S-state Cycle Research Articles

Tracking the first electron transfer step at the donor side of oxygen-evolving photosystem II by time-resolved infrared spectroscopy.

In oxygen-evolving photosystem II (PSII), the multi-phasic electron transfer from a redox-active tyrosine residue (TyrZ) to a chlorophyll cation radical (P680+) precedes the water-oxidation chemistry of the S-state cycle of the Mn4Ca cluster. Here we investigate these early events, observable within about 10ns to 10ms after laser-flash excitation, by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of 1310-1890cm-1 for oxygen-evolving PSII membrane particles from spinach. Comparing the IR difference spectra at 80ns, 500ns, and 10µs allowed for the identification of quinone, P680 and TyrZ contributions. A broad electronic absorption band assignable P680+ was used to trace largely specifically the P680+ reduction kinetics. The experimental time resolution was taken into account in least-square fits of P680+ transients with a sum of four exponentials, revealing two nanosecond phases (30-46ns and 690-1110ns) and two microsecond phases (4.5-8.3µs and 42µs), which mostly exhibit a clear S-state dependence, in agreement with results obtained by other methods. Our investigation paves the road for further insight in the early events associated with TyrZ oxidation and their role in the preparing the PSII donor side for the subsequent water oxidation chemistry.

Does Serial Femtosecond Crystallography Depict State-Specific Catalytic Intermediates of the Oxygen-Evolving Complex?

Recent advances in serial femtosecond crystallography (SFX) of photosystem II (PSII), enabled by X-ray free electron lasers (XFEL), provided the first geometric models of distinct intermediates in the catalytic S-state cycle of the oxygen-evolving complex (OEC). These models are obtained by flash-advancing the OEC from the dark-stable state (S1) to more oxidized intermediates (S2 and S3), eventually cycling back to the most reduced S0. However, the interpretation of these models is controversial because geometric parameters within the Mn4CaO5 cluster of the OEC do not exactly match those expected from coordination chemistry for the spectroscopically verified manganese oxidation states of the distinct S-state intermediates. Here we focus on the first catalytic transition, S1 → S2, which represents a one-electron oxidation of the OEC. Combining geometric and electronic structure criteria, including a novel effective oxidation state approach, we analyze existing 1-flash (1F) SFX-XFEL crystallographic models that should depict the S2 state of the OEC. We show that the 1F/S2 equivalence is not obvious, because the Mn oxidation states and total unpaired electron counts encoded in these models are not fully consistent with those of a pure S2 state and with the nature of the S1 → S2 transition. Furthermore, the oxidation state definition in two-flashed (2F) structural models is practically impossible to elucidate. Our results advise caution in the extraction of electronic structure information solely from the literal interpretation of crystallographic models and call for re-evaluation of structural and mechanistic interpretations that presume exact correspondence of such models to specific catalytic intermediates of the OEC.

Probing the proton release by Photosystem II in the S1 to S2 high-spin transition

The stoichiometry and kinetics of the proton release were investigated during each transition of the S-state cycle in Photosystem II (PSII) from Thermosynechococcus elongatus containing either a Mn4CaO5 (PSII/Ca) or a Mn4SrO5 (PSII/Sr) cluster. The measurements were done at pH 6.0 and pH 7.0 knowing that, in PSII/Ca at pH 6.0 and pH 7.0 and in PSII/Sr at pH 6.0, the flash-induced S2-state is in a low-spin configuration (S2LS) whereas in PSII/Sr at pH 7.0, the S2-state is in a high-spin configuration (S2HS) in half of the centers. Two measurements were done; the time-resolved flash dependent i) absorption of either bromocresol purple at pH 6.0 or neutral red at pH 7.0 and ii) electrochromism in the Soret band of PD1 at 440 nm. The fittings of the oscillations with a period of four indicate that one proton is released in the S1 to S2HS transition in PSII/Sr at pH 7.0. It has previously been suggested that the proton released in the S2LS to S3 transition would be released in a S2LSTyrZ• → S2HSTyrZ• transition before the electron transfer from the cluster to TyrZ• occurs. The release of a proton in the S1TyrZ• → S2HSTyrZ transition would logically imply that this proton release is missing in the S2HSTyrZ• to S3TyrZ transition. Instead, the proton release in the S1 to S2HS transition in PSII/Sr at pH 7.0 was mainly done at the expense of the proton release in the S3 to S0 and S0 to S1 transitions. However, at pH 7.0, the electrochromism of PD1 seems larger in PSII/Sr when compared to PSII/Ca in the S3 state. This points to the complex link between proton movements in and immediately around the Mn4 cluster and the mechanism leading to the release of protons into the bulk.

Redox Tuning via Ligand-Induced Geometric Distortions at a YMn3O4 Cubane Model of the Biological Oxygen Evolving Complex.

The function of proteins involved in electron transfer is dependent on cofactors attaining the necessary reduction potentials. We establish a mode of cluster redox tuning through steric pressure on a synthetic model related to Photosystem II. Resembling the cuboidal [CaMn3O4] subsite of the biological oxygen evolving complex (OEC), [Mn4O4] and [YMn3O4] complexes featuring ligands of different basicity and chelating properties were characterized by cyclic voltammetry. In the absence of ligand-induced distortions, increasing the basicity of the ligands results in a decrease of cluster reduction potential. Contraction of Y-oxo/Y–Mn distances by 0.1/0.15 Å enforced by a chelating ligand results in an increase of cluster reduction potential even in the presence of strongly basic donors. Related protein-induced changes in Ca-oxo/Ca–Mn distances may have similar effects in tuning the redox potential of the OEC through entatic states and may explain the cation size dependence on the progression of the S-state cycle.

Energetics and Kinetics of S-State Transitions Monitored by Delayed Chlorophyll Fluorescence.

Understanding energetic and kinetic parameters of intermediates formed in the course of the reaction cycle (S-state cycle) of photosynthetic water oxidation is of high interest and could support the rationale designs of artificial systems for solar fuels. We use time-resolved measurements of the delayed chlorophyll fluorescence to estimate rate constants, activation energies, free energy differences, and to discriminate between the enthalpic and the entropic contributions to the decrease of the Gibbs free energy of the individual transitions. Using a joint-fit simulation approach, kinetic parameters are determined for the reaction intermediates in the S-state transitions in buffers with different pH in H2O and in D2O.

Spin, Valence, and Structural Isomerism in the S3 State of the Oxygen-Evolving Complex of Photosystem II as a Manifestation of Multimetallic Cooperativity.

Photosynthetic water oxidation is catalyzed by a Mn4CaO5-cluster in photosystem II through an S-state cycle. Understanding the roles of heterogeneity in each S-state, as identified recently by the EPR spectroscopy, is very important to gain a complete description of the catalytic mechanism. We performed herein hybrid DFT calculations within the broken-symmetry formalism and associated analyses of Heisenberg spin models to study the electronic and spin structures of various isomeric structural motifs (hydroxo-oxo, oxyl-oxo, peroxo, and superoxo species) in the S3 state. Our extensive study reveals several factors that affect the spin ground state: (1) (formal) Mn oxidation state; (2) metal-ligand covalency; (3) coordination geometry; and (4) structural change of the Mn cluster induced by alternations in Mn···Mn distances. Some combination of these effects could selectively stabilize/destabilize some spin states. We found that the high spin state ( Stotal = 6) of the oxyl-oxo species can be causative for catalytic function, which manifests through mixing of the metal-ligand character in magnetic orbitals at relatively short O5···O6 distances (<2.0 Å) and long MnA···O5 distances (>2.0 Å). These results will serve as a basis to conceptually identify and rationalize the physicochemical synergisms that can be evoked by the unique "distorted chair" topology of the cluster through cooperative Jahn-Teller effects on multimetallic centers.

New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in Thermosynechococcus elongatus

The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg2+ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn4CaO5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1+PheoD1− radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of 1O2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to ChlD1 upon QA− formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 itself is the chlorophyll that is first damaged by 1O2, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.

Mechanism of Methanol Inhibition of Photosynthetic Water Oxidation As Studied by Fourier Transform Infrared Difference and Time-Resolved Infrared Spectroscopies.

Photosynthetic water oxidation is performed at the Mn4CaO5 cluster in photosystem II. In this study, we investigated the effect of methanol, an analogue of water, on the water oxidation reaction and its interaction site using Fourier transform infrared (FTIR) difference and time-resolved infrared (TRIR) spectroscopies. Flash-induced FTIR difference measurement of the S-state cycle showed that methanol decreases mainly the efficiency of the S3 → S0 transition. TRIR measurement further showed that methanol slowed the rates of the S2 → S3 and S3 → S0 transitions. FTIR difference spectra upon the S1 → S2 transition exhibited prominent methanol-induced changes in the amide I and II bands of the main chains, whereas little change was observed in the bands of carboxylate groups, histidine side chains, and a water network in the vicinity of the Mn4CaO5 cluster. Similar tendencies were also observed with ethanol and 2-propanol. The C-O stretching vibration of methanol was further identified in the S2-minus-S1 spectrum using 18O-labeled methanol. These results indicate that methanol and small alcohols are bound near the Mn4CaO5 cluster but with no direct interaction. They probably replace a water molecule in a water channel around the Mn4CaO5 cluster, possibly interacting with a main chain amide. It is thus suggested that this replacement of water with methanol or a small alcohol inhibits water/proton transfer during the S2 → S3 and S3 → S0 transitions, which in turn provides experimental support for the view that these two transitions involve the water uptake and proton release processes.

Fourier Transform Infrared Analysis of the S-State Cycle of Water Oxidation in the Microcrystals of Photosystem II.

Photosynthetic water oxidation is performed in photosystem II (PSII) through a light-driven cycle of intermediates called S states (S0-S4) at the water oxidizing center. Time-resolved serial femtosecond crystallography (SFX) has recently been applied to the microcrystals of PSII to obtain the structural information on these intermediates. However, it remains unanswered whether the reactions efficiently proceed throughout the S-state cycle retaining the native structures of the intermediates in PSII crystals. We investigated the water oxidation reactions in the PSII microcrystals using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. In comparison with the FTIR spectra in solution, it was shown that all of the metastable intermediates in the microcrystals retained their native structures, and the efficiencies of the S-state transitions remained relatively high, although those of the S2 → S3 and S3 → S0 transitions were slightly lowered possibly due to some restriction of water movement in the crystals.

D1-Asn-298 in photosystem II is involved in a hydrogen-bond network near the redox-active tyrosine YZ for proton exit during water oxidation

In photosynthetic water oxidation, two water molecules are converted into one oxygen molecule and four protons at the Mn4CaO5 cluster in photosystem II (PSII) via the S-state cycle. Efficient proton exit from the catalytic site to the lumen is essential for this process. However, the exit pathways of individual protons through the PSII proteins remain to be identified. In this study, we examined the involvement of a hydrogen-bond network near the redox-active tyrosine YZ in proton transfer during the S-state cycle. We focused on spectroscopic analyses of a site-directed variant of D1-Asn-298, a residue involved in a hydrogen-bond network near YZ We found that the D1-N298A mutant of Synechocystis sp. PCC 6803 exhibits an O2 evolution activity of ∼10% of the wild-type. D1-N298A and the wild-type D1 had very similar features of thermoluminescence glow curves and of an FTIR difference spectrum upon YZ oxidation, suggesting that the hydrogen-bonded structure of YZ and electron transfer from the Mn4CaO5 cluster to YZ were little affected by substitution. In the D1-N298A mutant, however, the flash-number dependence of delayed luminescence showed a monotonic increase without oscillation, and FTIR difference spectra of the S-state cycle indicated partial and significant inhibition of the S2 → S3 and S3 → S0 transitions, respectively. These results suggest that the D1-N298A substitution inhibits the proton transfer processes in the S2 → S3 and S3 → S0 transitions. This in turn indicates that the hydrogen-bond network near YZ can be functional as a proton transfer pathway during photosynthetic water oxidation.

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn4CaO5 cluster and QB reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to QA reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of QA− and P700+, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.

X-ray Free Electron Laser Radiation Damage through the S-State Cycle of the Oxygen-Evolving Complex of Photosystem II.

The oxygen-evolving complex (OEC) catalyzes water-splitting through a reaction mechanism that cycles the OEC through the "S-state" intermediates. Understanding structure/function relationsships of the S-states is crucial for elucidating the water-oxidation mechanism. Serial femtosecond X-ray crystallography has been used to obtain radiation damage-free structures. However, it remains to be established whether "diffraction-before-destruction" is actually accomplished or if significant changes are produced by the high-intensity X-ray pulses during the femtosecond scattering measurement. Here, we use ab initio molecular dynamics simulations to estimate the extent of structural changes induced on the femtosecond time scale. We found that the radiation damage is dependent on the bonding and charge of each atom in the OEC, in a manner that may provide lessons for XFEL studies of other metalloproteins. The maximum displacement of Mn and oxygen centers is 0.25 and 0.39 Å, respectively, during the 50 fs pulse, which is significantly smaller than the uncertainty given the 1.9 Å resolution of the current PSII crystal structures. However, these structural changes might be detectable when comparing isomorphous Fourier differences of electron density maps of the different S-states. One conclusion is that pulses shorter than 15 fs should be used to avoid significant radiation damage.

Infrared Determination of the Protonation State of a Key Histidine Residue in the Photosynthetic Water Oxidizing Center.

Photosynthetic water oxidation is performed at the Mn4CaO5 cluster in photosystem II (PSII). The protonation structures of amino acid residues and water molecules around the Mn4CaO5 cluster are crucial in water oxidation reactions. In this study, we determined the protonation state of a key His residue in water oxidation, D1-H337, that is directly hydrogen-bonded with the oxygen atom of the Mn4CaO5 cluster, using polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Flash-induced polarized ATR-FTIR difference spectra upon the S1 → S2 transition of oriented PSII membranes showed broad negative and positive features at about 2600 and 2900 cm-1, respectively, with large dichroic ratios, accompanied by several minor peaks attributable to the Fermi resonance of a His NH vibration. Quantum mechanics/molecular mechanics (QM/MM) calculations well reproduced the characteristics of these features as the NτH stretching vibrations of D1-H337 in its protonated cation form. The spectral features were reversed in the S3 → S0 transition, indicating that this His remains protonated during the S-state cycle. The redox potential (Em) of the Mn4CaO5 cluster in the S1 → S2 transition, which was estimated from the QM/MM calculations, was found to be comparable to that of water oxidation when D1-H337 is protonated cation. It was thus concluded that the positive charge on the protonated D1-H337 plays an important role in retaining a high Em value of the Mn4CaO5 cluster throughout the reaction cycle to enable water oxidation.

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.

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 the S2n → S3+ step (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) steps 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. 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.

Quantum mechanics/molecular mechanics simulation of the ligand vibrations of the water-oxidizing Mn4CaO5 cluster in photosystem II

During photosynthesis, the light-driven oxidation of water performed by photosystem II (PSII) provides electrons necessary to fix CO2, in turn supporting life on Earth by liberating molecular oxygen. Recent high-resolution X-ray images of PSII show that the water-oxidizing center (WOC) is composed of an Mn4CaO5 cluster with six carboxylate, one imidazole, and four water ligands. FTIR difference spectroscopy has shown significant structural changes of the WOC during the S-state cycle of water oxidation, especially within carboxylate groups. However, the roles that these carboxylate groups play in water oxidation as well as how they should be properly assigned in spectra are unresolved. In this study, we performed a normal mode analysis of the WOC using the quantum mechanics/molecular mechanics (QM/MM) method to simulate FTIR difference spectra on the S1 to S2 transition in the carboxylate stretching region. By evaluating WOC models with different oxidation and protonation states, we determined that models of high-oxidation states, Mn(III)2Mn(IV)2, satisfactorily reproduced experimental spectra from intact and Ca-depleted PSII compared with low-oxidation models. It is further suggested that the carboxylate groups bridging Ca and Mn ions within this center tune the reactivity of water ligands bound to Ca by shifting charge via their π conjugation.

Room-Temperature Energy-Sampling Kβ X-ray Emission Spectroscopy of the Mn4Ca Complex of Photosynthesis Reveals Three Manganese-Centered Oxidation Steps and Suggests a Coordination Change Prior to O2 Formation.

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kβ1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kβ1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kβ1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the S1 → S2 transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S2 → S3 transition, which could be oxidation of five-coordinated Mn(III) to six-coordinated Mn(IV). Conceivable options for the rearrangement of (substrate) water species and metal-ligand bonding patterns at the Mn4Ca complex in the S2 → S3 transition are discussed.

Native structure of photosystem II at 1.95Å resolution viewed by femtosecond X-ray pulses.

Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 ångström resolution, which revealed that the OEC is a Mn4CaO5-cluster coordinated by a well defined protein environment. However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation, and slight differences were found in the Mn-Mn distances determined by XRD, EXAFS and theoretical studies. Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanus in the S1 state at a resolution of 1.95 ångströms using femtosecond X-ray pulses of the SPring-8 ångström compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1-0.2 ångströms. The valences of each manganese atom were tentatively assigned as Mn1D(III), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation.

The first tyrosyl radical intermediate formed in the S2-S3 transition of photosystem II.

The EPR "split signals" represent key intermediates of the S-state cycle where the redox active D1-Tyr161 (YZ) has been oxidized by the reaction center of the photosystem II enzyme to its tyrosyl radical form, but the successive oxidation of the Mn4CaO5 cluster has not yet occurred (SiYZ˙). Here we focus on the S2YZ˙ state, which is formed en route to the final metastable state of the catalyst, the S3 state, the state which immediately precedes O-O bond formation. Quantum chemical calculations demonstrate that both isomeric forms of the S2 state, the open and closed cubane isomers, can form states with an oxidized YZ˙ residue without prior deprotonation of the Mn4CaO5 cluster. The two forms are expected to lie close in energy and retain the electronic structure and magnetic topology of the corresponding S2 state of the inorganic core. As expected, tyrosine oxidation results in a proton shift towards His190. Analysis of the electronic rearrangements that occur upon formation of the tyrosyl radical suggests that a likely next step in the catalytic cycle is the deprotonation of a terminal water ligand (W1) of the Mn4CaO5 cluster. Diamagnetic metal ion substitution is used in our calculations to obtain the molecular g-tensor of YZ˙. It is known that the gx value is a sensitive probe not only of the extent of the proton shift between the tyrosine-histidine pair, but also of the polarization environment of the tyrosine, especially about the phenolic oxygen. It is shown for PSII that this environment is determined by the Ca(2+) ion, which locates two water molecules about the phenoxyl oxygen, indirectly modulating the oxidation potential of YZ.

Biological Water Oxidation

Photosystem II (PSII), a multisubunit pigment-protein supercomplex found in cyanobacteria, algae, and plants, catalyzes a unique reaction in nature: the light-driven oxidation of water. Remarkable recent advances in the structural analysis of PSII now give a detailed picture of the static supercomplex on the molecular level. These data provide a solid foundation for future functional studies, in particular the mechanism of water oxidation and oxygen release. The catalytic core of the PSII is a tetramanganese-calcium cluster (Mn₄O₅Ca), commonly referred to as the oxygen-evolving complex (OEC). The function of the OEC rests on its ability to cycle through five metastable states (Si, i = 0-4), transiently storing four oxidizing equivalents, and in so doing, facilitates the four electron water splitting reaction. While the latest crystallographic model of PSII gives an atomic picture of the OEC, the exact connectivity within the inorganic core and the S-state(s) that the X-ray model represents remain uncertain. In this Account, we describe our joint experimental and theoretical efforts to eliminate these ambiguities by combining the X-ray data with spectroscopic constraints and introducing computational modeling. We are developing quantum chemical methods to predict electron paramagnetic resonance (EPR) parameters for transition metal clusters, especially focusing on spin-projection approaches combined with density functional theory (DFT) calculations. We aim to resolve the geometric and electronic structures of all S-states, correlating their structural features with spectroscopic observations to elucidate reactivity. The sequence of manganese oxidations and concomitant charge compensation events via proton transfer allow us to rationalize the multielectron S-state cycle. EPR spectroscopy combined with theoretical calculations provides a unique window into the tetramangenese complex, in particular its protonation states and metal ligand sphere evolution, far beyond the scope of static techniques such as X-ray crystallography. This approach has led, for example, to a detailed understanding of the EPR signals in the S₂-state of the OEC in terms of two interconvertible, isoenergetic structures. These two structures differ in their valence distribution and spin multiplicity, which has important consequences for substrate binding and may explain its low barrier exchange with solvent water. New experimental techniques and innovative sample preparations are beginning to unravel the complex sequence of substrate uptake/inclusion, which is coupled to proton release. The introduction of specific site perturbations, such as replacing Ca²⁺ with Sr²⁺, provides discrete information about the ligand environment of the individual Mn ions. In this way, we have identified a potential open coordination site for one Mn center, which may serve as a substrate binding site in the higher S-states, such as S₃ and S₄. In addition, we can now monitor the binding of the substrate water in the lower S-states (S₁ and S₂) using new EPR-detected NMR spectroscopies. These studies provided the first evidence that one of the substrates is subsumed into the complex itself and forms an oxo-bridge between two Mn ions. This result places important new restrictions on the mechanism of O-O bond formation. These new insights from nature's water splitting catalyst provide important criteria for the rational design of bioinspired synthetic catalysts.

Determination of the Miss Probabilities of Individual S-State Transitions during Photosynthetic Water Oxidation by Monitoring Electron Flow in Photosystem II Using FTIR Spectroscopy

Water oxidation by plants and cyanobacteria is performed via a light-driven cycle of five intermediates called S states (S(0)-S(4)) at the water oxidizing center (WOC) in photosystem II (PSII). The information about misses, i.e., the probabilities that the S-state transitions failed to advance, is crucial for detailed analysis of various spectroscopic data in investigations of the water oxidation mechanism. In this study, we have determined the miss probabilities of the individual S-state transitions using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The extent of S-state transitions in the WOC upon each saturating flash was monitored by detecting the flow of electrons from the WOC to ferricyanide, an exogenous electron acceptor, using the CN stretching bands of ferricyanide and ferrocyanide. Simulation of the oscillation pattern of the flash-number dependence of the signal amplitude provided the miss probabilities for the S(0) → S(1), S(1) → S(2), S(2) → S(3), and S(3) → S(0) transitions (α(0)-α(3), respectively) without any assumption about fitting parameters. The results for PSII preparations from Thermosynechococcus elongatus and spinach showed a general tendency of misses in the order, α(0) ≤ α(1) < α(2) < α(3), indicating that a more oxidized WOC has a higher miss probability. A very similar result observed for the Y(D)-less mutant (D2-Y160F) of T. elongatus confirmed that Y(D) does not affect the estimated misses. It was further shown that NO(3)(-) treatment specifically increased α(3), consistent with inactivation of the S(3) state reported previously. These results demonstrate the usefulness of this FTIR method for estimating individual miss probabilities in the S-state cycle in elucidation of the molecular mechanism of photosynthetic water oxidation.