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The understanding of light-induced biological water oxidation in oxygenic photosynthesis is of great importance both for biology and (bio)technological applications. The chemically difficult multistep reaction takes place at a unique protein-bound tetra-manganese/calcium cluster in photosystem II whose structure has been elucidated by X-ray crystallography (Umena et al. Nature 2011, 473, 55). The cluster moves through several intermediate states in the catalytic cycle. A detailed understanding of these intermediates requires information about the spatial and electronic structure of the Mn 4 Ca complex; the latter is only available from spectroscopic techniques. Here, the important role of Electron Paramagnetic Resonance (EPR) and related double resonance techniques (ENDOR, EDNMR), complemented by quantum chemical calculations, is described. This has led to the elucidation of the cluster's redox and protonation states, the valence and spin states of the manganese ions and the interactions between them, and contributed substantially to the understanding of the role of the protein surrounding, as well as the binding and processing of the substrate water molecules, the O-O bond formation and dioxygen release. Based on these data, models for the water oxidation cycle are developed., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Photosystem-II uses sunlight to trigger charge separation and catalyze water oxidation. Intrinsic properties of chlorophyll a pigments define a natural "red limit" of photosynthesis at ≈680 nm. Nevertheless, charge separation can be triggered with far-red photons up to 800 nm, without altering the nature of light-harvesting pigments. Here we identify the electronic origin of this remarkable phenomenon using quantum chemical and multiscale simulations on a native Photosystem-II model. We find that the reaction center is preorganized for charge separation in the far-red region by specific chlorophyll-pheophytin pairs, potentially bypassing the light-harvesting apparatus. Charge transfer can occur along two distinct pathways with one and the same pheophytin acceptor (Pheo D1 ). The identity of the donor chlorophyll (Chl D1 or P D1 ) is wavelength-dependent and conformational dynamics broaden the sampling of the far-red region by the two charge-transfer states. The two pathways rationalize spectroscopic observations and underpin designed extensions of the photosynthetically active radiation limit., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn 4 CaO x complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.

Heterogeneity in intermediate catalytic states of the oxygen-evolving complex (OEC) of Photosystem II is known from a wide range of experimental and theoretical data, but its potential implications for the mechanism of water oxidation remain unexplored. We delineate the consequences of structural heterogeneity for the final step of the catalytic cycle by tracing the evolution of three spectroscopically relevant and structurally distinct components of the last metastable S 3 state to the transient O 2 -evolving S 4 state of the OEC. Using quantum chemical calculations, we show that each S 3 isomer leads to a different electronic structure formulation for the active S 4 state. Crucially, in addition to previously hypothesized Mn(IV)-oxyl species, we establish for the first time, how a genuine Mn(V)-oxo can be obtained in the catalytically active S 4 state: this takes the form of a five-coordinate and locally high-spin (S Mn  = 1) Mn(V) site. This formulation for the S 4 state evolves naturally from a preceding S 3 -state structural intermediate that contains a quasi-trigonal-bipyramidal Mn(IV) ion. The results strongly suggest that water binding in the S 3 state is not prerequisite for reaching the oxygen-evolving S 4 state of the complex, supporting the notion that both substrates are preloaded at the beginning of the catalytic cycle. This scenario allows true four-electron metal-centered hole accumulation to precede OO bond formation and hence the latter can proceed via a genuine even-electron mechanism. This can occur as intramolecular nucleophilic coupling of two oxo units synchronously with the binding of a water substrate for the next catalytic cycle., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In biological water oxidation, a redox-active tyrosine residue (D1-Tyr161 or Y Z ) mediates electron transfer between the Mn 4 CaO 5 cluster of the oxygen-evolving complex and the charge-separation site of photosystem II (PSII), driving the cluster through progressively higher oxidation states S i ( i = 0-4). In contrast to lower S-states (S 0 , S 1 ), in higher S-states (S 2 , S 3 ) of the Mn 4 CaO 5 cluster, Y Z cannot be oxidized at cryogenic temperatures due to the accumulation of positive charge in the S 1 → S 2 transition. However, oxidation of Y Z by illumination of S 2 at 77-190 K followed by rapid freezing and charge recombination between Y Z • and the plastoquinone radical Q A •- allows trapping of an S 2 variant, the so-called S 2 trapped state (S 2 t ), that is capable of forming Y Z • at cryogenic temperature. To identify the differences between the S 2 and S 2 t states, we used the S 2 t Y Z • intermediate as a probe for the S 2 t state and followed the S 2 t Y Z • /Q A •- recombination kinetics at 10 K using time-resolved electron paramagnetic resonance spectroscopy in H 2 O and D 2 O. The results show that while S 2 t Y Z • /Q A •- recombination can be described as pure electron transfer occurring in the Marcus inverted region, the S 2 t → S 2 reversion depends on proton rearrangement and exhibits a strong kinetic isotope effect. This suggests that Y Z oxidation in the S 2 t state is facilitated by favorable proton redistribution in the vicinity of Y Z , most likely within the hydrogen-bonded Y Z -His190-Asn298 triad. Computational models show that tautomerization of Asn298 to its imidic acid form enables proton translocation to an adjacent asparagine-rich cavity of water molecules that functions as a proton reservoir and can further participate in proton egress to the lumen.

Multifrequency pulsed EPR data are reported for a series of oxygen bridged (μ-oxo/μ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-μ-oxo bridged complex and a bent, bis-μ-oxo-μ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the μ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.

First principle calculations of extended X-ray absorption fine structure (EXAFS) data have seen widespread use in bioinorganic chemistry, perhaps most notably for modeling the Mn4Ca site in the oxygen evolving complex (OEC) of photosystem II (PSII). The logic implied by the calculations rests on the assumption that it is possible to a priori predict an accurate EXAFS spectrum provided that the underlying geometric structure is correct. The present study investigates the extent to which this is possible using state of the art EXAFS theory. The FEFF program is used to evaluate the ability of a multiple scattering-based approach to directly calculate the EXAFS spectrum of crystallographically defined model complexes. The results of these parameter free predictions are compared with the more traditional approach of fitting FEFF calculated spectra to experimental data. A series of seven crystallographically characterized Mn monomers and dimers is used as a test set. The largest deviations between the FEFF calculated EXAFS spectra and the experimental EXAFS spectra arise from the amplitudes. The amplitude errors result from a combination of errors in calculated S0(2) and Debye-Waller values as well as uncertainties in background subtraction. Additional errors may be attributed to structural parameters, particularly in cases where reliable high-resolution crystal structures are not available. Based on these investigations, the strengths and weaknesses of using first-principle EXAFS calculations as a predictive tool are discussed. We demonstrate that a range of DFT optimized structures of the OEC may all be considered consistent with experimental EXAFS data and that caution must be exercised when using EXAFS data to obtain topological arrangements of complex clusters.

The photosynthetic protein complex photosystem II oxidizes water to molecular oxygen at an embedded tetramanganese-calcium cluster. Resolving the geometric and electronic structure of this cluster in its highest metastable catalytic state (designated S3) is a prerequisite for understanding the mechanism of O-O bond formation. Here, multifrequency, multidimensional magnetic resonance spectroscopy reveals that all four manganese ions of the catalyst are structurally and electronically similar immediately before the final oxygen evolution step; they all exhibit a 4+ formal oxidation state and octahedral local geometry. Only one structural model derived from quantum chemical modeling is consistent with all magnetic resonance data; its formation requires the binding of an additional water molecule. O-O bond formation would then proceed by the coupling of two proximal manganese-bound oxygens in the transition state of the cofactor., (Copyright © 2014, American Association for the Advancement of Science.)

The S2 state of the oxygen-evolving complex of photosystem II, which consists of a Mn4O5Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin ST = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca(2+)/Sr(2+)-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S2 state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, (55)Mn-ENDOR) and their first coordination sphere ligands ((14)N/(15)N- and (17)O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, (55)Mn and (14)N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH3 with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer MnA4 to the cuboidal unit Mn3O3Ca, resulting in predictable changes of the measured effective (55)Mn hyperfine tensors. Sr(2+) and NH3 replacement both affect the observed (17)O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522-3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55-60]}; (ii) ammonia displaces a water ligand to the outer manganese (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.

Protonation states of water ligands and oxo bridges are intimately involved in tuning the electronic structures and oxidation potentials of the oxygen evolving complex (OEC) in Photosystem II, steering the mechanistic pathway, which involves at least five redox state intermediates S(n) (n = 0-4) resulting in the oxidation of water to molecular oxygen. Although protons are practically invisible in protein crystallography, their effects on the electronic structure and magnetic properties of metal active sites can be probed using spectroscopy. With the twin purpose of aiding the interpretation of the complex electron paramagnetic resonance (EPR) spectroscopic data of the OEC and of improving the view of the cluster at the atomic level, a complete set of protonation configurations for the S(2) state of the OEC were investigated, and their distinctive effects on magnetic properties of the cluster were evaluated. The most recent X-ray structure of Photosystem II at 1.9 Å resolution was used and refined to obtain the optimum structure for the Mn(4)O(5)Ca core within the protein pocket. Employing this model, a set of 26 structures was constructed that tested various protonation scenarios of the water ligands and oxo bridges. Our results suggest that one of the two water molecules that are proposed to coordinate the outer Mn ion (Mn(A)) of the cluster is deprotonated in the S(2) state, as this leads to optimal experimental agreement, reproducing the correct ground state spin multiplicity (S = 1/2), spin expectation values, and EXAFS-derived metal-metal distances. Deprotonation of Ca(2+)-bound water molecules is strongly disfavored in the S(2) state, but dissociation of one of the two water ligands appears to be facile. The computed isotropic hyperfine couplings presented here allow distinctions between models to be made and call into question the assumption that the largest coupling is always attributable to Mn(III). The present results impose limits for the total charge and the proton configuration of the OEC in the S(2) state, with implications for the cascade of events in the Kok cycle and for the water splitting mechanism., (© 2011 American Chemical Society)

The electronic properties of the Mn(4)O(x)Ca cluster in the S(2) state of the oxygen-evolving complex (OEC) were studied using X- and Q-band EPR and Q-band (55)Mn-ENDOR using photosystem II preparations isolated from the thermophilic cyanobacterium T. elongatus and higher plants (spinach). The data presented here show that there is very little difference between the two species. Specifically it is shown that: (i) only small changes are seen in the fitted isotropic hyperfine values, suggesting that there is no significant difference in the overall spin distribution (electronic coupling scheme) between the two species; (ii) the inferred fine-structure tensor of the only Mn(III) ion in the cluster is of the same magnitude and geometry for both species types, suggesting that the Mn(III) ion has the same coordination sphere in both sample preparations; and (iii) the data from both species are consistent with only one structural model available in the literature, namely the Siegbahn structure [Siegbahn, P. E. M. Accounts Chem. Res.2009, 42, 1871-1880, Pantazis, D. A. et al., Phys. Chem. Chem. Phys.2009, 11, 6788-6798]. These measurements were made in the presence of methanol because it confers favorable magnetic relaxation properties to the cluster that facilitate pulse-EPR techniques. In the absence of methanol the separation of the ground state and the first excited state of the spin system is smaller. For cyanobacteria this effect is minor but in plant PS II it leads to a break-down of the S(T)=½ spin model of the S(2) state. This suggests that the methanol-OEC interaction is species dependent. It is proposed that the effect of small organic solvents on the electronic structure of the cluster is to change the coupling between the outer Mn (Mn(A)) and the other three Mn ions that form the trimeric part of the cluster (Mn(B), Mn(C), Mn(D)), by perturbing the linking bis-μ-oxo bridge. The flexibility of this bridging unit is discussed with regard to the mechanism of O-O bond formation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Twelve structural models for the S(2) state of the oxygen-evolving complex (OEC) of photosystem II are evaluated in terms of their magnetic properties. The set includes ten models based on the 'fused twist' core topology derived by polarized EXAFS spectra and two related models proposed in recent mechanistic investigations. Optimized geometries and spin population analyses suggest that Mn(iii), which is most often identified with the manganese ion at site D, is always associated with a penta-coordinate environment, unless a chloride is directly ligated to the metal. Exchange coupling constants were determined by broken-symmetry density functional theory calculations and the complete spectrum of magnetic sublevels was obtained by direct diagonalization of the Heisenberg Hamiltonian. Seven models display a doublet ground state and are considered spectroscopic models for the ground state corresponding to the multiline signal (MLS) of the S(2) state of the OEC, whereas the remaining five models display a sextet ground state and could be related to the g = 4.1 signal of the S(2) state. It is found that the sign of the exchange coupling constant between the Mn centres at positions A and B of the cluster is directly related to the ground state multiplicity, implying that interconversion between the doublet and sextet can be induced by only small structural perturbations. The recently proposed quantum chemical method for the calculation of (55)Mn hyperfine coupling constants is subsequently applied to the S(2) MLS state models and the quantities that enter into the individual steps of the procedure (site-spin expectation values, intrinsic site isotropic hyperfine parameters and projected (55)Mn isotropic hyperfine constants) are analyzed and discussed in detail with respect to the structural and electronic features of each model. The current approach performs promisingly. It reacts sensitively to structural distortions and hence may be able to distinguish between different structural proposals. Thus it emerges as a useful contributor to the ongoing efforts that aim at establishing correlations between the body of spectroscopic data available for the various S(i) states of the OEC and their actual geometric features.