Substrate water exchange in the S 2 state of photosystem II is dependent on the conformation of the Mn 4 Ca cluster

In photosynthesis, dioxygen formation from water is catalyzed by the oxygen evolving complex (OEC) in Photosystem II (PSII) that harbours the Mn 4 Ca cluster. During catalysis, the OEC cycles through five redox states, S 0 to S 4 . In the S 2 state, the Mn 4 Ca cluster can exist in two conformations, which are signified by the low-spin (LS) g = 2 EPR multiline signal and the high-spin (HS) g = 4.1 EPR signal. Here, we employed time-resolved membrane inlet mass spectrometry to measure the kinetics of H 218 O/H 216 O exchange between bulk water and the two substrate waters bound at the Mn 4 Ca cluster in the S LS2 , S HS2 , and the S 3 states in both Ca-PSII and Sr-PSII core complexes from T. elongatus . We found that the slowly exchanging substrate water exchanges 10 times faster in the S HS2 than in the S LS2 state, and that the S LS2 - S HS2 conversion has at physiological temperature an activation barrier of 17 (cid:2) 1 kcal mol (cid:3) 1 . Of the presently suggested S HS2 models, our findings are best in agreement with a water exchange pathway involving a S HS2 state that has an open cubane structure with a hydroxide bound between Ca and Mn1. We also show that water exchange in the S 3 state is governed by a diﬀerent equilibrium than in S 2 , and that the exchange of the fast substrate water in the S 2 state is unaﬀected by Ca/Sr substitution. These findings support that (i) O5 is the slowly exchanging substrate water, with W2 being the only other option, and (ii) either W2 or W3 is the fast exchanging substrate. The three remaining possibilities for O–O bond formation in PSII are discussed.


Introduction
Plants, algae and cyanobacteria harvest photons of visible light to convert solar light into chemical energy in a process known as oxygenic photosynthesis.The key reactions of this process are the extraction of electrons and protons from water and the reduction of carbon dioxide to carbohydrates.The final products, molecular oxygen and biomass, are essential for most life on Earth.2][3][4][5] Driven by light-induced charge separations in the reaction center of PSII, the OEC cycles through five intermediate states, S 0 through S 4 , where the subscript indicates the number of oxidizing equivalents stored. 6The S 4 state is highly reactive and converts within milliseconds into the S 0 state, releasing O 2 and rebinding one 'substrate water' (the term is used independent of the protonation state). 7If left in the dark, the OEC will eventually relax into the dark-stable S 1 state. 8he lowest oxidation state of the Mn 4 Ca cluster in the water splitting cycle, the S 0 state, was shown by 55 Mn-ENDOR spectroscopy to have the oxidation states Mn(III,III,III,IV). 9These overall 'high' oxidation states were recently confirmed by photoactivation experiments. 102][13][14][15][16][17][18][19] The S 3 -S 4 transition remains poorly understood and is suggested to lead to the formation of either an oxyl-radical, Mn(V,IV,IV,IV) or Mn(VII,III,III,III). 11,[20][21][22] The structure of the Mn 4 Ca cluster was first reported at high resolution (1.9 Å and 1.95 Å) for the S 1 state, 23,24 and recently also at resolutions between 2.0 Å and 2.1 Å for all S-states, except S 4 . 3(see also ref. 25).The S 0 , S 1 and S 2 states have similar structures, except that in S 0 the Mn3-Mn4 distance is longer indicating that the O5 bridge is protonated. 9,19,26This structure, often referred to as open cubane or A-type structure, is depicted schematically as S A 2 state in Scheme 1.8][29][30] The remaining coordination sites of the Mn 4 Ca cluster are completed by bridging carboxy ligands supplied by the D1 and CP43 proteins of PSII (not shown).The only exception is the Mn1 site, which features one histidine ligand and is five-coordinate. 3,11,31,32hile this S A 2 structure is the only one observed by crystallography, there is experimental and computational evidence for at least one additional conformation in the S 2 state.4][35] At near neutral pH values, the S LS 2 state is clearly dominant, and its structure is that of S A 2 (Scheme 1).The energy difference between the two S 2 states and the transition state barrier between them are small enough so that the normally less stable S HS 2 state can be enriched in many ways, for example by illumination of S 1 state samples at 130-140 K or by exposing the S LS 2 state to high pH (8.3-9.0),7][38] It is not clear if all S HS 2 states have the same structure, since slightly different g values in the range of 4.1-4.7 have been reported for the various conditions. 3930][40][41][42][43] Among these, the closed cubane S B 2 state is the most prevalent suggestion for the S HS 2 state.The S B 2 state may be reached from the open cubane S A 2 state by moving the central O5 bridge away from Mn4 so that it instead forms a bond with Mn1.This structural change makes Mn4 five-and Mn1 six-coordinate, and is suggested to be accompanied by a valence swap between Mn4 and Mn1. 30,40,45,46However, EXAFS experiments of samples in the S HS 2 state generated by 140 K illumination of S 1 state samples result in Mn-Mn distances that are inconsistent with the S B 2 state structure. 47,48It was recently argued that the S B 2 state also does not provide a rational for many of the treatments leading to the S HS 2 state formation. 42The latter study instead proposes a proton isomer of the S  2 ) that should otherwise insert during the S 2 -S 3 transition (see below). 39,41,43It is noted that the Mn-Mn distances of the S AW 2 state are likely also not in line with the above discussed EXAFS data of the S HS 2 state generated by 140 K illumination. 47The light-induced formation of the S 3 state from S 2 involves significant structural changes that include the binding of a water molecule in form of an additional oxo/hydroxo or oxyl bridge between Ca and Mn1 (S AW 3 in Scheme 1). 3,11,13,25Even so, the precise molecular sequence for the formation of this sixth bridge remains controversial.Both the rotation of the Ca-bound W3 ligand towards Mn1, and the addition of W3 or a protein ligated water to Mn4 in combination with a pivot or carrousel rearrangement of W1, W2 and O5 have been proposed. 32,45,49,50Thus, starting from the S A 2 state, many different pathways can be envisioned for the formation of this most stable form of the S 3 state (S AW 3 in Scheme 1).The structure of the S AW 3 state is well-characterized by X-ray crystallography and EPR spectroscopy. 3,13,25mportantly, EPR experiments additionally indicate the presence of an EPR-silent form of the S 3 state that under IR illumination converts into the EPR-detectable S 2 Y Z state. 36,39,513][54] The EPR silent S 3 state has been tentatively assigned to the S B 3 or S BW 3 structures. 39Additionally, a recent EDNMR study of the S 3 state identified a signal indicative of a five-coordinate Mn(IV) ion within the either the S A 3 or S B 3 structure. 55Furthermore, peroxidic states (S APO 3 , S BPO

3
][58] For determining the mechanism of O-O bond formation, which occurs during the S 3 -S 4 -S 0 transition, it is crucial to identify the two substrate waters.Presently, the only technique able to probe the binding sites of substrate water in PSII is timeresolved membrane inlet mass spectrometry (TR-MIMS). 7,59,60his method utilizes a rapid increase in H 2 18 O concentration of the bulk water to determine the exchange rates of the two bound substrates by measuring the isotopic composition of O 2 generated after various incubation times (Fig. 1).TR-MIMS measurements show that the two substrate waters exchange with different rates.The slow exchanging substrate water (W s ) is bound in all S-states.Its exchange rate slows 500-fold from S 0 to S 1 , increases 100-fold upon S 2 formation and remains about the same in the S 3 state despite the above described complexity of the S 2 -S 3 transition. 7,60Importantly, W s exchange is in all S states about 5-10 times faster in samples in which the natural Ca co-factor of the Mn 4 Ca cluster is replaced by Sr (Sr-PSII). 61n the S 2 and S 3 states, TR-MIMS measurements can also resolve the exchange of the faster exchanging substrate water, W f . 62,63This shows that both substrates are bound to the OEC in the S 2 state.Since protein-or Ca-ligated water molecules generally exchange at rates too fast for the present MIMS approach, this result indicates that W f is Mn-bound in the S 2 state and that the new water molecule binding in the S 2 -S 3 transition is not a substrate in the ongoing S-state cycle.Consequently, we suggested W2 as candidate for W f . 7,60,64As noted in these studies, this conclusion does not hold if the exchange of W f is limited by the diffusion of water through the protein channels.
Together with structural information available at the time, 26,65 the TR-MIMS data led to the proposal that the central oxygen bridge between Ca and two Mn ions, now referred to as O5, is the slow exchanging substrate W s that forms the O-O bond with W2 in a S B 2 like conformation. 64A related, more detailed proposal for the mechanism of water oxidation that involves a similar O-O bond formation mechanism, but utilizes a S AW 3 like conformation, was later made on the basis of DFT calculations. 66Importantly, subsequent advanced EPR measurements have demonstrated that O5 exchanges fast enough with bulk water to be compatible with W s exchange kinetics observed by TR-MIMS. 67Nevertheless, W2, W3 and O4 have been suggested by other groups to be the slow substrate water instead of O5. [68][69][70] Up to now, all TR-MIMS measurements were performed under conditions where the open cubane states are predominant.To compare the substrate water exchange rates in the two structural forms of the S 2 state, we followed in this study a recently developed protocol for enriching PSII core preparations from Thermosynechococcus elongatus (T.elongatus) in either the S LS 2 or the S HS 2 state. 38The data presented below provide unique insights into the pathway of substrate water exchange and the binding sites of W f and W s .

Experimental procedure
Photosystem II core preparation The T. elongatus DpsbA1DpsbA2 deletion mutant 71,72 was grown in Ca-or Sr-containing buffers, and the PSII core preparations were isolated and purified as described previously. 73,74After preparation, the PSII cores were washed with an aqueous solution of 1 M betaine, 15 mM CaCl 2 and 15 mM MgCl 2 , in an Amicon Ultra-15 centrifugal filter unit (cut-off 100 kDa) until the estimated residual MES concentration was smaller than 1 mM.Finally, the samples were frozen in liquid nitrogen until used.

Time-resolved membrane inlet mass spectrometry
For TR-MIMS measurements, an isotope ratio mass spectrometer (Finnigan Delta plus XP) was used.The spectrometer was connected to a membrane inlet rapid mixing cell (volume of 165 ml) via a steel pipe that runs through a cooling trap containing ethanol/dry ice. 59,62or each measurement, an aliquot of PSII cores was thawed on ice and then diluted 10-fold into an unbuffered solution containing 15 mM CaCl 2 , 15 mM MgCl 2 and 1 M Betaine.To fully oxidize tyrosine D, the samples were then exposed to a saturating xenon-flash (full width at half maximum E5 ms), followed by 60 min dark adaptation at 20 1C, during which the sample was loaded into the MIMS chamber.Five minutes before the measurements, the pH was adjusted by injecting 8 ml of 1 M buffer (see below) containing 2 mM of the artificial electron acceptor 2,6-dimethyl-1,4-benzoquinone (DMBQ), from a 50 mM stock solution in dimethyl sulfoxide.The final concentrations were 0.29 mg of Chl ml À1 , in 50 mM buffer (MES pH 6.0, TAPS pH 8.3 for Sr-PSII or TAPS pH 8.6 for Ca-PSII) and 100 mM DMBQ.The slightly lower pH used for Sr-PSII was chosen to ensure the integrity of the Sr-PSII samples.We note that the S LS 2 to S HS 2 conversion was nearly complete at this pH for Sr-PSII, while pH 8.6 was required to achieve a similar conversion in Ca-PSII. 38For measurements with ammonia, a final concentration of 50 mM NH 4 Cl was employed.
Rapid enrichment of the sample with H 2 18 O was achieved by means of a modified gas-tight syringe (Hamilton CR-700-200) that was driven by air-pressure via a fast-switching solenoid valve (k inj = 170 s À1 based on fluorescence rise after injections of fluorescein; see also ref. 59, 63 and 75).To minimize artifacts from dissolved oxygen, the syringe was loaded with 97% H 2 18 O in a N 2 -filled glove box.The H 2 18 O was further deoxygenated with a mixture of glucose/glucose oxidase (Sigma Aldrich, A. niger) and catalase (Sigma Aldrich, B. taurus). 59he measurement consisted of a series of saturating flashes and a single injection as shown in Fig. 1.After synchronization in the S 1 Y ox D state, the PSII samples were illuminated with one (S 2 ) or two (2 Hz; S 3 ) saturating flash(es) to advance the majority of the centers from the S 1 state to the desired S-state.This step was followed by a fixed delay (10.1 s for the S 2 and 30.1 s for the S 3 state) before the O 2 -generating flash(es) were given (two at 100 Hz for S 2 , and one in case of S 3 ) that advanced the enzyme via the S 4 state to the S 0 state.At various times t i before the O 2 -generating flash(es), the H 2 18 O was injected into the PSII sample resulting in the reported incubation times.After a delay of 400 s, which allowed all signals to return to baseline levels, the PSII samples were exposed to four more flashes given at 2 Hz.This signal was used for normalization, and for determining the relative flash-induced oxygen evolution activity, which was, at pH 8.6, 50% of that at pH 6.0, independent of the ammonia addition.
The mass-to-charge ratios m/z 34 and m/z 36 were monitored for determination of the flash-induced O 2 -production in PSII, while m/z 40 (Ar) was recorded as a reference.The H 2 18 O enrichment after complete mixing was calculated from the m/z 34/36 ratio of the four normalizing flashes to be E20%. 59,62ata points recorded at short incubation times that approached the mixing time were corrected for the change in isotopic enrichment and PSII concentration as described previously. 59,62,75netic modelling of substrate exchange Exchange rates (k f , k i , k s ) were determined by simultaneous fitting of the corrected 16,18 O 2 and 18,18 O 2 data to eqn (1) and ( 2). 59,62The pre-exponential a represents the ratio between fast and slow exchange in the 16,18 O 2 data.It was calculated from the initial H 2 18O enrichment (a in ; 0.07%), which was determined slightly higher than natural abundance due to a small leakage from the syringe tip, and the final (a f ) H 2 18O enrichment using eqn (3).The pre-exponential b represents the ratio between two distinct populations of slowly exchanging substrate waters.b was determined from an initial separate fit of the normalized 36 O 2 yield to eqn (2).Both parameters, a and b, were held constant in the final global fit of the m/z 34 and 36 data.
Activation energies were calculated according to the transition state theory: where R is the gas constant, T the temperature (T = 293 K), k B the Boltzmann constant and k the rate of the reaction.The exchange pathways I and II (Fig. 4) were modelled and compared to the best fits using an Excel spread sheet.

Results
Fig. 2 shows the results of the substrate water exchange experiments in the S 2 state of PSII core samples from T. elongatus containing the natural Ca cofactor in the OEC (Ca-PSII) or instead Sr (Sr-PSII).Each dot represents the normalized flashinduced yield of dioxygen produced after the exchange of one ( 16,18 O 2 ; m/z 34) or both ( rate k s , corresponding to the slow component of the m/z 34 rise in Fig. 2A.These results are consistent with a single conformation (S LS 2 = S A 2 ) for the S 2 state under these conditions, which is in line with EPR spectroscopy performed previously under the same conditions on the same type of samples. 38The data were thus fit employing eqn (1) and (2), using only two kinetic components (b was set to zero). 59,62The parameter of the best fits (solid lines in Fig. 2A and B) are given in Table 1.The results are fully consistent with previous measurements. 63,76,77he TR-MIMS data for Sr-PSII at pH 6.0 are displayed in Fig. 2C  and D (black points).It is clearly seen in Fig. 2D that the rise of the m/z 36 signal was biphasic, with the two phases having comparable amplitudes: a slow phase with a rate k s similar to that seen in Ca-PSII, and a 20-30 times faster phase, designated here as the intermediate phase, with rate k i (eqn (2) and Table 1).Such a biphasic behavior of the m/z 36 data was not reported previously.It can best be rationalized by the presence of two distinct forms of the S 2 state in Sr-PSII that are in slow equilibrium at room temperature.The proposed presence of two conformations in Sr-PSII is in agreement with recent low temperature EPR data showing that the S LS 2 and S HS 2 EPR signals coexist under these conditions (yet S HS 2 being present at lower ratio). 38 3), of which the parameters are given in Table 1.Given the observation of two rates for W s exchange, the m/z 34 rise was fit with three kinetic components according to eqn (1).The resulting W f exchange rate for the S 2 state was found to be similar to that measured in Ca-PSII at the same pH value.
At pH 8.6/pH 8.3, a strong acceleration in the exchange rate of the slow substrate was observed for both Ca-PSII and Sr-PSII samples (red dots and lines in Fig. 2).This rate was similar to k i observed at pH 6.0 in Sr-PSII, and accounted for 90% of the rise of the m/z 36 signal in the Ca-PSII samples, and for 100% in case of the Sr-PSII samples.This is fully consistent with the near total conversion of the multiline signal into the g = 4 signal observed by EPR under these conditions. 38Table 1 shows that the rate constants k f and k s are essentially insensitive to pH and Ca/Sr substitution.In Sr-PSII, k i is also essentially unaffected by pH, but the k i of Sr-PSII is larger by a factor of 2-3 compared to that of Ca-PSII.
It was recently demonstrated that addition of ammonia to PSII core complexes from T. elongatus in a pH 8.6 buffer leads to the quasi quantitative formation of the ammonia modified S 2 multiline signal (S LS 2 ) at the expense of the g = 4 signal (S HS 2 ). 38mploying this treatment we tested whether the accelerated water exchange is caused by the high pH or instead is related to the different structures of the Mn 4 Ca cluster in the S LS 2 and S HS 2 states (blue dots and lines in top panels of Fig. 2).It can be seen that ammonia addition essentially reverted the rates to those seen at pH 6.0 (black data points).This strongly suggests that the water exchange rates observed are a direct consequence of the conformation of the Mn 4 Ca cluster, rather than pH.In agreement with this conclusion, ammonia had very little effect on the substrate exchange kinetics at lower pH, where basically only the S A 2 state was present in Ca-PSII samples. 76n contrast to the slowly exchanging substrate water W s , only minor variations were observed for the exchange rate of the fast exchanging substrate W f in the S 2 state under all conditions (Fig. 2A, C and Table 1).The lack of any significant effect of the substitution of Ca by Sr in the S 2 state is especially notable, and indicates that W3 is either not a substrate (that possibly binds as Ox/O6 in the S 3 state), or its exchange at the Ca site is limited by factors other than breaking the bond to Ca/Sr, such as for example the diffusion of bulk water through water channels.
Fig. 3 shows the substrate water exchange in the S 3 state in Ca-PSII core complexes of T. elongatus at pH 8.6.These experiments revealed that the rate of W s exchange in the S 3 state is well described by a monophasic rise (red dots and line in Fig. 3).In stark contrast to S 2 , k s was in the S 3 state slower at pH 8.6 than observed previously at neutral pH (dashed black line in Fig. 3). 55,78This results in a 6-fold difference between the slow substrate water exchange rates of the S 2 and S 3 states at pH 8.6 (Table 1), indicating that the substrate exchange in the S 2 and S 3 states is governed by different exchange mechanisms and rate limiting steps.Thus, the previously found near identical exchange rate for these two S-states appears to be coincidental.

Mechanistic and energetic analysis
The fact that two different rates of W s exchange were measured under the same conditions (Fig. 2C) implies that the equilibrium between the two S 2 state conformations has a similar or higher barrier than substrate water exchange.Thus, two possibilities exist: (I) there are two independent exchange pathways for W s in the S LS 2 and S HS 2 states, of which the S HS 2 exchange has a lower barrier (exchange pathway I in Fig. 4), or (II) the S LS 2 conformation has to convert into the S HS 2 conformation so that water exchange can occur (pathway II in Fig. 4).In these two schemes, the rate k s corresponds to the exchange of W s that starts from the S LS 2 conformation; it thus reports either on the activation energy for the exchange process starting from this structure (pathway I), or on the energetic barrier for reaching the S HS 2 conformation (pathway II).Since k s is nearly pH and Ca/Sr independent (Table 1), it must be the energy difference between the S LS 2 and S HS 2 conformations that changes at high pH.Furthermore, as the HS state is stabilized at high pH, it is likely that a deprotonation is involved in the S LS 2 -S HS 2 conversion, as suggested previously. 38By contrast, the rate k i signifies in both pathways the W s exchange rate starting from the S HS 2 conformation.This rate is also nearly pH independent, but k i is about threefold larger in Sr-PSII than in Ca-PSII.
Employing the Eyring equation (eqn (4)), energy diagrams for the two exchange pathways were established for Ca-and Sr-PSII at both pH regimes (Fig. 4).The energy diagrams shown are not unique in all aspects, but rather the simplest ones we could conceive to explain our findings with minimal variations of parameters.As such, the relative energy levels of S LS 2 and S HS 2 were adjusted to reflect the percentages of centers undergoing intermediate and slow water exchange as reflected in the m/z = 36 data.
In the sequential exchange pathway II, shown in black lines in Fig. 4, the energy of the S HS 2 conformation is by 1.2 kcal mol À1 higher than that of the S LS 2 conformation in Ca-PSII at pH 6.This value is highly similar to that determined by previous DFT calculations that were based on the proposal that the S HS 2 conformation attains the S B 2 structure. 40,45Substitution of Ca by Sr makes the two conformations of the S 2 state iso-energetic at pH 6, while the increase of pH to 8.3/8.6 stabilizes the S HS 2 state by 2.3-2.5 kcal mol À1 in both samples.Within the sequential exchange pathway, k s is a direct measure of the activation energy of the S LS 2 -S HS 2 transition.A value of 15.8 kcal mol À1 was found for Ca-PSII at pH 6, while it was 16.9 kcal mol À1 under all other conditions tested here.This is higher than estimated in two previous DFT studies that modeled the HS to LS conversion to be a shift of O5 between Mn4 and Mn1 (6-11 kcal mol À1 for Ca and Sr). 40,45,79It is also more than twice the value (6.5 kcal mol À1 ) derived in one EPR study that followed the rate of S HS 2 to S LS 2 conversion in a temperature range between 150-170 K (see also ref. 100). 80However, the value is rather similar to the barrier (17.6 kcal mol À1 ) calculated by Siegbahn for the exchange of O5 in the S 2 state. 81imilar energy levels and barriers were obtained when examining the alternative parallel exchange pathway I (dashed lines in Fig. 4).The main difference is that the barrier between S LS 2 and S HS 2 must be higher to block water exchange of centers in the S LS 2 state via the S HS 2 route.

Discussion
In this study, we examined the exchange rates of the two substrate water molecules in the S LS 2 and S HS 2 conformations of PSII-core preparations of T. elongatus by pH shifts, ammonia addition and Ca/Sr substitution.We report for the first time that the slowly exchanging substrate water, W s , equilibrates 10 times faster in the S HS 2 state than in the S LS 2 (S A 2 ) state.While we employed a pH shift for switching between the two conformations of the S 2 state, 38 we excluded that the observed changes in rates are a consequence of the different proton concentrations by adding ammonia, which was previously shown to stabilize the S LS 2 configuration at high pH by directly binding to Mn. 38,76,[82][83][84] We also discovered that at alkaline pH the slow substrate water no longer exchanges with similar rate in the S 2 and S 3 states, and that the exchange rate of the fast exchanging substrate water is not only insensitive to Ca/Sr substitution in the S 3 state, as reported previously, but also in the S 2 state.
Below we discuss these three new findings in detail on the basis of present structural knowledge about the Mn 4 Ca cluster and with regard to the only detailed exchange pathway that has been proposed thus far for O5.The aim of the discussion is to both gain an improved understanding of the mechanism of substrate water exchange, and to scrutinize the presently favored assignments of W s to O5 and of W f and W2 or W3.This task is complicated by the fact that there is an ongoing vivid discussion regarding the structure of the S HS 2 state, with no less than 3 different proposals.This uncertainty in the field necessitates to discuss a variety of options.[87]

General considerations
Water exchange can follow an associative or dissociative pathway.In the former, a new water molecule binds first before the original water molecule is released into the bulk, while in the latter, the coordinated water molecule dissociates before a new water can bind.9][90][91][92][93][94] If water is bound in a deprotonated form, it needs to be protonated, and bridging oxygen's need additionally be brought into a terminal position before exchange with bulk water can occur.This implies that the exchange of O5 is a complex process that requires conformational changes of the Mn 4 Ca cluster, likely involving a number of the proposed structures summarized in Scheme 1.
Evaluation of O5 as the slowly exchanging substrate water W s On the basis of substrate water exchange experiments 64 and theoretical calculations, 11 it was postulated that O5 is the slowly exchanging substrate W s .The rational for the experimental assignment was twofold: firstly, the exchange rate of W s is dependent on both Ca/Sr substitution and S-state; thus W s was suggested to be a bridge between Mn and Ca. 61,64Secondly, this bridge was assigned to O5, 64 because EPR and EXAFS data are indicative of its deprotonation during the S 0 -S 1 transition, 9,11,18,19,26,27 matching the 500-fold decrease in substrate exchange rate between S 0 and S 1 . 95Subsequent EDNMR experiments have confirmed that O5 exchanges with bulk water within 15 s in the S 1 state, 67 which is unusually fast for a m-oxo bridge, 92 and this finding makes O5 a candidate for W s .However, a definitive assignment needs to await a higher time resolution that allows matching the EDNMR-based O5 exchange kinetics with those obtained for W s by TR-MIMS.
In 2013, Siegbahn proposed a mechanism for the exchange of O5 with bulk water. 81Starting from the S A 2 conformation (2A-1), the first step is the binding of a bulk water molecule (marked blue in Scheme 2A) to the open coordination site of Mn1.This step, which has a calculated barrier of 17.6 kcal mol À1 and is thus rate limiting for the O5 exchange, 81 results in a structure (2A-2) resembling S AW 2 , but with one additional proton on O5 and swapped oxidation states.Next, the newly inserted hydroxo swings into the O5 binding site and O5H becomes a terminal ligand of Mn4 (2A-3).The new bridging OH transfers its proton to form a fully protonated terminal O5 ligand on Mn4(IV).After a valence swap between Mn4 and Mn3 the Mn4(III)-O5H 2 conformation is reached (2A-4; a S BW 2 like structure with one additional proton) that allows O5 to exchange with bulk water, presumably via a dissociative mechanism.Thereafter, this multistep sequence reverses to yield back the S A 2 state, but with O5 exchanged from 16 O to 18 O.
It is important to note that the S BW 2 conformation reached via this Mn4-site exchange pathway is fundamentally different from the S B 2 conformation formed via the S A 2 2 S B 2 equilibrium proposed by the Pantazis, Guidoni and Yamaguchi groups. 32,40,96,97The important difference is that the original O5 (red) is bound terminally to Mn4(III), and not as a m 3 -oxo between Ca, Mn3 and Mn1 (Scheme 1).Therefore, the S B 2 conformation is not an intermediate of Siegbahn's Mn4-site exchange mechanism of O5.
Thus, if the frequently accepted proposals that, firstly, S B 2 is the structure of the S HS 2 conformation and, secondly, the Mn4-site exchange mechanism describes the exchange of W s are both correct, then it follows that the 10-fold faster exchange of W s in the S HS 2 conformation cannot be understood within a sequential exchange mechanism in which S A 2 converts first into S B 2 before water exchange can take place (pathway II in Fig. 4).Accordingly, a separate pathway starting from the S HS 2 state must be considered that can explain the 10-fold faster W s exchange in this conformation (Scheme 2B; Mn1-site O5 exchange pathway).The first step is water binding to Mn4, which induces a flip of bonds and charges akin to the pivot and carousel mechanisms describing water binding during the S 2 -S 3 transition (2B-1 to 2B-4). 7,32,49,98This is essentially the reverse of the Mn4-site exchange pathway (Scheme 2A), and places O5 in a S AW 2 like structure into a terminal position at Mn1(III), where it may exchange with bulk water, possible via Ca.However, since it is not obvious why this pathway would have a lower barrier than the Mn4 exchange pathway, we presently disfavor this option.Looking at the two other structural proposals for the S HS 2 state (Scheme 1), it is noted that the S AW 2 conformation may provide an explanation for the faster exchange of O5 in the S HS 2 state, since it resembles the first intermediate of the Mn4 exchange pathway (2A-2; note the different oxidation state assignments and the extra proton).Indeed, the energy barrier determined here for the S LS 2 -S HS 2 conversion is with 16 to 17 kcal mol À1 similar to that calculated by Siegbahn for the first step of the Mn4-site exchange pathway for O5 (17.6 kcal mol À1 ). 81Similar values for water binding to Mn1 (in the S 2 -S 3 transition) were obtained by Guidoni and Pantazis. 32,99By contrast, the theoretical estimates for S A 2 -S B 2 (6-11 kcal mol À1 ) are significantly lower, 40,45,46 as are previous experimental determinations of the barrier for the S HS 2 to S LS 2 conversion that gave values of 6.7 AE 0.5 kcal mol À1 and 7.9 AE 1.4 kcal mol À1 , respectively. 80,100These previous experimental barriers were obtained by generating the S HS 2 state from S 1 by illumination at 130-135 K in spinach PSII membrane fragments, and measuring the temperature dependence of the conversion of the g = 4.1 signal into the S 2 multiline signal in the temperature range of 150-170 K. Thus, the experimental conditions are highly different from the ones in the present study, where for the first time this barrier was determined at physiological temperatures that facilitate protonation state and structural changes, including water binding.By contrast, such changes are inhibited in T. elongatus PSII samples at cryogenic temperatures, as indicated by the experiments by Boussac, in which he needed to warm the samples to room temperature for a few seconds to allow the conversion of the S LS 2 state into the S HS 2 state after a 200 K illumination at alkaline pH. 38As such it seems likely that the S HS 2 signal obtained at cryogenic conditions has a different structure and hence a different barrier for the conversion of the S HS 2 state into the S LS 2 state than found here at physiological temperature.Alternatively, the discrepancies to the earlier experimental data are due to species differences.
Since the assignments of the W s exchange rates to the S HS 2 and S LS 2 states is solid, and the conversion of these rates into energetic barriers is straight forward, we regard our determination of the energetic barrier to be relevant for T. elongatus PSII core preparations at physiological temperatures, and to be a strong support for (i) the Mn4 exchange pathway for O5 proposed previously based on DFT calculations, 81 and (ii) the identification of W s as O5.It is also in line with the idea that the high pH induced S HS 2 state has an S AW 2 like structure, 39,41,43,47 but other water/hydroxide-bound conformations as for example S BW 2 cannot be excluded.S BW 2 is similar to intermediate 2A-3 (Scheme 2A), which was calculated to have a total energy 4.6 kcal above S A 2 , 81 thus not too far from the level expected for S HS 2 (Fig. 4).Additional constraints for structure and oxidation states of the S HS 2 state comes from a recent report of Mino and Nagashima, in which they utilized the orientation dependence of the S HS 2 EPR signal to identify that (i) Mn4 is the only Mn(III) ion in the S HS 2 state, and (ii) there needs to be a strong coupling (short distance) between Mn4 and Mn3 to simulate their data within a four-spin coupling scheme. 101change of O5 in the S 3 state For the S 3 state, Siegbahn proposed that water exchange requires the back-donation of one electron from Y Z to the Mn 4 Ca cluster in order to reduce one of the four Mn(IV) ions to Mn(III), 81 which would allow S 2 -type water exchange.In this S 2 Y Z state, the Mn 4 Ca cluster would likely reside in the S AW 2 structure, and could thus exchange O5 with the rate k i .If one then assumes that the transition state for the reduction of the Mn 4 Ca cluster by Y Z has a similar barrier to the water exchange starting from S A 2 , this would resolve a major criticism of Siegbahn's Mn4-site exchange proposal for O5.This criticism relates to the experimental finding that, at neutral pH, W s exchanges in the S 2 and S 3 states with very similar rates, while the equilibrium between S 3 Y Z and S 2 Y Z would be expected to slow down the O5 exchange in the S 3 state, given that the S 2 Y Z population must be very low, as this state has not been experimentally observed at neutral pH.The situation is, however, very different at pH 8.6.Here, the S 2 Y Z state is clearly observed by EPR and hence significantly populated. 102Thus, one may expect that substrate water exchange in the S 3 state at pH 8.6 should occur fast (with rate k i ) in a significant fraction of centers, resulting in a bi-phasic exchange curve as observed in Fig. 2D for the S 2 state.9][30]104 We have previously excluded W1 from being W s as it can be replaced by ammonia in the S 2 state with only minor effects on the substrate exchange rates (at pH 7.5). 76This leaves W2 as a possible alternative candidate for W s .][107][108][109][110][111][112] Water exchange in the S LS 2 = S A 2 state would then occur via the equilibrium with the S B 2 conformation (Scheme 3 and pathway II in Fig. 4).
To scrutinize the alternative W2 proposal, we used the S-state dependence of W s exchange.For this, we extended pathway II (Fig. 4) to the other S-states by proposing, in line with previous suggestions, 7,56 as well as experimental data and theoretical calculations, 29,30,38,110,113 that all S-states can exist in A-and B-type conformations, and that the barriers between these conformations are S-state dependent.In addition, we assume that W2, if bound to a non-JT-axis at a six coordinated Mn(III) ion, exchanges much slower as compared to when it is bound to a five-coordinated Mn(III) ion.
In the S 0 state of Ca-PSII, where all Mn ions ligating O5 are in oxidation state (III) and O5 is protonated, the energy difference between the A and B structures and the transition state barrier between them should be small (long arrows of equal length in Scheme 3).Consequently, the S B 0 form, containing a 5-coordinated Mn4(III) site, should be easily attainable, resulting in the fastest W s exchange of all S-states.Consistent with the idea that W s exchange occurs in the S 0 , S 1 and S 2 states at a fivecoordinated Mn(III) site, and that the barrier for reaching this state is low in the S 0 state, the rate k s in the S 0 state is with about 10-20 s À1 (in spinach) 75,95 nearly identical to the rate k i measured here for the water exchange in the S HS 2 state (Table 1).Oxidation of Mn3 during the S 0 -S 1 transition strongly stabilizes the S A 1 state, making it the clearly dominant conformation.Thus, the exchange rate measured for W s in the S 1 state, which is about 500 times slower than in the S 0 state, may either reflect the exchange of W2 at the six-coordinate Mn4(III) ion, or the barrier for reaching the S B 1 conformation, in which W2 is bound to a five-coordinated Mn4(III) ion facilitating rapid exchange.
Further oxidation of the Mn 4 Ca cluster into the S 2 state is expected to increase the exchange rate of W2, since the B-type state can now be stabilized by locating the additional oxidizing equivalent on Mn1.Thus, the barrier for reaching the fast exchanging S B 2 state can be assumed to be lower than in the S 1 state, explaining the 100-fold faster k s exchange rate.In the S 3 state, water exchange can then occur as described above, either by the reduction of the Mn 4 Ca into the S 2 state by Y Z , or via the fivecoordinate Mn4(IV) site of the S B 3 state.The main shortcoming of this proposal is the mismatch of the activation barriers described above, that in our view favors the assignment of S HS 2 to a water/ hydroxide bound conformation of the S 2 state, such as S AW 2 .We anticipate that all the arguments above are exactly the same if W2 were a water molecule instead of a hydroxide.

O4 as possible alternative assignment for W s
The S API 2 structure for the S HS 2 state involves the protonation of O4.Thus, if O4 were W s , this would likely result in a faster Scheme 3 Proposed substrate exchange mechanisms for the S 0 , S 1 and S 2 states assuming that W2 (labelled red) is the slow substrate water and that S HS 2 has the S B 2 structure.Mn oxidation states are labelled green for oxidation state III and black for oxidation state IV.In all three S-states the generally more stable S A 2 conformation (the energy difference in S 0 is proposed to be small) must first convert into the S B 2 conformation before a water molecule (blue), here suggested to be W3, binds to Mn4.This water donates a proton to W2, which then detaches, after which the Mn 4 Ca cluster returns to the S A 2 conformation with the new water in the W2 position.
Paper PCCP  69,70 However, we were not able to propose a scheme for the exchange of O4 that appeared consistent with the water exchange data.Additionally, the assignment of W s to O4 would be in conflict with the EDNMR assignment of O5 as the only exchangeable oxo bridge of the Mn 4 Ca cluster, 67 and with the recent polarized EPR data of the S HS 2 state. 101e fast exchanging substrate W f The most significant finding of this study regarding the exchange of W f in the S 2 state is the invariance of k f towards the substitution of Ca by Sr.This is important, since in recent proposals for the S 2 -S 3 transition it is frequently assumed that W3 is W f , which would be bound to Ca in the S 0 , S 1 and S 2 states, but to Mn1 or Mn4 in the S 3 state.The lack of Ca/Sr dependence in the present S 2 state data thus disfavors that W3 is a substrate.However, this option cannot be excluded until firm data for the rate of diffusion of substrate water to the catalytic site are obtained.While it generally would be assumed to be unlikely that water access is limiting the fast water exchange, it cannot be excluded a priori since present calculations indicate that all channels have barriers in the range of 10 kcal mol À1 , 109,114 and a NMR proton relaxation study indicates a distance of 10 Å from the spin center of the Mn 4 Ca cluster to the protons that rapidly exchange with the protons of bulk water. 115n case that there are no significant access barriers for W f exchange, the previously proposed W2 assignment remains the best option for W f , and the reported low barrier of the S A 2 2 S B 2 equilibrium may provide the means for fast W2 exchange (similar to Scheme 3).Given these presently equally likely options for W f (in case W s = O5), detailed mutational studies aiming to increase or decrease the access of water through the known channels connecting the OEC with bulk water will be needed for a final decision.Such experiments are beyond the scope of the present study.

Possible mechanisms of water oxidation in PSII
The energetics for W s exchange determined here for samples in the S LS 2 state agree well with those calculated by Siegbahn for the O5 exchange starting from the S A 2 state, and thereby strongly support the earlier assignment of W s to O5 by Messinger and Siegbahn. 64,66The present data favor that a S AW 2 -like conformation is both an intermediate in the exchange of O5 and the structure of the S HS 2 state (Fig. 5A).However, in case that the S HS 2 state adopts the closed cubane conformation (S B 2 ), we are unable to exclude W2 as the slow substrate water, since a consistent proposal for water exchange could be made for both O5 and W2 (Fig. 5B and C).For a final assignment further studies will be required, such as 17  With regard to W f , both W2 and W3 remain options until the possible role of water accessibility to the catalytic site on the rate of fast water exchange is clarified by mutational studies in combination with substrate water exchange.By contrast, all other options can be excluded.
On that basis, O-O bond formation mechanisms via radical coupling involving O5 as W s are strongly favored by our new results and may occur in either an A-type or B-type conformation of the Mn 4 Ca cluster (Scheme 4A and B). 7,11,12,63,64,66,85,116,117he only difference would be that the radical coupling in the S BW 3 state would require first a structural rearrangement starting from S AW 3 , possibly in line with the lag phase observed after the S 3 Y Z formation. 118,119Interestingly, the origin of the two substrate oxygen's would vary depending on the water insertion pathway.Assuming that the S 2 -S 3 transition would involve W3 binding to Mn1 or Mn4, 3,45,50,120,121 then in both cases the O-O bond would be formed between the former W3 and O5, but the origin of oxygen's in the O5 and Ox/O6 positions would be swapped depending on the insertion site.If, however, water is inserted during the S 2 -S 3 transition via the pivot mechanism, 32 then mechanisms A and B (Scheme 4) would involve O-O bond formation between W2 and O5. 7,13,64y contrast, if Ox/O6 originates from W3, but is not a substrate, W3 would be 'parked' in the S 3 state between Ca and Mn1 to replace O5 during the S 3 -S 4 -S 0 transition, while the O-O bond would be formed between W2 and O5 via geminal coupling at the Mn4 site (Scheme 4C).Geminal coupling at this site was proposed first by Kusunoki on the basis of DFT calculations that resulted in a B-type structure for the Mn 4 Ca cluster. 113The proposal was further inspired by his analysis of substrate water exchange data from Hillier and Wydrzynski. 60He suggested that, in the S 3 state, there is a significant correlation between the exchange of W f and W s , indicating that both must be bound at the same Mn ion to allow them to swap places.At the time the proposal was made, water addition to the Mn 4 Ca cluster during the S 2 -S 3 transition was not established, and thus the different S-state dependence of W f and W s seemed to exclude this idea.In addition, the demonstration that ammonia binds to the W1 site in the S 2 state without significantly affecting the exchange of the two substrate waters argued against this proposal. 76,122owever, in the light of the recent data suggesting the binding of W3 to Mn1 during S AW 3 formation, geminal coupling is now consistent with present experimental results.Zhang and Sun recently proposed that this type of O-O bond formation involves the transient formation of a Mn4(VII) species obtained via disproportionation within the Mn 4 Ca cluster. 22hile we cannot yet distinguish between the three options displayed in Scheme 4, we exclude nucleophilic attack of a Ca-bound (W3) water onto W2, 59,65,94,[123][124][125][126] since W3 would need to serve a dual role: firstly it would need to fill the open coordination site of Mn1 for preloading the new O5, and secondly the successor water ligand at the W3 site would need to be the fast exchanging substrate W f .Notably, in case of the pivot pathway for filling the Ox/O6 site, both nucleophilic attack of W3 onto W2, and geminal coupling at the Mn4 site are excluded.Thus clarifying the pathway for water insertion in the S 2 -S 3 transition is another requirement for deriving at an experimentally confirmed mechanism.Such studies are ongoing in the field, so that the discussion provided here can serve as blueprint for identifying the substrate once this independent problem is solved.

Conclusion
Substrate water exchange experiments provide a unique and independent view on the water oxidation mechanism.In this study, we have advanced this approach significantly by providing unique new experimental results.By combining these new data with emerging knowledge about the structures of various conformers of each S-state, together with earlier DFT calculations regarding O5 exchange, we have derived molecular interpretations of substrate water binding and its exchange with bulk water not previously attainable.The present analysis along with future investigations of the temperature dependence for the barrier between LS and HS states, provides the basis for the interpretation of ongoing TR-MIMS experiments utilizing point mutations, Ca/Sr-exchange and H/D-labelling, which together with other outlined experiments have the potential to resolve the mechanism of water oxidation.

Fig. 1
Fig. 1 Flash and injection scheme for TR-MIMS measurements in the S 2 -state (top) and the S 3 -state (bottom).Vertical lines indicate saturating flashes and the downward pointing arrows indicate injection of 18 O-labelled water.The first flash was given to synchronize the samples in the S 1 Y ox D state, while the final group of four flashes is employed for normalization.

Fig. 2
Fig. 2 H 2 18 O substrate exchange of Ca-PSII (A) and (B) and Sr-PSII (C) and (D) in the S 2 -state.(A) and (C) represent the normalized flash yields of singlelabelled dioxygen (m/z 34), while (B) and (D) represent the normalized flash yields of double-labelled dioxygen (m/z 36).Black dots represent measurements performed at pH 6.0, while red dots are data from measurements taken at pH 8.6 for Ca-PSII and at pH 8.3 for Sr-PSII.Blue dots signify the results of experiments with Ca-PSII at pH 8.6 in presence of 50 mM NH 4 Cl.Lines are fits according to eqn (1)-(3), of which the parameters are given in Table1.

Fig. 3
Fig. 3 H 2 18 O substrate exchange of Ca-PSII at pH 8.6 in the S 3 state.Red dots represent the results from single time points.Red lines are fits according

Fig. 4
Fig. 4 Kinetic models (top panel) and energy diagrams (lower panels) for the exchange ('ex') of the slow substrate water W s in the S LS 2 and S HS 2 conformations of photosystem II.The barriers were calculated from the rates listed in Table 1 using transition state theory (eqn (4)).They are given in kcal mol À1 .Dashed lines correspond to pathway I, where S LS 2 and S HS 2 exchange independently, while solid lines represent the sequential pathway II, in which the S LS 2 conformation has to convert first into the S HS 2 conformation before water exchange can occur.Where lines overlap, only the solid line is visible.The length of the arrows in the top panel correspond to the rates of W s exchange in Ca-PSII at pH 6.0.

Scheme 2
Scheme 2 Possible exchange pathways for O5 starting from the S LS 2 state (panel A) and the S HS 2 state (panel B). (A) Mn4 site O5 exchange mechanism (redrawn after ref. 81).A bulk water or W3 (blue) binds to Mn1 in the S A 2 conformation, leading to a valence flip between Mn4 and Mn3, and the transfer of one proton from the new water to O5 (red).The final conformation has a water-bound S B 2 -type structure, in which Mn4 has the oxidation state III (green), allowing the exchange of O5 before returning to the S A 2 conformation by reversing the sequence.(B) Proposal of a Mn1-site O5 exchange pathway starting from the S B 2 conformation.A water (blue) binds to the five coordinated Mn4(III) in the S B 2 conformation, which induces a proton transfer and valence flip that leads to the formation of a water-bound S A 2 conformation, in which O5 is bound to the five-coordinated Mn1(III) site, where water exchange may occur.
see also ref. 75.The recent experimental evidence for the S B 3 conformation 55 allows proposing an alternative exchange pathway for O5 in the S 3 state.As shown in Scheme 1, S B 3 may be reached from the dominant S AW 3 conformation via S BW 3 .After the loss of the O5-water molecule, a new water may bind leading to the re-formation of S AW 3 containing a new O5.Thus, also the new S 3 state substrate water exchange data are consistent with O5 being the slowly exchanging substrate water W s .W2 as possible alternative assignment for W sWe evaluated the structures of the Mn 4 Ca-cluster to see if W s = O5 is the only option to explain our data.One possible alternative was found assuming that the S our experimental conditions, we discuss this option since it emphasizes the importance of a unique structural resolution of the S HS 2 state for deriving the mechanism of water oxidation.Aside from the different position of O5 in the S A 2 and S B This journal is © the Owner Societies 2020 Phys.Chem.Chem.Phys., 2020, 22, 12894--12908 | 12903 O-EDNMR experiments with high-enough time and spectral resolution to allow monitoring the time course of 17 O/ 16 O-exchange of both W2 and O5, 67 thus allowing the comparison of the W2 and O5 exchange rates with those of W s determined by TR-MIMS experiments.In addition, obtaining room temperature crystal structures of the high pH S HS 2 state would allow removing the remaining uncertainties.

Fig. 5
Fig. 5 Possible substrate water exchange pathways in the S 2 state.The energy diagram shown as solid line indicates the case for Sr-PSII at pH 6. Dashed lines indicate changes in the relative energies of S LS 2 and S HS 2 due to pH and/or substitution of Ca by Sr.

Table 1
18mmary 2 and S 3 states were performed at 20 1C and the indicated conditions (NH 3 signifies addition of 50 mM NH 4 Cl).The rate k f describes the fast exchange phase with the amplitude a in m/z 34, which is assigned to the fast exchanging water (W f ), while k i describes the intermediate phase, which is resolved in some of the m/z 36 data with the amplitude b.The parameter k s describes the slowest exchange rate resolved in the m/z 36 data with the amplitude 1 À b.The rate constants k i and k s are both assigned to the slow exchanging water W s .The amplitude a varies due to small differences in the final H 218O enrichment This of parameters extracted from the global fits of the mass-to-charge ratio signals m/z 34 ( 16,18 O 2 ) and m/z 36 ( 18,18 O 2 ) displayed in Fig. 2 by employing eqn (1)-(3).The H 2 18 O substrate exchange measurements of Ca-PSII and Sr-PSII core preparations from T. elongatus in the S journal is © the Owner Societies 2020 Phys.Chem.Chem.Phys., 2020, 22, 12894--12908 | 12899 Open Access Article.Published on 27 April 2020.Downloaded on 9/16/2023 3:17:02 AM.This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.