Time-resolved Structural and Mechanistic Studies of Water Oxidation in Photosystem II
Water here, water there, water everywhere
Casper de Lichtenberg
Department of Chemistry Umeå 2020
Detta verk är skyddat av svensk upphovsrätt (Lag 1960:729) Avhandling för filosofie doktorsexamen
ISBN: 978-91-7855-344-0
Energy landscape of Ws exchange in the S2 state of photosystem II. First published as inside front cover in PCCP, 2020,22, 12834-12834.
Elektronisk version tillgänglig på: http://umu.diva-portal.org/
Tryck: Print & Media, Umeå Universitet Umeå, Sverige 2020
Til min søn Balder, du er mit livs lys og til Lisa for at bringe lyset ind i mit liv
Jeg elsker jer meget højt
The second son of Odin is Baldur, and good things are to be said of him.
He is best, and all praise him;
he is so fair of feature, and so bright, that light shines from him.
Gylfaginning XXII, Brodeur's translation
i
List of papers ... iii
Author contributions ... v
Abstract ... vi
Abbreviations ... viii
Enkel sammanfattning på svensk ... x
Introduction / Background ... 1
Learning to survive on sunshine and water ... 1
The first step in photosynthesis ... 2
Photosystem II ... 4
The structure the Mn4Ca-cluster ... 7
The EPR spectroscopic signatures of the Kok-cycle ... 10
Substrate waters of the Kok-cycle ... 14
Materials and Methods ... 20
List of buffers: ... 20
Mutagenesis of PSII in Synechocystis sp. PCC 6803 ... 21
Preparation of PSIIcc from Synechocystis PCC. 6803 ... 22
Cell culturing ... 22
Isolation of thylakoid membranes ... 22
Purification of PSIIcc ... 23
Time-resolved membrane-inlet mass spectrometry ... 24
Sample preparation ... 26
Estimation of injection and mixing time ... 27
Correction of signal artifacts from H218O-injection ... 28
Kinetic fits of the TR-MIMS data ... 30
Considerations about substrate water exchange from the Mn4Ca-cluster ... 32
Electron paramagnetic resonance (EPR) spectroscopy ... 33
General introduction to EPR ... 33
CW-EPR measurements of PSIIcc ... 34
17O ELDOR-detected NMR ... 35
Measurement of nuclear transitions with pulsed EPR ... 35
Sample preparation by rapid freeze quench ... 36
EDNMR measurements ... 38
PSII serial crystallography and X-ray emission spectroscopy ... 39
Crystallization of PSII ... 39
Determination of crystal turnover efficiency ... 39
Droplet on tape - Ensuring consistent sample delivery and illumination ... 39
XFEL data processing ... 42
Results and Discussion ... 44
What are the differences between the PSII structure at cryogenic temperatures and the structure of PSII at room temperature? How does the structure of the Mn4Ca-cluster change between the individual semi-stable intermediate states of the Kok cycle? (Papers I & II) ... 45
Results and Discussion ... 45
Summary ... 48
What is the sequence of the structural and oxidative changes in the S2 to S3 transition?
(Paper III) ... 50
Results and Discussion ... 50
Summary ...55
Is substrate binding different in the two conformers of the S2-state? (Paper IV) ... 56
Results and discussion ... 56
Summary ... 62
What are the effects of the D1- V185N mutation on the Mn4Ca-cluster and substrate binding? (Paper V) ... 63
Results and discussion ... 64
Summary ... 69
How are the slow and fast exchanging waters bound in the S2-state? Lessons from the D61A and E189Q mutants (Paper VI) ... 70
Results and discussion ... 71
Summary ... 75
Can WS be assigned with a combination of TR-MIMS and freeze quench 17O-EDNMR? (Paper VII) ...76
Results and Discussion ...76
Summary ...79
Conclusion ... 82
Future experiments ... 84
Acknowledgements... 85
References ... 90
iii
List of papers
This thesis is based on the following papers and manuscripts I. Young, I., Ibrahim, M., Chatterjee, R. et al.
Structure of photosystem II and substrate binding at room temperature.
Nature 540, 453–457 (2016).
II. Kern, J., Chatterjee, R., Young, I.D. et al.
Structures of the intermediates of Kok’s photosynthetic water oxidation clock.
Nature 563, 421–425 (2018).
III. Ibrahim, M., Fransson, T., Chatterjee, R., Cheah, M. H. et al.
Untangling the sequence of events during the S2 → S3 transition in photosystem II and implications for the water oxidation mechanism
Proc. Natl. Acad. Sci. U.S.A., May, 1-12 (2020) IV. de Lichtenberg, C. & Messinger, J.
Substrate water exchange in the S2-state of photosystem II is dependent on the conformation of the Mn4Ca cluster Phys. Chem. Chem. Phys. 129, 13421–13435 (2020)
V. de Lichtenberg, C., Avramov, A. P., Zhang, M., Mamedov, F., Burnap, R. L., Messigner, J.
The D1-V185N mutation alters substrate water exchange by stabilizing alternative structures of the Mn4Ca-cluster in photosystem II
Manuscript in review
VI. de Lichtenberg, C. Kim, C. J., Debus, R. J., Messinger, J.
Effects of the D61A and E189Q mutations on the exchange of substrate water in photosystem II
Manuscript in preparation
VII. de Lichtenberg, C., Rapatskiy, L., Reus, M., Heyno, E., Schnegg, A., Novaczyk, M. M, Lubitz, W., Messinger, J. & Cox, N.
Assignment of the slow exchanging substrate water of Nature’s water splitting cofactor
Manuscript in preparation
List of papers not presented in the thesis
In addition to the manuscripts listed above, I am co-author on the following manuscripts that were published during my PhD as part of my thesis work (or are still in preparation), but are not covered here as individual chapters. Even so, these are highly relevant to the work presented here and the results obtained in these papers/manuscript have influenced the conclusions or are extended discussions of the work that I present.
Fuller, F., Gul, S., Chatterjee, R. et al.
Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.
Nat. Methods 14, 443–449 (2017).
Chatterjee, R., Lassalle, L., Gul, S., Fuller, F.D., Young, I.D., Ibrahim, M., de Lichtenberg, C., Cheah, M.H., Zouni, A., Messinger, J., Yachandra, V.K., Kern, J.
and Yano, J.,
Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?
Physiol. Plantarum, 166: 60-72 (2019).
de Lichtenberg, C., Pham, L. V., Walter, K., Liang, F., Kim, C. J., Magnusson, A., Ho, F., Debus, R., Lindblad, P., Messinger, J.
Is exchange rate of the fast substrate water in the S2 state of photosystem II limited by diffusion through the water channels ? Lessons from mutations in the O1 channel
Manuscript in preparation
v
Author contributions
I. The author assisted with data collection during beam times (LCLS) and performed control experiments of sample turnover outside beamtimes. Additionally the author participated in discussions regarding the experiment and provided input and comments to the manuscript
II. The author assisted with data collection during beam times (LCLS) and performed control experiments of sample turnover outside beamtimes. Additionally the author participated in discussions regarding the experiment and provided input and comments to the manuscripts.
III. The author assisted with data collection during beam times (LCLS &
SACLA) and performed control experiments of sample turnover outside beamtimes. Additionally the author participated in discussions regarding the experiment and provided input and comments to the manuscript.
IV. The author performed the experiments, analyzed the data, prepared most of the figures and participated in writing of the manuscript.
V. The author devised the experimental strategy, performed the experiments, analyzed the data, prepared all figures and wrote the manuscript with input from the other authors.
VI. The author concieved and planned the project, participated in isolation of the samples, performed the experiments, analyzed the data, made the figures and written the manuscript with input from the other authors.
VII. The author performed the TR-MIMS experiments, assisted in developing the sample preparation protocol for the EDNMR freeze quench, assisted in preparation of samples for the 17O-EDNMR and performed some of the measurements at the Max Planck Institute in Mülheim. The author also analyzed the TR-MIMS data, prepared a figure and participated in writing the manuscript.
Abstract
Oxygenic photosynthesis is undisputedly one of the most important chemical processes for human life on earth as it not only fills the atmosphere with the oxygen that we need to breathe, but also sustains the accumulation of biomass, which is not only used as nourishment but is also present in almost every aspect of our lives as building material, textiles in clothes and furniture, or even as living decorations to name a few.
The photosynthetic water-splitting mechanism is catalyzed by a water:plastoquinone oxido-reductase by the name of photosystem II (PSII), which is embedded in the thylakoid membranes of plants, algae and cyanobacteria. As it is excited by light, charge separation occurs in the reaction center of the protein and an electron is extracted by oxidation of Mn4Ca-cluster, that constitutes the active site for the water splitting reaction in PSII. When the Mn4Ca-cluster has been oxidized 4 times, it forms an oxygen-oxygen bond between two water derived ligands bound to the Mn4Ca-cluster and returns to the lowest oxidation state of the catalytic cycle. Understanding what ligands of the cluster that are used in the water splitting reaction is the key to unlocking the underlying chemical mechanism.
In this thesis I describe investigations, with room temperature X-ray diffraction (XRD) and X-ray emission spectroscopy (XES) on PSII microcrystals, of how the active site looks in all the stable intermediate oxidation states. Furthermore I describe how we uncovered the sequence of events that lead to insertion of an additional water ligand in the S2-S3 state transition of the catalytic cycle.
Furthermore, through time-resolved membrane-inlet mass spectrometry (TR- MIMS) measurements of the isotopic equilibration of the substrate waters with the bulk in conditions that induce different electron magnetic resonance (EPR) spectroscopic signatures, I present evidence that the exchange of the slowly exchanging substrate water Ws is controlled by a dynamic equilibrium between conformations in the S2-state that give rise to either the low-spin multiline (LS- ML) signal or the high-spin (HS) signal. Based on the crystal structures and litterature suggestions for the conformation of the HS state different scenarios were presented for the assignment of Ws and how it exchanges. This analysis is discussed in the context of all semi-stable intermediate oxidation states in the Kok cycle.
To further the understanding of this equilibrium, I also studied a selection of mutants positioned at strategic places in the vicinity of the different proposed substrates and at points that were suggested to be critical for substrate entry.
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With the combination of TR-MIMS and EPR, I reached the conclusion that by mutating valine 185 to asparagine, the water bound A-type conformation was stabilized, meanwhile in the mutant where aspartate 61 was mutated to alanine I observed that the barrier of the equilibrium between the exchanging conformations was so high that the interchange between them was arrested at room temperature. Additionally the retardation of the substrate exchange rates in the S3-states fit best with D61 being in the vicinity of the fast exchanging water.
With this information we found the data best explained in a scenario where the water insertion of the S2-S3 transition was determining the if O-O bond formation occurred between the waters that were W2 and W3 or W2 and O5 in the S2 state.
In addition, by mutation of glutamate 189 to glutamine that this residue is not important for the exchange of substrate waters in the S2 or the S3 states.
Finally I use a combination of substrate labelling with TR-MIMS and time resolved labelling of the waters that ligate the Mn4Ca-cluster to show that the briding oxygen O5 is exchanging with a near identical rate to Ws, further supporting the assignment that Ws=O5.
In conclusion, O-O bond formation most likely occurs between W2 (Wf) and O5 (Ws) via an oxo-oxyl radical coupling mechanism. The newly inserted water thus represents the slow exchanging water of the following S-state cycle.
Abbreviations
PSII Photosystem II
XRD X-ray diffraction
XES X-ray emission spectroscopy
TR-MIMS Time-resolved membrane-inlet mass spectrometry
LS Low spin
ML Multiline
HS High spin
TW Terrawatts
MTOE Million Tonnes of Oil Equivalents
ROS Reactive oxygen species
CBB Calvin-Benson-Bassham
D Photosystem I
Chl chlorophyll
Pheo Pheophytin
P Pigment
YZ Tyrosine Z; D1-Tyrosine 161
W Water
Wf Fast-exchangeing water
Ws Slow-exchangeing water
OEC Oxygen Evolving Cluster
YD Tyrosine D
EXAFS Extended X-ray Absorption Fine Structure XANES X-ray Absorption Near-Edge Structure
DFT Density Functional Theory
LS Low Spin
HS High Spin
ML Multiline
LO Low Oxidation
HO High Oxidation
IR InfraRed
NIR Near InfraRed
FTIR Fourier Transform InfraRed
NMR Nuclear Magnetic Resonance
ELDOR ELectron DOuble Resonance
ENDOR Electron Nuclear DOuble Resonance
EDNMR ELDOR-Detected NMR
ESEEM Electron Spin Echo Envelope Modulation
TR-MIMS Time-resolved membrane inlet mass spectrometry Cw-EPR Continuous wave – electron paramagnetic resonance
TM Thylakoid Membrane
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PSIIcc Photosystem II core complex
BBY Berthold-Babcock-Yocum
OD Optical Density
PPBQ Phenyl-p-benzoquinone
MES 2-(N-morpholino)ethanesulfonic acid
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid TAPS [tris(hydroxymethyl)methylamino]propanesulfonic acid
PI Proton Isomer
Enkel sammanfattning på svensk
Syrebildande fotosyntes är tveklöst en av de viktigaste kemiska processerna för mänskligt liv på jorden. Fotosyntes förser inte bara vår atmosfär med syrgas som vi behöver för andning, men är också den drivande kraften för nybildande av biomassa som vi finner i nästan alla aspekter av våra liv från föda till byggmaterial och textilier för kläder, möbler och dekoration.
Den fotosyntetiska vattenspjälkningsmekanismen är katalyserad av ett vatten:plastoquinonoxidoreduktas vid namn fotosystem II (PSII) som är inbäddat i tylakoidmembran hos växter, alger och cyanobakterier. När PSII exciteras av ljus sker så kallad laddningsseparering i enzymets reaktiva center, och en elektron extraheras genom oxidering av ett Mn4Ca-kluster – det aktiva sätet för vattenspjälkning hos PSII. När Mn4Ca-klustret har oxiderats fyra gånger, bildar det en syre-syrebindning mellan två syreatomer från två vattenligander till klustret, som då återvänder till sitt lägsta oxidationstillstånd. Förståelse för vilka ligander som finns på Mn4Ca-klustret är av stor vikt för att förstå den grundläggande kemiska mekanismen för vattenspjälkning i PSII.
I denna avhandling beskriver jag undersökning av det aktiva sätet i PSII, med hjälp av rumsteperatursröntgendiffraktion (XRD) och röntgenutstrålningsemissionsspektroskopi (XES) på mikrokristaller av PSII.
Dessutom beskriver jag hur vi uppdagade de kemiska steg som sker i PSII som leder till införandet av en ytterligare vattenligand i S2-S3-övergången i den katalytiska cykeln.
Vidare, genom mätningnar med tidsupplöst membraninsläpp-mass-spektroetri (TR-MIMS) på den isotopa jämvikten hos substrat-vattnet med övrigt omgivande vatten under förhållande som inducerar olika elektronparamagnetisk resonans (EPR) spektroskopiska signaler, presenterare jag här belägg för att uybytet av det långsamt utbytande substratvattnet Ws kontrolleras av en dynamisk jämvikt mellan olika strukturer i S2-tillståndet ger upphov till antingen en låg-spinn multilinjesignal (LS-ML) eller en hög-spinnsignal (HS). Baserat på kristallstrukturer och information från litteratur på de strukturer av HS- tillståndet, kunde olika scenarion presenteras för tillskrivningen av Ws- tillståndet, och hur det utbyts. Denna analys diskuteras i kontexten av alla semi- stabila intermediära oxidationstillstånd i Kok-cykeln.
För att främja förståelsen av denna jämvikt, har jag även studerat ett urval av mutantioner positionerade på strategiska platser i närheten av de olika förslagna substraten, och vid punkter som föreslogs vara kritiska för substratinträde till cykeln. Med kombinationen av TR-MIMS och EPR har jag nått slutsatsen att
xi
genom mutation av valin 185 till asparagin kunde vatten bundet till A-strukturen stabiliseras. När aspartat 61 (D61) muterades till alanin observerade jag att jämviktsbarriären mellan de utbytande strukturerna var så hög att utbytet mellan dem stannade av i rumstemperatur. Dessutom passar substratets utbytestakt I S3-tillståndet bäst när D61 var i närheten av det snabbt utbytande vattnet. Med denna information fann vi att datan bäst förklarades av ett scenario då vatteninföring i S2-S3-övergången var hastighetsavgörande om O-O-bindingen bildades mellan vattenmolekylerna W2 och W3 eller W2 och O5 i S2-tillståndet.
Vidare, genom mutering av glutamat 189 till glutamin kunde jag se att denna aminosyra ej var avgörande för utbytet av substrat-vattnet i S2 och S3-tillstånden.
Slutligen använde jag en kombination av substratmärkning med TR-MIMS och tidsupplöst märkning Mn4Ca-klustrets vattenligander för att visa att det överbryggande syret O5 utbyter med en nästan identisk hastighet som Ws, vilket vidare stöttar tilldelningen Ws = O5.
Som slutsats kan jag dra att O-O-bindningsbildningen mest sannolikt sker mellan W2 (Wf) och O5 (Ws) genom en oxo-oxylradikal-kopplande mekanism. Den nyligen införda vattnet representerar då den långsamt utbytande vattenmolekylen i den följande S-tillståndscykeln.
Water, water, every where, And all the boards did shrink;
Water, water, every where, Nor any drop to drink.
Samuel Taylor Coleridge From The Rime of the Ancient Mariner
Introduction / Background
Learning to survive on sunshine and water
During the industrial revolution, human society has come to rely on fossil fuels as its main energy source and 70% of the total world energy consumption in 2016 was from fossil fuels as reported by the international energy agency[1].
Apart from coal, which is burned as it is, fossil fuels are essentially biomaterials converted to a variety of high energy organic compounds that release a large amount of energy in the form of heat when burnt. This energy can then be used to drive an engine, heat up a house, run a generator or similar things.
Unfortunately, our large consumption of fossil fuels leads to the release of huge quantities of CO2 into the atmosphere. The CO2 essentially traps heat in the atmosphere as it absorbs the infrared radiation from the earth, leading to the infamous greenhouse effect.With the current projections it seems increasingly unlikely that we avoid a scenario in which the global temperature increases more than 2°C compared to pre-industrial levels [2].
The current annual energy consumption rate sits at 12.5 TW (converted from the 9384 MTOE consumption of 2015) [1]. Of this, consumption of coal, oil and gas make up approximately 10%, 40% and 15% of the total energy consumption respectively. This energy consumption is expected to increase drastically in the coming years. Within the last 40 years the worlds energy consumption doubled, and it is expected to do so again within the next 40 years [3, 4]. Such an increase in energy consumption is incompatible with the stores of oil and gas that have been projected to diminish massively in the coming years as less is being discovered [4-6]. Futhermore, this is obviously incompatible with the ambition to decrease the global emissions of greenhouse gasses that was agreed upon in the 2015 Paris Climate Agreement in order to diminish global warming [7]. An agreement that today is ratified by no less than 189 countries.
Clearly, alternatives to fossil fuels are needed. There are many suggested solutions on how to move from fossil to more sustainable energy sources. Many of these will carry some of the energy burden during the transition to sustainable alternatives, drawing from different sources of energy. Ultimately, there is only a single energy source with a high enough abundance and energy density to sustain the human race, while minimizing pollution. The sun. Quite simply we need to take our lesson from plants and figure out how to live on sunshine, water and CO2, by making light our primary energy resource.
The first step in photosynthesis
Before the oxygenation of Earths atmosphere, the World was populated by anaerobic chemolitoautotrophic and anoxic photoautotrophic organisms harvesting their electrons from small inorganic compounds (H2, H2S, S2O32- NO2-
, NH3) organic molecules (CO, CH4) or metal (Mn2+, Fe2+)[8-11]. To many of these organisms, oxygen was detrimental to their catalytic function as many enzymes in the cells can react with the oxygen and be either directly inhibited or be inhibited through reactions with reactive oxygen species (ROS). ROS are highly toxic and the cells existing at the time would have had no mechanisms to deal with them. With the emergence of oxygenic photosynthesis, the oxygenic photoautotrophs completely changed the atmosphere through what is known as the great oxygenation event [12-14]. For billions of years since then, cyanobacteria, algae and plants have been harvesting energy from sunlight and water through a process known as oxygenic photosynthesis. Importantly photosynthesis can be divided into two regimes: The light driven photosynthetic regime and the dark (light independent) photosynthetic regime. Light driven photosynthesis is a multistep process that harvests the energy of photons and stores it in high energy compounds for the cell to use in the dark reactions or other chemical processes. Several proteins embedded in the thylakoid membranes of plants, algae or cyanobacteria catalyze the overall light driven process (see Figure 1). Meanwhile, the dark processes of photosynthesis accumulates biomass by sequestration of CO2 in the Calvin-Benson-Bassham (CBB) cycle. Some photosynthetic organisms even include the machinery for nitrogen fixation as part of their “dark” photosynthetic reactions.
The first electron transfer reaction in the light driven chain of photosynthetic reactions is known as water-splitting and is catalyzed by the protein pigment complex photosystem II (PSII). Here water is split into its elemental building blocks as shown in the reaction scheme (1).
Figure 1: Schematic overview of the proteins embedded in the thylakoid membrane and the chemical processes that they catalyze. Image kindly provided by Dmitry Shevela
Figure 2: Z-scheme of the linear energy transfer from H2O to NADP+ during light-driven photosynthesis, ploted as a function of redox midpoint potentials at pH 7.0, as presented in [15]. The figure was kindly provided by Dmitry Shevela.
2𝐻2𝑂 + 4ℎ𝜈 → 𝑂2+ 4𝐻++ 4𝑒− (1) The protons from the water molecules are released to the luminal side of the thylakoid membrane and contribute to the generation of the proton gradient across the membrane that is used to drive the production of ATP. Meanwhile the electrons are stored by reducing plastoquinone molecules (along with an equal amount of protons) that diffuse to the next protein in the photosynthetic sequence, the cytochrome b6f complex, where they are re-oxidized. Again, the protons are released to the luminal side of the membrane while the electrons are delivered to plastocyanin, which transmits the electrons to photosystem I (PSI) to fill electron holes in P700. In PSI, the electron holes in P700 are produced by light induced charge separation, where the excited electron is transferred to NADP+ which is reduced to NADPH. Both NADPH and ATP are high energy molecules that work as coenzymes in a wide variety of metabolic reactions and are absolutely essential for the cells.
The energetic profile of the electron as it passes through the electron transport chain in the light driven processes is shown in Figure 2 [15]. In its original form this scheme resembles a Z when it is rotated 90° clockwise. As a result, it is still known as the Z-scheme today. Here we see that the charge separation event at P680 in PSII leads to a large energy potential that drops as the electron is transported away from the electron hole. When the electron reaches PSI, the energy potential has dropped quite significantly and an additional charge separation is necessary to generate enough energy for NADPH production. These
drops in energy may seem wasteful, but are essential as driving force for efficient forward transfer of the electron and as a preventive mechanism from forming long lived excited state chlorophylls that may react with oxygen to generate singlet oxygen [16, 17]. Additionally, the separation between protein moieties help to decrease the risks of backreactions. For example, as the plastoquinones leave PSII the risk of backreactions is greatly diminished.
The theoretical maximum for solar to chemical energy conversion by photosynthetic water oxidation has been estimated at approximately 16%
(without taking the proton motive force into account) [18]. As mentioned, the energy rich compounds produced in photosynthesis are then used to further drive the molecular machinery in a series of dark reactions building biomass. The final efficiency from solar energy to biomass is estimated to have a theoretical maximum of 4.6% for C3-photosynthetic organisms and 6% for C4-photosynthetic organisms [19]. While the highest reported efficiencies for C4-crops lie at around 2-4% for short-term growth, the final efficiency typically falls at 0.2-1% annually [20-23]. Hence the transferral of the energy into an energy carrier such as H2, that could either be used as fuel directly or together with CO, for the production of carbon-based fuels (CH4, CH3OH, C2H5OH etc.), at an early stage in the photosynthetic process would be more favorable than simply harvesting the biomass. Others attempt to engineer the metabolisms of plants and cyanobacteria in ways that they would produce and excrete high value compounds such as fuels or precursors for medicine or other chemical reactions that would be otherwise difficult to synthesise [24-26].
Photosystem II
PSII is a water:plastoquinone oxido-rectuctase with a molecular weight of ~350 kDa. It is a transmembrane pigment-protein complex consisting of at least 20 protein subunits (in the monomer) and a myriad of inorganic and organic cofactors, including chlorophylls (Chl), carotenoids, porphyrins, non-heme iron, chloride and of course the ‘manganese’ cluster (Mn4Ca-cluster). This cluster consists of four manganese ions and one calcium ion interconnected by oxygen bridges and is the catalytic site for water splitting in PSII. The Mn4Ca-cluster is also broadly referred to as the oxygen evolving cluster (OEC) [27-30].
Chlorophylls in the antennae of PSII absorb photons in the 300-780 nm range [18, 31, 32]. This produces an excited state that is transferred between chlorophylls in the antennae through via excitonic couplings.
Figure 3: Schematic of water splitting inside a PSII monomer sitting in the membrane. Chlorophylls PD1 and PD2 are colored dark green and labelled as P680. Chlorophylls D1 and D2 are colored light green and pheophytins are collered orange. The plastioquinones QA and QB are colored red. The Mn4Ca-cluster and tyrosine Zare also shown
Eventually the excitation reaches a special pair of chlorophylls (PD1 & PD2) in the reaction center of PSII, known as P680, where P stands for pigment and the subscript indicates the wavelength that it absorbs [33-35]. Another suggestion is that P680 should rather be considered as a weakly coupled multimer of all four chlorophylls and the two pheophytins in the reaction center [36]. When the excitation reaches P680, this produces the excited state *P680, in which the excitation may also be delocalized across ChlD1 and ChlD2. *P680 can then transfer the excited electron to a neighboring electron acceptor. This has been reported to either lead to formation of PD1+ ChlD1- or ChlD1+ PheoD1- [37], although it is generally referred to as formation of P680+Pheo- [38, 39] The electron is then quickly transferred through the PheoD1 to the plastoquinone QA and finally to plastoquinone QB (reviewed in [40]). When QB has accepted 2 electrons and protons it is fully reduced. In this form the QBH2 it leaves its binding site and is replaced by a fully oxidized plastoquinone. P680+ is a highly oxidizing species with an oxidizing potential of ~1.2-1.3 V [41, 42]. Thus, it will take an electron from the closest electron source, namely the nearby tyrosine 161 (YZ) in the D1 subunit [43]. Finally, YZox oxidizes the nearby Mn4Ca-cluster . The electrons are eventually extracted from waters bound to the Mn4Ca-cluster (see Figure 4). After four oxidation events of the Mn4Ca-cluster, it is prepared for O=O bond formation. That water splitting is a cyclic redox process was realized by Bessel
Kok and co-workers and who, based on flash induced oxygen production patterns measured by Pierre Joliot, rationalized that the water splitting reaction cycles through a series of five intermediate states with a period of four light induced events (see also Figure 4) [44, 45].
Thus the catalytic cycle of PSII can be divided into four light induced transitions with the semi-stable intermediate states S0-S3. The S3 state is here the last stable intermediate state before O=O bond formation. After O2 is formed, PSII returns to the S0-state. The subscript indicates the number of accumulated oxidizing equivalents, which is increased by one with every charge separation. Since an additional charge separation event is required to transition the OEC from the S3
to S0 state, it seems intuitive that a transient intermediate state (S4) where the Mn4Ca-cluster is oxidized should exist between the S3 state and O=O bond formation, before the system returns to the S0-state [46] (see Figure 4). The cycle is today commonly referred to as the Kok-cycle after one of its discoverers. If PSII is left in the dark, back reactions will conveniently occur between the Mn4Ca- cluster and QB- [27, 47, 48] or YD [49-51] or through the oxidation of S0 by YDOX
[52]. This eventually synchronizes the Mn4Ca-cluster in the S1-state and it is now common practice to pre-illuminate PSII samples to generate 100% YDOX which drastically reduces the rate of reduction of the Mn4Ca-cluster during a flash train.
For this reason, the S1-state is generally referred to as the dark-stable state, although some percentage may also be in the S0-state.
Figure 4: The Kok cycle of photosystem II. Each semi-stable intermediate is represented by a green circle (note that S4 is only a transient intermediate state). Light induced transitions are indicated by green arrows, while the spontaneous decay from S4 to S0 is indicated by a dark green arrow.
Electron transfer to YZ and proton release to the lumen is indicated for each S-state transition.
The structure the Mn4Ca-cluster
To better understand the chemical mechanism of enzymes and catalysts, it is generally helpful to study their structures as it often will provide valuable insight into what functional groups may interact. With regard to PSII and the mechanism of O=O bond formation, it would be particularly interesting to see what water moieties are bound and what structural changes may occur in the Mn4Ca cluster, the surrounding water molecules and amino acids during the Kok cycle.
Insights from X-ray spectroscopy
Before the first high resolution crystal structures of PSII started emerging, the best source of structural information about the Mn4Ca-cluster would come from X-ray spectroscopy. This method can both be used to detect the overall oxidation state of the cluster as it progresses through the S-state cycle (X-ray emission spectroscopy; XES & X-ray absorbtion spectroscopy in the near-edge region;
XANES) and it can be used to detect Mn-Mn, Mn-O and Mn-Ca distances (extended X-ray absorption fine structure; EXAFS), reviewed in [53, 54].
Through data from these methods, some proposals for the structure of the Mn4Ca-cluster managed to arrive fairly close to the crystallographic model [54- 57] . In addition, both XES and EXAFS have on more than one occasion served as very important tools that help verify the validity of the structural models obtained from X-ray diffraction (XRD) experiments. As the X-ray spectroscopy measurements are generally recorded with much lower X-ray doses, the risk of radiation damage to the OEC is much smaller. Thus these methods have proven themselves to be essential in the pursuit of damage free models, since it was shown that a fairly low X-ray dose, even at cryogenic temperatures may damage the Mn4Ca-cluster [58, 59]. Furthermore, crystallographic models will most of the time represent either a single static state or conformation. Since such conformational differences can be highly dependent on the buffering conditions [60, 61] (see also paper IV), EXAFS as a tool, may have access to conformational spaces of the OEC that are not accessible with crystallography, due to incompatability with the crystallization conditions. Importantly, XES remains an essential tool during time-resolved room temperature crystallographic measurements, since it provides the opportunity to make direct correlations between atomic movements in the crystal structure and the oxidation state changes associated with S-state changes. The XES measurements performed simultaneously with XRD measurements provide in situ evidence for efficient sample turnover (see papers I-III).
Crystallographic structures of PSII
High-resolution crystallographic models of PSII have been pursued over the last 20 years since Zouni et al. published their 3.8 Å structure of cyanobacterial PSII [62-67].
Figure 5: Development of the S1-state model of the Mn4Ca-cluster and the interatomic distances.
Bonds are shown where they were interpreted to exist in the corresponding publication. A is a representation of the structure as presented in reference [66]; B is a representation of the damage free cryogenic-structure as presented in reference [69]; C is a representation of the structure as presented in Paper I & II (distances not presented in the paper were measured in WinCoot, PDB:
6DHE) [70, 71].
In these structures, the overall architecture of the protein was revealed, along with the placement of the essential pigments involved in the charge separation process and other essential co-factors. In these structures, it was also possible to roughly position the 4 manganese ions. In 2011 the first high resolution (1.9 Å) cryo-straucture of PSII in the S1 state was released, showing the characteristic distorted chairlike structure of the OEC which is shown in Figure 5A [68]. Mn1, Mn2, Mn3 and Ca make up a cuboid as the bridging oxygens O1, O2, O3 and O5 interconnect them. Furthermore there is a ‘dangler manganese’, namely Mn4 which is connected to the cuboidal shape through bonds to O4 (which connects with M3) and O5. In this first high-resolution structure of PSII, O5 was modelled to be roughly equidistant from Mn1 and Mn4 and was assigned to be a µ4-oxo bridge (Figure 5A) [68]. Moreover, the Mn4Ca-cluster habors 4 terminal water ligands of which two are bound to Mn4 (W1 & W2), while the other two are bound to the calcium ion (W3 & W4). Later it was shown through computations and comparisons to interatomic distances in the EXAFS data that this structure likely had suffered radiation damage [54, 69, 70], leading to a quite significant reduction of the manganese ions (oxidation state of the cluster was likely S-3), most likely due to a high X-ray dose. A later cryogenic structure resolved the issue with X-ray damage and found that O5 was shifted further towards Mn4 than Mn1, albeit still as a µ4-oxo (Figure 5B) [71].
Recently, we solved the PSII structures of all the semi-stable intermediate states to observe the changes in the OEC (see Figure 6; paper I & II) [72, 73]. From these structures we also see that O5 is closer to Mn4 than Mn1, which we interpret as O5 being a µ3-oxo bridge (Figure 5C). We also observed that W20 disappears already in the S1 to S2 transition, which may be important for the events in the S2
to S3 state transition, where we see the insertion of extra water moiety (Ox) as a bridging oxygen, likely as a hydroxo, between the Ca2+-ion and Mn1. This suggests that Mn1 is now a MnIV as indicated previously by DFT calculations and spectroscopic evidence [74-76].
Figure 6: Structures of the OEC in the all the stable intermediate states of the Kok-cycle. Mn are purple, bridging oxygens are shown in red and Ca is shown in green. Mn-Mn and Mn-Ca distances indicated by the dashed lines are given in Ångström (see paper II) [71].
This naturally raises the question of how the new water is inserted? There are two suggestions for this. The insertion could either occur by a mechanism where waters are rotated around the Mn4 so that W2 becomes the new O5 and O5 becomes the Ox as shown in Figure 7A This option has come to be known as either the carrousel or the pivot insertion mechanism [77, 78]. Alternatively, water insertion could occur from the calcium. In this case W3 would either be inserted on Mn1 and itself become Ox (Figure 7B) or it could be inserted on Mn1, forcing O5 to shift at become Ox, while taking over the O5 position (see Figure 7C). This mechanism has been suggested on several occasions and is perhaps at the moment also the leading mechanism [79-81] (see also paper III-V). Another question that arises is whether the new water is a substrate in O-O bond formation of the current cycle or is it there to fill the void of a substrate water (likely O5), as O2 is released? These questions will be addressed in later chapters.
Finally, as PSII was advanced to the S0-state Ox was gone. The Mn1 – M4 disctance remained large than the S1-state which indicates that both Mn1 and Mn4 are MnIII and fits well with a protonated O5 as described previously [82].
Figure 7: Models for the transition between the S2- and S3-states. A represents insertion of W2 through the carrousel or pivot mechanisms [75, 76]. B & C represent insertions of W3 onto either Mn1 or Mn4 [77-79].
The EPR spectroscopic signatures of the Kok-cycle
Electron paramagnetic resonance (EPR) can be used to monitor unpaired electrons (see Methods for more detail). In this way, it is an ideal tool for monitoring the electron transport in PSII as every electron carrier in PSII can potentially generate a signal in the EPR spectrometer under the right conditions.
These electron carriers include the Mn4Ca-cluster, the two tyrosines YZ and YD, the different pigments (such as ChlD1 and pheophytin), the plastoquinones and non-heme iron. All of these signals and their relaxation properties have played a large role in the understanding of PSII function. For reviews on the EPR signals in PSII read [83-85].
Manganese is a nucleus with a spin of I = 5/2 which in its soluble form will lead to an EPR signal at g ~ 2 with a hyperfine splitting into six lines. The Mn4Ca- cluster in PSII contains four highly coupled manganese ions with varying oxidation states that change as PSII transitions through the different intermediate states in the catalytic cycle. Since the 1980’s EPR signals from the Mn4Ca-cluster have been found in all of the intermediate states [83].
The first EPR signal of the Mn4Ca-cluster to be discovered was the S2 state low spin (LS) multiline signal (See Figure 8). Dismukes and Siderer first observed this signal in chloroplasts from spinach in 1981 [86]. With this discovery came the insight that the catalytic site for water oxidation contained what had to be an either di- or tetranuclear manganese complex with an antiferromagnetic coupling between a MnIII and a MnIV. It was also noted that in the case of a tetranuclear manganese complex this could arise from either a MnIII3MnIV or a MnIIIMnIV3.
Figure 8: Light-dark EPR spectrum of PSII membrane particles from Arabidopsis at X-band, showing the S2-state LS-multiline (g~2) and HS (g~4) EPR signals. The spectrum was kindly provided by Fikre Mamedov.
This led to an ongoing dispute in the field, as the overall valence state of the Mn4Ca-cluster could be rationalized in either a low oxidation (LO; S0 = MnIIMnIII3) state regime or a high oxidation (HO; S0 = MnIII3MnIV) state regime [87]. Although there are still proponents of the LO regime [88-90], the PSII community have generally converged on HO-model as this is now supported by EPR, XAS, XES and photocounting experiments [74, 82, 91-94]. Therfore I will be using this model in my analysis of the substrate exchange measurements. For the most recent scientific development of this controversy, see [91, 95].
In addition to the S2-state multiline, the S0-state can also produce a multiline signal in normal mode EPR spectroscopy [96, 97]. No such signal can be observed in the S1- or S3-state, as they have integer spins (S=0 and S=3 respectively). This shows clearly that the oxidation events occurring in the S0 to S1 and the S1 to S2
transitions are manganese centered as an unpaired electron is present in the S0- and the S2-state, but not in the S1-state. Pulsed EPR measurements on the S0- state indicates that O5 is likely bound as a briding hydroxyl in this S-state [82].
In the S2-state, some chemical and physical treatments of the PSII samples are shown to give rise to a variety of high spin (HS) signals, with the most prominent being the g ~ 4 signal. Based on DFT calculations and EPR measurements it was shown that the two conformations that give rise to these signals are almost isoenergetic with a barrier of 6-7 kcal/mol [95, 98-101]. This barrier was estimated based on induction of the HS signal with NIR illumination at 130K followed by annealing at 200K to see the rate of conversion from the HS to the LS signal. Recent experiments show that in core complex preparations from Thermosynechococcus elongatus a shift to pH values higher than 8 or with the exchange of the calcium in the cluster for strontium can almost entirely convert the multiline signal to the g~4 signal [102]. In 2012, Pantazis showed that the g ~ 4 signal can be rationalized by a change in the bonding of O5 in conjunction with a valence flip between Mn1 and Mn4, leading to a closed cubane structure rather than open cubane structure that is observed with crystallography. In this thesis, open type conformations of the Mn4Ca-cluster will be referred to as A-type conformations (e.g. S2A is the open structure determined by crystallography; see Figure 9), while closed conformations will be referred to as B-type conformations (e.g. S2B in Figure 9) [95, 98]. For a while the open-closed rationalization was the accepted model for the g~2 to g~4 equilibrium, but in recent studies there have been a number of alternative proposals, such as water bound structures (labelled W) or proton isomers (labelled PI), which are shown in Figure 9. Particularly the pH shift measurements described above are in favor of a conformation that requires structural rearrangement, as a brief annealing step to room temperature is required to convert the signal from g ~ 2 to g ~ 4. This indicates that the conversion between the low spin multiline and the high spin g ~ 4 signal at high pH could be associated with an event such as water binding (S2AW in Figure 9)
[102]. This is further supported by experiments, showing that when the g ~ 4 signal is generated by high pH conversion, PSII can transition at lower temperatures to the S3 state, producing a broad EPR signal. From the S2LS
multiline state one needs to go to higher temperatures to see the transition to the S3-state [103]. In this way, the hypothesis that the S2HS signal conformer is an intermediate between S2 and S3 (a hypothesis that dates all the way back to 1984 [104]) has gained some traction. A third hypothesis, which is strictly supported by DFT calculations, suggests that the g ~ 4 signal in the S2-state arises from the protonation of the O4 µ-oxo bridge (S2API in Figure 9).
The S3-state EPR signal is produced by an integer spin system. It is very broad and has features appearing in the range from g ~ 10 to g ~ 0.5, which were modelled as arising from a ground state of S = 3 [105]. The S3 state spectrum measured at W-band (~94 GHz) was then later used to show that all manganese ions in this S-state were six-coordinate MnIV ions, which supported theoretical calculations by Siegbahn predicting that an additional water moiety was bound to Mn1 in the S3-state [74]. This was later shown by crystallography to be a bridging oxygen between Mn1 and Ca [73, 80, 106, 107]. Latest, it was shown that by addition of 3% methanol or by substitution of calcium with strontium, a significant fraction of centers in the S3-state could be stabilized in a conformation where one of the manganese was still five-coordinate, suggesting that no water binding event had taken place, corresponding to either the S3A or S3B
conformations in Figure 9 [61].
Another signal in PSII that can be detected by EPR through the different S-states, is the so-called split signal. This signal arises from the spin-coupling between the Mn4Ca-cluster and the YZ• and can be induced in all S-states by visible light illumination (reviewed in [83]). Meanwhile in the S3-state the signal can also be induced by NIR illumination even though there is no charge separation that leads to the formation of YZ•. This is thought to be possible as the manganese ions may be able to absorb photons in the NIR regime [108]. In the same paper this also led to the conclusion that charge separation is not necessary for the generation of the split signal in the S3-state.
Thus this type of measurement can inform about the coupling between the Mn4Ca-cluster and the tyrosine as it was concluded that the signal likely arises from the same interaction in all S-states [109]. A connection that has some bearing for certain proposals of how water can exchange in the S3-state as will be discussed in later sections.
Figure 9: Scheme of known and suggested isomers of the S2- and S3-states in photosystem II as presented in [110]. The structures of the S2A and S3AW states were determined by X-ray crystallography [71, 78, 104, 105]. All other states are proposed on the basis of EPR spectroscopy and DFT calculations. Open cubane structures are labelled A, while closed cubane structures are labelled B. W indicates binding of an additional hydroxo group, while PI designates a proton isomer and PO designates the formation of a peroxidic intermediate. S2A was assigned to the S2LS state, while there are three proposals for the conformation of the S2HS state: the closed cube S2B state[95], the hydroxo bound S2AW state [111] (see ref. [103, 112] and for related proposals), and the proton shift isomer S2API [113]. The S2BW state is shown in brackets since it is a proposed intermediate in the S2 - S3 transition [61, 114] and for water exchange [115]; It is noted that the position of the Mn(III) valence and protonation states of oxygen ligands and bridges differ among the various suggestions.
Evidence for the S3B or S3A states comes from EDNMR experiments indicating the presence of a five coordinate Mn(IV) ion under conditions preventing water binding [61]. The peroxide bound S3 states are consistent with early proposals by Renger [116], and recent DFT calculations by the Yamaguchi group[117]. Labelling of atoms referred to in the text is provided in the S2A structure. Mn(IV) ions are shown in purple, Mn(III) in green, Ca in yellow and oxygen in orange.
Substrate waters of the Kok-cycle
As determined by the product of the water splitting catalyzed by PSII, there must be two waters that act as substrates during the formation of the O=O bond that occurs at the Mn4Ca-cluster. Several different mechanisms for this O=O bond formation have been suggested over the years. Some of the more recent ones are shown in Figure 10 (For reviews see [53, 57, 118, 119]).
Both the oxyl-oxo radical coupling mechanisms shown in Figure 10A & B and the nucleophilic attack mechanism shown in Figure 10D have been largely favored in the PSII literature (reviewed in [57, 119]), but Siegbahns oxyl-oxo mechanism is presently the leading suggestion (Figure 10A)[76]. Meanwhile, the oxyl-oxo radical coupling between W1 and O4 in Figure 10C is a more recent suggestion [120]. The coupling between two bridging oxygens which seen in some molecular catalysts [121, 122](Figure 10E) was suggested only once or twice and can be largely excluded as it is incompatible with a lot of the isotope labelling data that exists for the water substrates (elaborated below). Geminal coupling of two terminal ligands on a manganese (Figure 10F) was also suggested on occasion [123, 124].
From these different suggestions, it is clear that there are different water ligands in play for the different water splitting mechanisms and since they are chemically very similar to the solvent, it has proven quite the challenge to probe them directly. It is essentially equivalent to looking for a specific drop of water in the ocean. Presently, there are only a few techniques that directly probe the waters bound to the Mn4Ca-cluster. These include X-ray crystallography, 17O-ELDOR detected NMR (EDNMR), to some extent FTIR spectroscopy and finally time- resolved membrane inlet mass spectrometry (TR-MIMS). Of these, the latter is the only technique that specifically probes the substrate waters, as it detects the oxygen that is produced by PSII [125-127] (see also methods section).
In 1995 it was shown for the first time through time-resolved 16O/18O substrate water exchange measurements in the S3-state, that one of the substrate waters was bound to the active site cluster with a relatively high affinity [128]. At the time, this was a very important finding as the active site was thought, by some, to be dry until the S4 state was reached as a protective mechanism against oxygen radical formation at the cluster. In 1998 it was shown that the second substrate was bound less tightly in the S3-state, such that the exchange rates of the two substrates vary by more than an order of magnitude [129]. The substrates have thus been named the fast exchanging water (Wf) and the slow exchanging water (Ws).
Figure 10: Overview of the O=O bond formation mechanisms that have been suggested over the years. A) Oxyl-oxo radical coupling in A-type conformation. B) Oxyl-oxo radical coupling in B-type conformation. C) Oxyl-oxo radical coupling between W1 and O4. D) Nucleophilic attack mechanism between a Ca-bound water and a water bound to Mn4. E) Coupling of two oxygen bridges. F) Geminal coupling between two terminal water ligands on Mn4.
The exchange rate of Wf has only been measured in the S2 and in the S3 state [130, 131], while the exchange rate of Ws can be measured through the entire Kok-cycle.
The inability to measure Wf in the S0- and the S1-states, is likely a result of the necessary dark time between flashes that is required in each S-state for PSII to turn over efficiently. In the S0 state the exchange rate of Ws was measured to ~20 s-1. As PSII progresses from the S0 to the S1-state this decelerates ~100 fold. When the system progresses to the S2-state an Ws exchange accelerates 10 fold and remains the same in the S3-state. On the other hand, Wf exchanges with a rate of
~120 s-1 in the S2 state, which decreases about 3 times to ~40 s-1 in the S3-state [132]. This shows that as PSII cycles through the S-states, the exchange rates of the two substrate waters vary independently of each other. These rates were measured in thylakoid membranes from spinach at 10 °C in HEPES (pH 6.8) with 400 mM sucrose. Meanwhile, there are indications that the substrate exchange rates might also be affected by different factors, such as: the organism of origin (plant/algae/cyanobacterium, mesophilic/thermophilic) preparation type (thylakoid / BBY / PSIIcc) and buffer composition (succrose/betaine/glycerol, pH, small molecule addition) [110, 130, 132, 133] (see also papers IV-VII). Later it was found that as PSII is progressed from the S3-state to the S3+YZ• state substrate exchange stops [134].
Substitution of the Ca2+ to Sr2+ only leads to an acceleration in the exchange rate of Ws [110, 135] (see also Ca2+ vs Sr2+ data in Paper IV & VI). This seems like a clear sign that Ws must be bound to calcium. In conjunction with the S-state dependence of the Ws exchange, a bridging position between Mn and Ca becomes the most likely option for Ws [57, 135]. Within the framework of the current
structural models it follows that this would be O5 [57]. The main argument against this hypothesis has been that the exchange of a bridging oxygen is very slow in model complexes (~10-6-10-4 s-1) compared to the exchange rate of Ws
(~10-2 - 2s-1), especially when it is coordinated to at least one MnIV [136].
Measurements of the ligated waters in PSII with 17O-EDNMR in H217O-enriched PSII-samples have shown that one of the oxygen bridges in the Mn4Ca-cluster was exchangeable within 15 s [137]. This oxygen bridge was assigned to O5 [138, 139]. Exchange of a bridging oxygen of course requires a large degree of structural rearrangements as the bridge would need to position itself in a terminal position.
For this reason, it is clear that any substrate exchange mechanism involving this oxygen bridge would have to include a series of intermediates conformations for this to occur (see Paper IV-VII for discussions on the exchange of oxygen ligands of the cluster).
Another popular water in terms of Ws assignment is W2. For this hypothesis, it has been particularly difficult to explain the dependence of Ws on the S-state cycle in terms of the Mn4 oxidation state which would be III;III;IV;IV in the S0, S1, S2
and S3-states respectively. If W2 is Ws, one would then intuitively expect fast exchange rates in S0 and S1, but slow exchange rates in S2 and S3, following the oxidation state of the Mn4. This is not the case, as described above. For a rationalization, the exchange rate of Ws is instead more dependent on distortions along the O4-Mn4-W2 axis. In the case of such a distortion along a Jahn-Teller axis, it is likely that that the binding affinity of the ligands along this axis decrease significantly. If the Mn4Ca-cluster exists in a conformational equilibrium with an open/closed conformation [95, 98] of the Mn4Ca-cluster, this could potentially lead to distortions that would allow dissociative water exchange of W2 from Mn4.
Thus this option also implies that the S2B conformation corresponds to the the S2HS state (as described in Paper IV).
Since the exchange of Wf is only measurable in the S2 and S3-states not much can be said about the binding of the water in the S0- and S1-states. As we shown in Paper II, the structure of the Mn4Ca-cluster does not change in terms of bound waters between these S-states (see also Figure 21c). Since a time dependence can be measured for isotope incorporation of Wf in the S2-state it is reasonable to assume that if Wf is bound to the cluster, the exchange rate should be the same or perhaps even faster in the S0 and S1-states. Based on these observations, we can speculate about which water moieties would exchange with the observed S2- and S3-rates and couple that to the proposed Ws candidates. Intuitively, Wf is most likely a terminal water ligand due to the high exchange rate in the S2-state.
Furthermore, due to the oxidation state dependence on said exchange rate, the simplest option would be if it is a terminal ligand at a manganese ion, i.e. leaving either W1 or W2 to be the fast exchanging water. From these two W1 is the least likely; as it was found that it can be replaced with ammonia without significantly
affecting the substrate exchange rate or the overall activity [72, 138, 140] (see also paper IV). W2 has been suggested to be Wf in scenarios where O5 is Ws, since the deceleration of the exchange rate from the S2-to S3-state would follow an expected trend for the oxidation of the manganese ion. Even so, one might also imagine that the Ca-bound W3 is Wf and the decelerated exchange rate upon S2 to S3
transition stems from insertion of W3 as a bridging oxygen between calcium and Mn1. The main argument against W3 has been that exchange from a Ca-ion should occur much faster than the timescale of the water-substrate exchange measurements, making the exchange rate that is measured in the S2-state incompatible with this hypothesis. In addition, the absence of an effect from Ca/Sr substitution on the Wf exchange rate decreases confidence in this assignment.
As a counterargument, it has been suggested that the observed exchange rate in the S2-state may actually be a result of mass transport limitations imposed by the protein matrix, rather than the actual exchange of the substrate at the Mn4Ca- cluster. The channels that lead to the OEC from the bulk will be discussed in this thesis as the O1-channel, the O4-channel and the Cl-channel as depicted in Figure 11. Indeed, computational studies have found areas in all of these water channels that have estimated barriers of 10 kcal/mol or more for waters to pass [141, 142].
Currently, the field seems to favor the O1-channel as the substrate delivery pathway over both the O4 channel and the Cl-channel [73, 80, 107, 143]. This would clearly have implications for the insertion of Ox. This favors an option where an incoming water is inserted from the O1-channel and onto the calcium- ion. This would force W3 to become Ox and either the freshly bound water or W4 would then become the new W3 [79, 81] (Figure 7A&B). The alternative is that a new water would be inserted on the Mn4 ion from either the O4-channel or the Cl-channel, leading to a carrousel/pivot rearrangement of the waters, such that O5 becomes Ox and W2 becomes the new O5 [77, 78] (Figure 7C).
Figure 11: Water channels that are suggested to act as substrate supply channels or product removal channels for the OEC. Manganese ions are shown in magenta, calcium is yellow, chloride is shown in green, waters are shown in red, while potential substrate waters are displayed in orange. The O1-channel, O4-channel and Cl-channel are shown as pink, blue and green clouds respectively, while arrows indicate the direction of the channel. (PDB-model 6DHE)
I often put boiling water in the freezer
Then whenever I need boiling water, I simply defrost it.
- Gracie Allen
