Nicholas Cox

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 natures water splitting catalyst provide important criteria for the rational design of bioinspired synthetic catalysts.

Electronic structure of the oxygen-evolving complex in photosystem II prior to O-O bond formation

Setting the stage for release of oxygen Plants transform water into the oxygen we breathe using a protein-bound cluster of four manganese (Mn) ions and a calcium ion. Cox et al. now establish the precise electronic structure in that cluster immediately before formation of the O-O bond (see the Perspective by Britt and Oyala). Using the technique of electron paramagnetic resonance spectroscopy, they confirm a hypothesis that all four Mn ions are octahedrally coordinated and in the 4+ oxidation state. Such clues to the efficiency of the photosynthetic process, so essential to life on Earth, may also facilitate the development of artificial waters-plitting catalysts. Science, this issue p. 804; see also p. 736 Electron paramagnetic resonance spectroscopy reveals that photosynthetic production of O2 proceeds from four MnIV ions. [Also see Perspective by Britt and Oyala] 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.

Two Interconvertible Structures that Explain the Spectroscopic Properties of the Oxygen-Evolving Complex of Photosystem II in the S2 State†

Using models derived from the X-ray structure of photosystem II, it is shown that the oxygen evolving complex in the S(2) state exists in two energetically similar and interconvertible forms. A longstanding question regarding the spectroscopy of the catalyst is thus answered: one form corresponds to the multiline g=2.0 EPR signal (see picture, right; O red, Mn purple, Ca yellow), and the other to the g≥4.1 signals (left).

Theoretical evaluation of structural models of the S2 state in the oxygen evolving complex of Photosystem II: protonation states and magnetic interactions.

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.

Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy.

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

Reflections on substrate water and dioxygen formation.

This brief article aims at presenting a concise summary of all experimental findings regarding substrate water-binding to the Mn4CaO5 cluster in photosystem II. Mass spectrometric and spectroscopic results are interpreted in light of recent structural information of the water oxidizing complex obtained by X-ray crystallography, spectroscopy and theoretical modeling. Within this framework current proposals for the mechanism of photosynthetic water-oxidation are evaluated. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Charge separation in Photosystem II: A comparative and evolutionary overview☆

Our current understanding of the PSII reaction centre owes a great deal to comparisons to the simpler and better understood, purple bacterial reaction centre. Here we provide an overview of the similarities with a focus on charge separation and the electron acceptors. We go on to discuss some of the main differences between the two kinds of reaction centres that have been highlighted by the improving knowledge of PSII. We attempt to relate these differences to functional requirements of water splitting. Some are directly associated with that function, e.g. high oxidation potentials, while others are associated with regulation and protection against photodamage. The protective and regulatory functions are associated with the harsh chemistry performed during its normal function but also with requirements of the enzyme while it is undergoing assembly and repair. Key aspects of PSII reaction centre evolution are also addressed. This article is part of a Special Issue entitled: Photosystem II.

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (μ-oxo) of the manganese tetramer

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.

A Tyrosyl−Dimanganese Coupled Spin System is the Native Metalloradical Cofactor of the R2F Subunit of the Ribonucleotide Reductase of Corynebacterium ammoniagenes

The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 A. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli . Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled Mn(III)Mn(III) dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (Mn(III)Mn(III)Y(*)) generation is proposed. H(2)O(2) (HO(2)(-)) instead of O(2) is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H(2)O(2) dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.

The electronic structures of the S(2) states of the oxygen evolving complexes of photosystem II in plants and cyanobacteria in the presence and absence of methanol

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.