Sequential and Coupled Proton and Electron Transfer Events in the

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Sequential and coupled proton and electron transfer events in the S2#S3 transition of photosynthetic water oxidation revealed by time-resolved X-ray absorption spectroscopy Ivelina Zaharieva, Holger Dau, and Michael Haumann Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01078 • Publication Date (Web): 21 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016

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Sequential and coupled proton and electron transfer events in the S2→S3 transition of photosynthetic water oxidation revealed by time-resolved X-ray absorption spectroscopy

Ivelina Zaharieva, Holger Dau*, Michael Haumann*

Freie Universität Berlin, Department of Physics, 14195 Berlin, Germany

Funding Information H.D. was supported by the Deutsche Forschungsgemeinschaft (DFG) within the Berlin collaborative research center “Protonation Dynamics in Protein Function” (SFB 1078, project A4). M.H. thanks the DFG for support within the Unicat Cluster of Excellence Berlin and the German Bundesministerium für Bildung und Forschung for funding in the Röntgen-Angström Cluster (grant 05K14KE1).

*Correspondence to: Michael Haumann, Holger Dau Freie Universität Berlin, Institut für Experimentalphysik, Arnimallee 14, 14195 Berlin, Germany, Phone: +49 30 838 56101/53581, Fax: +49 30 838 56510, Email: [email protected], [email protected]

Keywords: photosynthetic water oxidation, manganese complex, time-resolved X-ray absorption spectroscopy, proton and electron transfer

Running title: PT and ET in S2→S3 revealed by time-resolved XAS

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Dedication: We dedicate this work to the memory of Fabrice Rappaport for his seminal timeresolved investigations on photosynthetic water oxidation.

Abbreviations: ET, electron transfer; Mn4Ca, water oxidizing complex of PSII; PCET, proton-coupled electron transfer; PSII, photosystem II; PT, proton transfer; Si, intermediate states of the water oxidation cycle; YZ, redox-active tyrosine residue (Tyr161) in the D1 subunit of PSII; XANES, X-ray absorption near edge structure; XAS, X-ray absorption spectroscopy


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Abstract The choreography of electron transfer (ET) and proton transfer (PT) in the S-state cycle at the manganese-calcium (Mn4Ca) complex of photosystem II (PSII) is pivotal for the mechanism of photosynthetic water oxidation. Time-resolved room-temperature X-ray absorption spectroscopy (XAS) at the Mn K-edge was employed to determine the kinetic isotope effect (KIE = τD2O/τH2O) of the four S-transitions in a PSII membrane particle preparation in H2O and D2O buffers. We found a small KIE (1.2-1.4) for manganese oxidation by ET from Mn4Ca to the tyrosine radical (YZ+) in the S0n→S1+ and S1n→S2+ transitions and for manganese reduction by ET from substrate water to manganese ions in the O2-evolving S3n→S0n step, but a larger KIE (~1.8) for manganese oxidation in S2n→S3+ (subscript, number of accumulated oxidizing equivalents; superscript, charge of Mn4Ca). Kinetic lag phases detected in the XAS transients prior to the respective ET steps were assigned to S3+→S3n (~150 µs, H2O; ~380 µs, D2O) and S2+→S2n (~25 µs, H2O; ~120 µs, D2O) and attributed to PT events according to their comparatively large KIE (~2.4, ~4.5). Our results suggest that proton movements and molecular rearrangements within the hydrogen-bonded network involving Mn4Ca and its bound (substrate) water ligands and the surrounding amino-acid/ water matrix govern to different extents the rates of all ET steps, but affect particularly strongly the S2n→S3+ transition, assigned as proton-coupled electron transfer (PCET). Observation of a lag phase in the classical S2→S3 transition verifies that the associated PT is a prerequisite for subsequent ET, which completes Mn4Ca oxidation to the all-Mn(IV) level.


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Photosynthetic water oxidation is catalyzed at a manganese-calcium (Mn4Ca) complex bound to the proteins of photosystem II (PSII).1-4 Unraveling the mechanism of the most efficient oxidation of two water molecules to yield four electrons, four protons, and one O2 molecule by PSII is pivotal for development of improved synthetic catalysts for solar fuel production 5-8. Reaching this goal requires a comprehensive understanding in particular of the interplay between electron transfer (ET) and proton transfer (PT) reactions at Mn4Ca in the course of the catalytic cycle.9-14 The molecular structure of the Mn4Ca complex presumably poised in its dark-stable resting state has been resolved to about 1.9 Å resolution in seminal protein crystallography experiments.15 The available structures show four water species bound to manganese or calcium, five bridging oxygen species, as well as about 40 water molecules in a ~7 Å diameter sphere around the Mn4Ca-oxo core, which form an extended hydrogenbonded network15-18 (Fig. 1A). All these water and oxygen species might participate in the ET and PT events during the catalytic cycle. The water oxidation cycle includes four semi-stable intermediates (S-states), denoted S0 to S3, and S-state interconversion in purified PSII preparations is achieved by application of single-turnover (laser) flashes of visible light.19 The S1 state is stable in the dark and flashes 1, 2, 3, and 4 induce the S1→S2, S2→S3, S3→S0, and S0→S1 transitions with O2 evolution occurring during S3→S0. Extensive characterization of the PSII reactions has shown that during each of the three lower S-transitions, manganese is oxidized in microseconds by a tyrosyl radical (Tyr161 in the D1 subunit of PSII, YZ+). YZ+ is created by nanosecond electron transfer to the primary electron donor chlorophylls (P680) after formation of the charge-separated P680+QA- radical pair (QA, first plastoquinone acceptor at the reducing side of PSII) (see, e.g., refs. 11, 20-22 for review). These events result in the following redox states: S0, MnIII3MnIV; S1, MnIII2MnIV2; S2, MnIIIMnIV3; and S3, MnIV4 (Fig. 1B).22-28 Manganese reduction in milliseconds by electrons from the two substrate water molecules during the S3→S0 transition parallels O2 release.29 In the S3→S0 transition, the S3YZ+ state 4 ACS Paragon Plus Environment

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spontaneously converts to S0 without formation of any kinetically resolvable reaction intermediate, thereby closing the oxidation-reduction cycle. The above basic S-state scheme has previously been extended by us to account for the results of time-resolved experiments using X-ray absorption spectroscopy (XAS), optical absorption or infrared spectroscopy, as well as photoacoustic and photothermal techniques (see refs.

11, 14, 30-32

and references therein). These experiments, which included determination

of activation energies, pH-dependences, and H/D kinetic isotope effects (KIE) of the time constants of the S-transitions, have suggested a reaction cycle with alternating PT and ET events. In this scheme (Fig. 1B), the classical S1→S2 transition corresponds to the Mn4Ca→YZ+ ET during the S1n→S2+ transition (+, up-charging of Mn4Ca by one unit compared to S1; n, neutral state with the same charge of Mn4Ca as in S1), the S0→S1 transition was dissected into an ET step (S0n→S1+) followed by a slower PT event (S1+→S1n), and during the O2-evolving transition (S3n→S0n), completion of a preceding PT event (S3+→S3n) allows the subsequent ET and O-O bond formation chemistry to occur.30 For the S2→S3 transition, the situation has remained less clear. Our earlier photothermal beam deflection (PBD) experiments have suggested that the Mn4Ca→YZ+ ET is preceded by a kinetic phase with an exceptionally large KIE, which hence was attributed to a PT event.30, 33 According to the PBD results, the time constant of the PT step is smaller than the time constant of the ET step, showing that the PT precedes the ET step. However, these results do not prove that completion of the PT is an essential prerequisite for the ET; the two events might be mechanistically unrelated. In the present investigation, we provide evidence that PT and ET in the S2→S3 transition are sequential reaction steps, meaning that the PT event also mechanistically precedes the ET step. A kinetic component assignable to a preceding PT event in the S2→S3 transition has not been detected in, e.g., time-resolved UVvis experiments for tracking Mn4Ca oxidation state changes.29,

34-41

Possible reasons are

related to technical or signal quality limitations and to the overlay of spectral components due 5 ACS Paragon Plus Environment

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to the redox reactions of Mn4Ca, YZ, P680, and QA. In this respect, time-resolved X-ray spectroscopy17, 24, 42-45 at the Mn K-edge is advantageous because its strict element-specificity facilitates site-selective monitoring of structural and oxidation state changes of the manganese ions of Mn4Ca.24, 42, 46-51 In this study, time-resolved XAS was employed to determine the kinetics of the Stransitions for PSII membrane particles suspended in H2O or D2O buffers. While small KIE values were observed for ET during S0n→S1+, S1n→S2+, and S3n→S0n, a larger KIE for S2n→S3+ suggested direct coupling of the ET to a PT event. In the O2-evolution transition (S3→S0), as well as in S2→S3, a lag phase preceding the oxidation state changes of manganese ions was detected, verifying a sequential reaction scheme with priming reactions preceding the respective ET step. Inter alia based on their pronounced KIE, these priming reactions can be assigned to proton removal from the active site of water oxidation (Mn4Ca), which may be associated with structural rearrangements of the metal complex and its proteinwater environment.

Materials and Methods PSII membrane particles from spinach (activity of ~1200 µmol O2 mg chlorophyll-1 h-1) were prepared as previously described.52 Prior to XAS sample preparation, PSII membranes (~130 µg Chl mL-1) were washed twice in 30 mL volumes of buffers (100 mM NaCl, 5 mM CaCl2, 1 M betaine, 10 mM MES) dissolved in H2O (pH 6.3) or D2O (~97 %, pH-meter reading of 5.9) to give the same pL of 6.3 and membranes were pelleted and resuspended in buffers containing 100 µM PPBQ as an artificial electron acceptor at a chlorophyll concentration of ~12 mg mL-1. 10 µL of PSII suspensions were loaded into each of the 20 cavities of Teflon sample holders, each sample spot was illuminated by a single saturating flash of visible light (of microsecond duration) provided by a Xenon lamp. Thereby S1 state enrichment was achieved. Subsequently the samples were dark-adapted and partially 6 ACS Paragon Plus Environment

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dehydrated (loss of ~30 % of the initial water content) by drying in a desiccator for 2 h over silica gel at 0.5 bar air pressure at 4° C and then frozen in liquid nitrogen. About 400 mounts with 20 PSII samples each (8000 PSII samples) were prepared. Samples were handled during preparation and XAS experiments under dim green light. These procedures followed our previously established protocols.24, 42 X-ray absorption spectroscopy experiments at the Mn K-edge were carried out at the undulator beamline ID26 at the European Synchrotron Radiation Facility (ESRF) at Grenoble (France) using a Si[111] double-crystal monochromator and a large-area scintillation detector (at about 5 cm distance from the sample and shielded by 6 µm Cr foil against scattered Xrays) for X-ray fluorescence monitoring (sample temperature of 20±1 °C). The used experimental protocol concurs with our previously developed techniques for time-resolved XAS at room temperature.24, 42 The three undulators of ID26 were adjusted such that a flat intensity profile of the exciting X-ray beam resulted in the energy region of the Mn K-edge (maximal X-ray flux ~1013 photons s-1, 5-fold attenuated by stacked Al foils for XANES measurements, spot size 1x1.5 mm2 set by slits). The sample mounts, each loaded with 20 PSII samples, were mounted on a motorized stage (sample positioning accuracy in the X-ray beam of ~4 µm); neighboring samples were protected against scattered light by using an Al aperture. In one set of measurements, the PSII samples were excited by 0-4 laser flashes (Continuum Inlite-II, 532 nm, pulses of 5 ns and maximally 50 mJ spaced by 700 ms, last flash applied 100 ms prior to data acquisition) prior to a rapid monochromator scan (~1 s, 0.1 eV steps) over the Mn K-edge for collection of XANES spectra. About 100 scans each on a fresh PSII sample were averaged for signal-to-noise ratio improvement; IF/I0 spectra were normalized to an amplitude of 1.28 at 6564 eV. In a second set of measurements, 0-10 laser flashes were applied to PSII samples with the last flash given at 5 ms after opening of a fast shutter (τopen < 1 ms) and the start of X-ray irradiation and fluorescence data collection. X-ray absorption time courses were collected at a constant excitation energy of 6552 eV (middle of 7 ACS Paragon Plus Environment

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the edge rise) for a total measurement time of 20 ms and digitized with a time resolution of 1 µs and an electrical bandwidth set by an amplifier to 1 MHz. Monochromator scans, sample positioning, laser flashes, fast shutter operation, and data acquisition were synchronized using appropriate trigger electronics. About 700 measurements each on a fresh PSII sample for each S-transition were averaged to improve the signal-to-noise ratio. X-ray fluorescence transients were smoothed by adjacent averaging over 10 data points and interpolation to a step size of 20 µs per data point and normalization of the first flash (F1) transient in H2O or D2O to the same amplitude of the steady-state F1-F0 XANES difference (and of the F2-F10 transients in H2O or D2O by the same respective scaling factors) for visualization. Deconvolution of X-ray transients to yield the transients due to the pure S-transitions was performed using miss and double-hit parameters as determined with the Kok model30,

33

from flash patterns of

millisecond kinetic components in the X-ray transients. Kinetic X-ray transients were analyzed using single-exponential functions or a function including a lag phase (Eq. 1;53 τ1,2, apparent time constants for two sequential reaction steps, Amax = maximal amplitude):

 τ −1 exp(−t / τ 2 ) − τ 2−1 exp(−t / τ 1 )  A(t ) = Amax 1 + 1  τ 2−1 − τ 1−1  

(1)

.

Results Time-resolved XAS was employed to monitor the redox reactions of the Mn4Ca complex during the S-state cycle via shifts of the Mn K-edge, as shown in Fig. 2. The XANES spectrum of dark-adapted PSII (zero flash samples, 0F) was typical for the Mn4Ca complex in the S1 state (in H2O buffer).27, 54-56 After application of 1, 2, 3, or 4 laser flashes prior to XANES collection, which induced predominantly the S1→S2, S2→S3, S3→S0, or S0→S1 transition, the K-edge was shifted to higher energies (1F, 2F, 4F) due to Mn oxidation, or it was shifted to lower energies (3F) due to Mn reduction during the O2-evolving transition, both in agreement with previous results.23,

27, 54-56

At an excitation energy of ~6552 eV, 8

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maximal relative K-edge absorption changes during the S-transitions were observed (Fig. 2A). This energy was used to monitor changes in the S-transitions in real time. X-ray fluorescence changes of manganese in a series of 10 laser flashes for PSII in H2O or D2O buffer are shown in Fig. 2B. Similar rapidly decreasing fluorescence level changes on flashes 1, 2, and 4, a slower and larger fluorescence increase on flash 3, and a pronounced quaternary pattern of the signal amplitudes were observed in H2O and D2O, which indicated similar and effective flash-induced progression through the S-states cycle in both buffers.24, 42 The damping of the amplitude oscillations results from 'miss events', that is, a fraction of PSII centers not progressing to the next S-state upon flash excitation.57 The thirdflash transient revealed pronounced manganese reduction during the O2-evolving transition with a typical time constant (τ) of about 1.5 ms. Exponential curve-fitting of the individual Xray transients (not shown) revealed the amplitude patterns of this millisecond phase in the flash series (Fig. 2B). The pattern on the first six flashes in H2O was well described using the Kok model and a small miss factor (12.0 %), which was only slightly increased (to 13.5 %) in D2O. A very small (1 %) double-hit factor (PSII centers progressing by two steps in the Sstate cycle upon a flash) improved the fit and likely reflected imperfect shielding of PSII on neighboring locations of the sample holder against the laser flashes.23 More pronounced damping of the signal oscillations on higher flash numbers was explained by limitations in plastoquinone (QA) reoxidation at the reducing side of PSII between the flashes, presumably due to slow diffusion of the artificial electron acceptor in the partially dehydrated membrane particles. These results closely resembled our previous observations and facilitated deconvolution of the flash transients to yield the XAS transients of the pure S-transitions.24, 42 XAS transients of the pure S-transitions of PSII in H2O or D2O buffer are shown in Fig. 3. Similar amplitude patterns and quantitative reversal of the fluorescence changes in the S0n→S1n, S1n→S2+, and S2+→S3+ transitions during the O2-evolving S3+→S0n transition revealed similar processes at Mn4Ca in H2O and D2O (Fig. 3A). Their time constants were 9 ACS Paragon Plus Environment

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determined by exponential curve-fitting of the kinetic transients (Fig. 3B, Table 1). For the S0n→S1+ transition, time constants of about 50 µs in H2O and 65 µs in D2O and a small kinetic isotope effect (KIE = τH2O/τD2O) of ~1.3 reflected manganese oxidation due to electron transfer (ET) from Mn4Ca to the oxidized tyrosine acceptor (YZ+). A slower kinetic phase due to the S1+→S1n transition with a larger KIE attributed to proton transfer (PT) has been resolved in previous time-resolved experiments using a photothermal technique30, 33 (Table 1). This kinetic phase is not expected to be related to manganese oxidation and thus likely was invisible in the X-ray transients. For S1n→S2+, typical time constants of ~90 µs in H2O and ~110 µs in D2O and a small KIE of ~1.2 again reflected the Mn4Ca→YZ+ ET step. For the O2-evolving S3+→S0n transition, single-exponential simulations of the X-ray transients were insufficient to describe the kinetic behavior (Fig. 3B). In this case, a lag phase prior to the fluorescence rise in milliseconds was observed both in H2O and D2O. Satisfactory simulation of the S3+→S0n transients was achieved including the lag phase behavior according to Eq. 1, which describes a sequential reaction scheme. The fits revealed apparent time constants of the lag phase of ~150 µs in H2O and ~380 µs in D2O and thus a significant KIE of ~2.4. This KIE was similar to the value previously determined for the lag phase53, 58 (Table 1). Based on complementary recombination fluorescence data, its pH dependence and larger KIE compared to the ET steps, analysis of electrochromism, time-resolved O2 polarography on genetically modified PSII, and further circumstantial evidence, the lag phase has been attributed to proton removal from Mn4Ca.11, 14, 24, 31, 37, 53, 58, 59 The τ-values of ~1.55 ms (H2O) and ~2.20 ms (D2O) of the step causing manganese reduction during S3→S0 correspond to a moderate KIE of ~1.4, suggesting that the rate of the O2-evolving reaction itself was not severely limited by PT events. For the S2+→S3+ transition, a single-exponential simulation of the XAS transient in H2O within noise limits reasonably described the kinetic behavior (Fig. 3B). However, the transient in D2O was insufficiently described by a single-exponential decay. The fit residuals 10 ACS Paragon Plus Environment

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showed that such a simulation approach did not account for a signal fraction occurring shortly after the laser flash (Fig. 3B). This signal fraction of the S2+→S3+ transition in D2O was rather described by including a lag phase in the fit approach (Eq. 1) with an apparent time constant of ~115 µs. We attribute this lag phase to the S2+→S2n transition, as previously assigned in a photothermal experiment showing a similar time constant.30, 33 Using Eq. 1 also for simulation of the S2+→S3+ transient in H2O yielded a τ-value of ~25 µs and thus a particularly large KIE of ~4.5 for the lag phase (Table 1), which also agreed with our earlier result.30, 33 We note that in H2O buffer, the presence of a lag phase could not be concluded from the XAS data in an unambiguous way, but the XAS data clearly is compatible with a time constant close to the value determined for a PT step by means of our previous PBD experiments.30,

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The

combined evidence of the XAS and PBD data in D2O and H2O suggested that a lag phase behavior could be assigned to PT in the S2+→S2n transition, with an especially large H/D isotope effect. An alternative approach for determination of the lag phase duration during the S2+→S3+ and S3+→S0n transitions is shown in Fig. 4. Plotting the difference between the maximal signal amplitudes and the time-dependent signal amplitudes on a logarithmic scale transformed the exponential parts of the transients into straight lines, which when graphically extrapolated to the baseline levels, revealed lag phase durations of ~150 µs (H2O) and ~380 µs (D2O) and a KIE of ~2.4 for the S3+→S3n transition and of ~25 µs (H2O) and ~120 µs (D2O) and a KIE of ~4.8 for the S2+→S2n transition (Table 1). This graphical approach further supported the particularly large KIE of S2+→S2n. The main kinetic phase of the S2+→S3+ transients due to manganese oxidation showed time constants of ~320 µs (H2O) and ~570 µs (D2O) and a KIE of ~1.8, which was significantly larger compared to ET during S0n→S1+ and S1n→S2+, in agreement with previous results.34, 41, 53 Accordingly, the ET in S2n→S3+ likely was affected by coupling of the ET step to (local) PT events. 11 ACS Paragon Plus Environment

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Discussion Time-resolved XAS has revealed the kinetics of manganese oxidation or reduction during the S-transitions of PSII in H2O or D2O buffers. The present data confirm the time constants and small KIE values (1.1-1.3) of ET during S0n→S1+ and S1n→S2+ previously derived using various spectroscopic methods.33, 34, 41, 53, 60 The KIE is small, but exceeds unity, which suggests that primary H/D isotope effects, meaning breaking for example of an O-H bond, likely are not involved.61-64 Secondary effects, such as proton movements within hydrogen bonds,65-68 are more likely to contribute to limitations of the velocity of manganese oxidation during S0n→S1+ and S1n→S2+. This is supported by the negligible pH-dependence and small activation energies (Ea) of the S0n→S1+ and S1n→S2+ ET steps.33, 41, 53 14, 30, 33, 41, 53, 69 The present and earlier data show that manganese reduction during the O2-evolving transition is preceded by a kinetic (lag) phase.14,

17, 24, 30, 33, 37, 41, 53, 58, 70, 71

Because of its

relatively large KIE (~2.4) and of its significant pH-dependence and more sizeable Ea, the lag phase is attributed to PT during S3+→S3n. Previous analyses of the preceding phase have revealed its at least biphasic character, meaning that two or more consecutive PT steps are involved.12, 14, 32, 71 Proton movements from (substrate) water species bound at Mn4Ca, among amino acids close to Mn4Ca, and along proton channels extending towards carboxylate group clusters at the protein surface, as well as proton release into the bulk prior to YZ+ reduction likely relate to the S3+→S3n transition.11,

72-74

These reactions prepare the system for

manganese reduction by electrons from water and O2 formation. The relatively slow manganese reduction parallels O2 release, final proton release, and YZ+ reduction during the S3n→S0n transition, its time constant showing a small KIE (1.2-1.4), moderate Ea, and negligible pH-dependence.29, 30, 33, 34, 53, 58, 69, 71, 72 These findings may suggest a mixed kinetic limitation with contributions from ET, protonation changes of (metal-bound) water species and structural changes of Mn4Ca and the protein/water matrix, reverting the changes during the lower S-transitions, O=O bond formation and O2 release, and binding and partial 12 ACS Paragon Plus Environment

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deprotonation of new substrate water molecules. Various scenarios for the molecular sequence of events during S3n→S0n have been discussed (see, e.g., refs.

1, 2, 11, 22, 26, 75

).

Characterization of further intermediates in the reaction course is required for final elucidation of the O=O bond formation mechanism. Our XAS data directly show that a lag phase (~120 µs duration, S2+→S2n) precedes manganese oxidation during S2n→S3+ in PSII in D2O buffer. For PSII in H2O buffer, the lag phase was at the detection limit in the XAS transient and its maximal duration may be estimated as ~25 µs. The KIE thus was ~4.5, but also larger values are compatible with the XAS data. In earlier kinetic studies, we have determined similar τ-values, a KIE of 4.3-5.6, as well as the largest Ea and pH-dependence compared to the other S-transitions for a similar kinetic phase.30,

33

S2+→S2n is further accompanied by proton release into the bulk in the

presence of YZ+.72, 76 We therefore attribute this transition to a PT event (Fig. 5). The kinetic parameters suggest that for example the breaking of an O-H bond (and transfer of the proton to a neighboring group) gives rise to a primary H/D isotope effect and governs the S2+→S2n transition. The relatively large KIE (1.8-2.2), activation energy, and pronounced pH-dependence imply that S2n→S3+ may be considered as the only truly proton-coupled electron transfer (PCET) in the reaction cycle of photosynthetic water oxidation. In this case, proton movements for example between the terminal water ligands at Mn4 (W1 or W2) or Ca (W3 or W4) and the hydrogen-bonded network may occur concomitantly with binding of an additional oxygen species at Mn1(IV), thereby generating a fourth octahedral Mn(IV) ion in S3+ (Fig. 5).23 Alternative or even more intricate sequences of proton/water movements and charge transfer certainly are conceivable.21, 26, 75, 77-80 In conclusion, we propose that proton movements within the hydrogen-bonded network involving the Mn4Ca complex and its bound water ligands and surrounding amino


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acids, as well as water species and related nuclear rearrangements (structural changes) determine to different extents the rates of all ET steps in the S-state cycle and in particular the S2n→S3+ transition assigned as a PCET reaction. PT during S1+→S1n and S2+→S2n and thus prior to manganese oxidation facilitates three sequential manganese oxidation steps (S0n→S1+, S1n→S2+, S2n→S3+), inter alia by maintaining roughly constant relative redox potentials of YZ+ and Mn4Ca in each oxidation step of the catalytic cycle.11, 81, 82 The PT during S3+→S3n induced by YZ+ primes the active site for water oxidation and O=O bond formation.

Acknowledgements We thank the team of P. Glatzel at ID26 of ESRF for excellent technical support and L. Gerencsér for help in PSII sample preparation and XAS data collection.


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Table 1: Kinetic parameters of the S-transitions from time-resolved XAS.a

transition: assigned to: τ(H2O) [µs] τ(D2O) [µs] KIE (XAS)

S0n→S1+ ET 52(8) 66(6) 1.3±0.2

S1+→S1n PT -

S1n→S2+ ET 89(4) 108(4) 1.2±0.1

KIE (OAS) KIE (PBD)

1.3 -

2.9

1.2 1.3

a

S2+→S2n PT 26(7) 117(57) 4.5±2.0 [4.8] 4.9

S2n→S3+ PCET 317(23) 568(39) 1.8±0.2 1.7 1.9

S3+→S3n PT 153(35) 380(46) 2.5±0.7 [2.5] 2.4 -

S3n→S0n ET 1538(55) 2208(75) 1.4±0.1 1.2 1.3

Time constants (τ) stem from exponential curve-fitting of the X-ray transients in Fig. 3 (fit

error in parenthesis), values in brackets were derived from graphical analysis of transients (Fig. 4). KIE values from XAS (reasonable error ranges were estimated from the τ-errors) are compared to values from kinetic optical absorption spectroscopy (OAS)53 or photothermal beam deflection (PBD) experiments.30, 33 Annotations and assignments of S-transitions follow our previous studies.33


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Legends to Figures

Figure 1: Crystal structure and S-state cycle of the Mn4Ca complex of PSII. (A) The shown structure (PDB entry 4UB6, 1.95 Å resolution) was attributed to the S1 state (MnIII2MnIV2).15 Amino acids are denoted by standard abbreviations; magenta, direct amino acid ligands to Mn or Ca (except for Glu354 from the CP43 subunit, all amino acid ligands belong to the D1 subunit of PSII); W, red dots, and bluish shadings mark locations of water species within van der Waals radii; O5 denotes a specific bridging oxygen between Mn1 and Mn4. Mn2, Mn3, and Mn4 possess six O-ligands in distorted octahedral sites, one O-ligand at Mn1 is replaced by a N(His) ligand. The Mn1-O5 distance (blue dashes) is much longer than the other MnO/N bonds so that Mn1 in effect is 5-coordinated. Tyr161 (YZ) is the electron transfer partner of Mn4Ca. Atom color code: purple, Mn; green, Ca; light green, Cl; red, O; blue, N; grey, C; protons were not resolved in the structure. Dotted lines connect likely hydrogen-bonded donor/acceptor atoms (mostly O or N) separated by less than ~3 Å. (B) Extended S-state cycle proposed on the basis of previous results (n, same charge of Mn4Ca as in S1n; +, Mn4Ca upcharged by one unit compared to S1n)30 and its relation (dashes) to the classical S-states.19 Experimental support for the hypothetical (transient) S4+ state is still missing; evidence for PT after O2 release and substrate water binding to form S0+ comes from earlier experiments.14, 41

Figure 2: Time-resolved XAS at the Mn K-edge. (A) XANES spectra of PSII (at room temperature and in H2O buffer) after 0-4 laser flashes (predominantly S1, S2, S3, S0, and S1 states populated). Vertical dots mark the excitation energy of 6552 eV for time-resolved experiments in (B). Inset (a): XANES spectra around half-height (50 %) level in magnification and edge energy shifts due to flashes 1-4 (arrows). Inset (b): XANES difference spectra and X-ray florescence intensity changes at 6552 eV (arrows). (B) X-ray fluorescence transients at 6552 eV for PSII in H2O or D2O buffer in a series of 10 laser flashes. Inset: fitted 22 ACS Paragon Plus Environment

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amplitudes of the millisecond rising phase due to the O2-evolution transition, S3→S0, versus the flash number (solid circles) and simulations (lines) using the Kok model with the following parameters: initial S1 = 100 %, 10 % centers undergoing the S1→S2 transition only on flash 1, 1 % double-hits, and miss factors of 12.0 % in H2O and 13.5 % in D2O.

Figure 3: Kinetic analysis of XAS transients. (A) Deconvoluted XAS transients due to the four S-transitions (derived from data in Fig. 2 and shown at a resolution of 20 µs per point). (B) XAS transients of the four S-transitions in magnification and simulations (smooth lines, parameters in Table 1) using single-exponential functions (S0→S1, S1→S2) or a function (Eq. 1) including a lag phase (S2→S3, S3→S0). Dark-yellow (H2O) and green (D2O) curves for S2→S3 and S3→S0 represent single-exponential simulations. (C) Fit residuals (Savitzky-Golay 3rd-order polynomial smoothing over 10 data points) for single-exponential simulations (darkyellow and green curves) or simulations including a lag phase (blue and red curves); note deviations from baseline levels around t = 0 indicating lag-phase behavior.

Figure 4: Graphical lag phase determination. Amplitudes of XAS transients in Fig. 3B were transformed as indicated on the y-axis (Amax is the fitted maximal amplitude at t = 10 ms after the flash relative to the baseline at zero level in Fig. 3B) and plotted on a logarithmic amplitude scale. Straight lines represent extrapolations of fit curves including a lag phase to the baseline levels (dashes) for estimation of lag durations (dashed lines show exponential fits without a lag phase).

Figure 5: PT and ET during the S2→S3 transition. Starting from the YZ+S2+ state containing a 5-coordinated Mn1(III) ion,15, 23 the S2+→S2n transition accounts for a kinetic lag phase in the X-ray absorption transient due to manganese oxidation, an apparent PSII volume expansion (structural change),30, 33 both showing a particularly large (~4.5) KIE, and for rapid 23 ACS Paragon Plus Environment

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proton release to the bulk in the presence of YZ+,72 which facilitate the subsequent manganese oxidation by YZ+. The latter event is coupled to proton rearrangements, for example due to PT between a water ligand at Mn or Ca and the hydrogen-bonded network, and may involve ligand binding at the newly formed Mn1(IV) (structural change),23 which determines the rate of the proton-coupled electron transfer (PCET) reaction S2n→S3+. Ligand binding at Mn1 to reach a Mn(IV)3L6Mn(III)L6 state prior to the PCET step is a further conceivable option.


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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Table of Contents Entry D2O/H2O isotope effects in the water oxidation cycle of photosystem II, detected by timeresolved XAS, revealed proton transfer preceding manganese oxidation in S2→S3, priming the active site for subsequent PCET.

Table of Contents Graphic


Sequential and Coupled Proton and Electron Transfer Events in the

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