of the first EPR signals ever detected from chloroplasts;56 consequently, it is one of the most thoroughly studied systems

within PSII. This is enabled by the stability of the TyrD◦ state, which only decays slowly via electron donation from the WOC in the S0 state.54,57TyrD appears to play a regulatory role in

the oxidation state of the WOC, as S3and S2may be reduced by TyrD to S2and S1respectively,

while overreduced states (e.g. S−1) can be oxidised by TyrD◦.58,59This feature of TyrD may be relevant in the oxidative assembly of the Mn4CaO5cluster.60However, TyrD does not appear to

be essential for PSII function, as the redox reactions of PSII are not affected by the replacement

of TyrD with Phe.57,61A notable reduction in the rate and quantum efficiency of WOC assembly

was observed, nonetheless.60

The oxidation of TyrD by P680+involves the concomitant loss of a proton (i.e. proton-coupled

electron transfer (PCET)), which leads to TyrD◦a neutral oxidation product.62TyrD oxidation is also strongly pH dependent; it occurs with t1/2>150µs at pH 6.5, but becomes much faster at pH 8.5, with a t1/2of∼190 ns.52 This pH-dependent behaviour titrates with a pKa of 7.7,

suggesting that the kinetics of TyrD oxidation are governed by the protonation state of TyrD

or some neighbouring residue. FTIR studies however, indicate that it is not TyrD or its

(24)

pH, TyrD was able to undergo oxidation at liquid helium temperatures, which came as a surprise

since it was commonly assumed that electron transfer from the tyrosines was thermally inhibited

at∼250 K.64Furthermore, it was found that the TyrD◦radical formed at liquid helium temper-atures is highly electropositive and undergoes thermal relaxation at 77 K and above, suggesting

that some intermediate TyrD◦state is formed at ultralow temperatures.65

Saitoet al.have proposed that the oxidation of TyrD occurs through proton transfer from TyrD

to D2-Arg180, via a mobile water that functions as a proton carrier; the proton that was removed

from TyrD then travels along a chain of hydrogen-bonded waters to D2-His61, and that the chain

may continue further into bulk water.66This is supported by a recent FTIR study by Nakamura

et al., who have found that a proton is indeed, released into the bulk upon TyrD oxidation.67

While the QM/MM study by Saitoet al. has not been fully extended to cover the high pH

sce-nario, the authors proposed that it is the mobile water that undergoes deprotonation, which leads

to TyrD donating a H-bond to D2-His189 instead of the mobile water; this is a reversal of the

low pH scenario, where TyrD accepts a H-bond from D2-His189.66The resulting configuration

is then similar to that observed for the TyrZ system, where a strong H-bond also exists between

TyrZ and D1-His190.35,53 The proton egress pathway for TyrD in the high pH configuration is

remains uncertain.

As for the highly-electropositive TyrD◦ radical that is formed at liquid helium temperatures, O’Malley and Hart have proposed that it is an imidazolium complex, having found a good fit

between QM/MM simulations of an imidazolium complex and the experimental EPR data.68

Ultralow temperature FTIR experiments would enable a comparison to be made between the

in-termediate and relaxed states of the TyrD◦radical. It would be easy to verify whether the inter-mediate is indeed, an imidazolium complex, as they exhibit substantial IR absorption differences

compared to imidazole complexes.69It would also be interesting to observe the TyrD◦/TyrD IR difference spectra at cryogenic temperatures, since all protein and water movements are

ther-mally inhibited – in particular, the water proposed to act as a mobile proton carrier for TyrD

should be immobilised. The resulting difference spectrum is therefore likely to be different

from the well-known room temperature signal, thus providing an opportunity for novel research

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1.4.1.2 TyrZ

TyrZ has the fastest proton coupled electron transfer that occurs in photosynthesis, with the

pro-ton shifting within the hydrogen bond in ∼20 ns.53,70 The reduction of TyrZ◦ by the WOC is somewhat slower and occurs in the microseconds to milliseconds time scale, but nevertheless,

the very fast kinetics of TyrZ make it a difficult system to study. TyrZ redox reactions are

gen-erally slower in the absence of the Mn4CaO5 cluster, but this also alters the bonding of TyrZ,

causing it to interact with several hydrogen bond acceptors instead of having only one well

de-fined hydrogen bond to D1-His190.53

TyrZ oxidation by P680+ is multiphasic, occurring dominantly with nanosecond kinetics but

having an additional microsecond component in intact systems.53,70,71 Oxidation is fastest in

the S0 and S1 states, where the dominant fast kinetic phase has a half time of ∼20 ns. Like

TyrD, TyrZ oxidation kinetics are also pH-dependent, as the fast phase decreases both in rate

and amplitude under acidic conditions (pKaof 4.5-5.3), but is essentially constant from pH 5.5

to 8.0.72,73In the absence of the Mn4CaO5cluster, TyrZ oxidation becomes even more strongly

pH dependent; the electron donation from TyrZ rate increases at high pH with a pKa6.9-8.3,

and is reduced a hundred-fold at low pH.74–77The currently preferred mechanism of PCET at

TyrZ is a so-called “ proton-rocking” model, where the phenolic proton of TyrZ is transferred to

D1-His190 upon its oxidation by P680+via a strong H-bond, and transferred back upon TyrZ

reduction by the WOC.35,53

Following the discovery of cryogenic TyrD oxidation, it was soon shown that TyrZ could also

be undergo oxidation at liquid helium temperatures.78,79Under these conditions, the TyrZ◦ dis-plays illumination-dependent kinetics; if it is induced using visible light (400-700 nm), the

TyrZ◦ radical decays within minutes. However, if it is induced using near-infrared light (720-780 nm) in samples poised in higher S-states (S2and S3), it becomes indefinitely stable.80–82It

is then possible to study TyrZ◦in intact systems using “slower” spectroscopies such as EPR and FTIR. Currently, all ultralow temperature studies have been performed exclusively using EPR.

(26)

Parallel FTIR experiments at ultralow temperatures may provide further information on the

in-teraction between the WOC and TyrZ, and perhaps by proxy, structural information on the WOC.

1.4.2 Interpreting WOC FTIR Data through Model Complex Calculations

FTIR difference spectroscopy has been extensively employed to probe the S-states of the WOC,

beginning as early as 1992.85 By 2001, complete sets of mid-frequency (1000 – 2000 cm−1) Sn+1-minus-SnFTIR difference spectra had been reported. These spectra contained a wealth of

spectral features; on the basis of samples labeled with13C or15N, a number of features between

1500 and 1700 cm−1 were assigned to amide I and amide II modes, with the remaining modes being assigned to asymmetric carboxylate stretches. Features appearing between 1300 and 1450

cm−1 were assigned to symmetric carboxylate stretches.13,86 Samples grown with13C-alanine resulted in shifts in the carboxylate symmetric stretching region of the S2/S1 difference

spec-trum, and enabled the assignment of these shifted features to D1-Ala344, a carboxylate residue

that binds Mn2 of the WOC (see Figure 1.3).87 A feature near 1113–1114 cm−1 was also as-signed to the C-N stretching mode of the Mn1-ligating histidine, D1-His332. This feature is

positive in S1/S0, negative in S2/S1and S3/S2 spectra, and absent from the S0/S3 spectrum – it

was thus concluded the vibrational mode changes that occur during the S0 to S1 transition are

reversed during the S1to S2and S2to S3transitions.13,88

Based on the 1.9 ˚A and 1.95 ˚A X-ray structures (PDB ID 3ARC and 4BU8, respectively), the

WOC has six direct (first-shell) carboxylate ligands, namely Asp170, CP43-Glu354,

D1-Glu189, D1-Glu333, D1-Asp342 and D1-Ala344 (see Figure 6.1).30,34In an effort to assign the

difference signals of the remaining carboxylate ligands of the WOC, a number of PSII mutants

with mutated WOC ligands have been studied with FTIR difference spectroscopy.

Unexpect-edly, many mutations to first-shell ligands produced S-State difference spectra that were all but

identical to the wildtype within the mid-frequency region (1000 — 2000 cm−1), with the ex-ception of CP43-Glu354, leading to some confusion whether the ligands targeted for mutation

(27)

altered by the mutations.90–92

To explain these predominantly null results, one suggestion is that the deprotonation of water

or hydroxo ligands suppresses oxidation-driven shifts in the carboxylate stretching modes of the

WOC’s first-shell ligands, rendering these shifts too difficult to detect in conventional FTIR

ex-periments. In this thesis, we study the vibrational frequency changes of a number of manganese

and transition metal complexes with carboxylate ligation upon simultaneous deprotonation with

oxidation. In the final chapter of this thesis, we report the vibrational frequencies of WOC

models proposed by various authors, and monitored the effect of deprotonation on the WOC’s

(28)
(29)

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Materials and Methods

In this chapter, we describe some aspects of the experimental methods and materials used in the

research conducted for this thesis, particularly those concerning the spectroscopic study of PSII.

A brief theoretical background on the spectroscopic and computational methods employed in

this thesis is also given here.

2.1

PSII Samples

The spectroscopic studies in this thesis were performed on purified PSII core and membrane

samples, isolated from spinach or cyanobacteria (T.vulcanus). The preparation of these samples

is a highly demanding task in itself, and I would like to sincerely express my gratitude to Dr.

Paul Smith and Professor Jian-Ren Shen, for providing the spinach andT.vulcanusPSII cores

used in this study. Some of the PSII membrane material used in this study were prepared by

myself. The isolation procedure for PSII membranes is described below.

2.1.1 Isolation of PSII Membranes from Spinach

The isolation procedure used in this work is from the PhD thesis of Dr. Paul Smith, which is a

modified version of the BBY procedure,1and is also briefly outlined in the work by Smith et al.2

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The spinach used for this preparation was obtained from local markets. On the night prior to

the isolation of PSII membranes, the plants were kept in the dark and at 4 – 5◦C. This causes the plant to consume the starch that is stored in the leaves, and reduces the amount of starch

contaminating the PSII sample. The major vein along the abaxial surface of the leaf was also

re-moved, as it makes it more difficult to blend the leaves. The de-veined leaves were then washed

in double-distilled water, and wrapped in fresh paper towels wetted with distilled water to

main-tain leaf turgor.

Five different buffers were used in this procedure, the compositions of which are detailed below:

1. Buffer 1, blending buffer, pH 7.5: 0.1 M sucrose, 0.05 M KH2PO4, 0.05 M Na2HPO4, 0.2

M NaCl, Bovine Serum Albumin (BSA) added to a content of 2 g L−1

2. Buffer 2, wash/incubation buffer, pH 6.0: 0.4 M sucrose, 0.015 M NaCl, 0.005 M MgCl2,

0.05 M 2-[N-Morpholino] ethane sulfonic acid (MES), 0.005 M ethylenediaminetetraacetic

acid (EDTA) 0.005 M, BSA added to a content of 2.5 g L−1

3. Buffer 3, solubilisation buffer, pH 6.0: The same composition of Buffer 2, with 0.2 kg

L−1of Triton X-100 detergent.

4. Buffer 4, post-detergent wash buffer, pH 6.0: 0.05 M NaCl, 0.005 M MgSO4, 0.05 M

MES

5. Buffer 5, storage buffer, pH 6.0: 0.4 M sucrose, 0.015 M NaCl, 0.01 M MgCl2, 0.02 M

MES

The preparation method described is based on a 100 g sample of spinach leaves.

1. Sets of de-veined, washed leaves were shredded by hand and loaded into a blender, in 50 g

sets. 100 mL of Buffer 1 was added for each 50 g set, and the leaves were blended at high

speed for∼15 seconds. The homogenate was loaded onto filtration material, consisting

of one layer of cheesecloth and 4 layers of muslin. After the blending the second 50 g

set of leaves, 50 mL of Buffer 1 was added to rinse excess homogenate, and this was also

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2. The filter material was parceled around the homogenate and squeezed by hand, to facilitate

drainage of the homogenate (very little of the homogenate drains without mechanical

assistance). The filtrate was then centrifuged at 6500 rpm (5000 x g) in an SS34 rotor for

10 minutes.

3. The supernatant was decanted and discarded, the pellet homogenised in Buffer 2. The

homogenised pellet was centrifuged at 6500 rpm in an SS34 rotor again, for 10 minutes.

4. The supernatant was discarded once more, and the pellet was homogenised in Buffer 2

again to obtain a concentration of approximately 4 mg Chl mL−1. The homogenate was left in the dark on ice for an hour, to allow the thylakoid membrains to undergo stacking

and lateral segregation into granal (PSII containing) and stromal regions, without any

mechanical agitation. Note that steps 1 to 4 (up to the beginning of dark incubation) were

undertaken quickly (within 75 minutes) to minimise degradation of the PSII material.

5. After the period of dark incubation, Buffer 3 was added to obtain a Triton X-100 detergent

to ratio of 20:1 w:w. The solubilised thylakoid suspension was rapidly stirred to enhance

mixing and centrifuged at 18300 rpm (35000 x g) for 30 minutes. The contact time of

Triton X-100 in the thylakoid suspension is typically less than 2 minutes. The detergent

solubilises the single membrane layer (stromal) regions and the outer membrane of the

granal multi-lamellar regions. The granal membranes reseal, forming inverted thylakoidal

micelles.

6. The supernatant was discarded, leaving a pellet containing the granal membrane regions

and starch from within the thylakoid. The pellet was re-homogenised in Buffer 4 and

centrifuged at 9000 rpm (9700 x g) for 1 minute; the resulting pellet, which consists of≥

90% starch was discarded. The supernatant was transferred to a clean centrifuge tube and

centrifuged at 18300 rpm for 30 minutes.

7. The supernatant following the final centrifugation was discarded, and the pellet suspended

and partially homogenised in the centrifuge tube using minimal amounts of Buffer 5

(typi-cally∼0.5 mL). The concentration of the homogenate is redetermined, typically yielding

a concentration of∼15 – 18 mg Chl mL−1. It is important to maintain a high concentra-tion of PSII, if the membrane sample is to be used for the isolaconcentra-tion of PSII core samples.

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2.2

Infrared Spectroscopy

2.2.1 Principles of Infrared Spectroscopy

Since molecules are never at rest relative to each other, this gives rise, in a general sense, to

vi-brational modes. A non-linear molecule with N atoms has 3N-6 degrees of freedom, which gives

the number of ways that the atoms may vibrate, i.e. the number of vibrational modes. At room

temperatures, a vibration may be approximated as a harmonic oscillator, with a characteristic

frequency,vi. The vibrational energy states,Vivmay then be described by the equation:

Viv=hvi(νi+

1

2) (2.1)

wherehis Planck’s constant,vithe characteristic frequency of the mode, andνithe vibrational

quantum number of theith mode (νi=0,1,2,...). The energy difference between the ground state

(νi=0) and the first excited state (νi=1) usually corresponds to the energy of radiation in the

mid-infrared, 400-4000 cm−1 range. However, a vibrational mode is only infrared-active (i.e. being able to absorb light) if the dipole moment of the molecule changes during the vibration,

as the absorption strength of a vibrational mode is proportional to the change in dipole moment.

In many cases however, vibrational motions are not strictly harmonic, and must be described by

an anharmonic potential function instead. Equation 2.1 then becomes:

Viv=hvi(νi+

1

2) +hvixi(νi+ 1 2)

2 (2.2)

wherexi is the anharmonicity constant. xi is dimensionless and may vary between -0.001 and

-0.02, depending on the mode. The effect of anharmonicity is to allow absorption bands other

than those that correspond to fundemental transitions (∆νi=1), such as overtone (∆νi=2,3,...)

and combination (∆νi=1;∆νj=1, wherejrepresents a different mode) bands. These bands tend

to absorb more weakly in the mid-infrared spectrum, compared to fundamental bands.

Addi-tionally, Fermi resonance may occur if two vibrational bands have nearly the same energy and

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vibration together with an overtone or combination band. The effect of this interaction is that

the band intensities are “equalised”, and the energy (i.e. frequency) splitting between the two

bands increases. In this way, overtone or combination bands can also show significant intensity

in IR spectra.

While vibrational motions in principle, involve all atoms of a molecule, many modes only give

rise to large displacements in a few atoms while leaving the rest of the molecule almost

sta-tionary, and may thus be approximated as ‘local’ modes that arise due to the vibrations of some

functional group. This allows for correlations to be drawn between infrared spectra and the

presence of certain chemical groups, e.g. the carbonyl C=O stretching vibration absorbs in the

1665-1760 cm−1region. Extensive spectra-structure correlation tables (often known as Colthup charts) have been developed to allow the absorption bands in a given infrared spectrum to be

assigned to vibrational mode(s) associated with a certain functional group.

Stretching vibrations arising from polar groups, e.g. C=O, are the strongest absorbing modes

in the mid-infrared region. Assuming that the vibration is mostly harmonic, the frequency of a

stretching mode,ν, is given by:

ν= 1 2πc

s

k(m1+m2)

m1m2

(2.3)

wherekis the force constant of the molecular bond, withm1andm2the masses of the two atoms participating in the stretching vibrations. Consequently, a vibrational frequency is a direct

mea-sure of the molecular bond strength,k, which is in turn sensitive to the chemical environment in which the group lies in. It may be altered by electrostatic effects, hydrogen bonding interactions,

or molecular orbital effects, e.g. electron back-donation from a metal centre into anti-bonding

orbitals. The vibrational frequency also depends on the atomic masses of the participant atoms,

so it is possible to chemically alter a vibrational frequency through isotopic labeling. For

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2.2.2 The Effect of Environmental Interactions on IR Absorptions

In the following section, the effects of environmental interactions commonly found in proteins

on vibrational mode frequencies are briefly discussed.

2.2.2.1 Hydrogen Bonding

Hydrogen bonds are ubiquitous in proteins, and play important roles in catalytic reactions and

the stabilisation of protein structure. These bonds have binding energies that range from 4 to

50 kJ mol−1, much weaker than a covalent bond, but stronger than most other intermolecular forces.4Disordered and extended networks of hydrogen bonds form in liquids such as water and

alcohol, which gives rise to many of these liquids’ unique chemical and physical properties.

In the context of mid-IR spectroscopy, hydrogen bonding usually causes frequency downshifts in

stretching modes, often accompanied by substantial spectral broadening, as well as an increase

in absorption intensity. The downshift (typically∼20 cm−1for a single hydrogen bond to a CO group5) is due to the weakening of covalent bonds associated with the hydrogen-bonded atom,

and increased vibrational mode anharmonicity.4The downshift generally reflects on the

hydro-gen bonding strength.6,7 Hydrogen bond-induced spectral broadening has a number of likely

causes, such as anharmonic coupling of the high-frequency stretching mode to low-frequency

modes, Fermi resonances with overtone and combination tone levels of fingerprint modes,

vibra-tional dephasing, and inhomogenous broadening due to different hydrogen bonding geometries

in the molecular ensemble. The increased absorption intensity is caused by changes in the

elec-tronic structure.4

In contrast to stretching modes, bending modes are slightly up-shifted upon hydrogen bonding,

usually without significant changes in the lineshape.4This is due to the hydrogen bond providing

Figure

FIGUREtransitions have been plotted as a function of the rhombicitym 2.11: Rhombogram for an S = 5/2 system
FIGUREtion 1 (Red): A – B – C, Subtraction 2 (Black): A – B + D. The peaks that are present in both spectra are 3.6: TyrD◦/TyrD at 80 K and 10 K, obtained using two different spectral subtractions
TABLE 4.2: Frequency shifts of the carboxylate stretching modes of B upon oxidation (cm−1)
TABLE 6.1: The oxidation state, formal charge, multiplicity and the identity of O5, W2 and O3 in eachof the clusters investigated in this study, in their initial state.

References

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