Absence of Mn-centered oxidation in the S(2) --> S(3) transition: implications for the mechanism of photosynthetic water oxidation.
ABSTRACT: A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) --> [S(4)] --> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) --> S(3) transition, in contrast to the S(0) --> S(1) and S(1) --> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) --> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.
Project description:In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 ? S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 ? S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (? of ?350 µs) during the S2 ? S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
Project description:The oxygen-evolving complex of Photosystem II in plants and cyanobacteria catalyzes the oxidation of two water molecules to one molecule of dioxygen. A tetranuclear Mn complex is believed to cycle through five intermediate states (S0-S4) to couple the four-electron oxidation of water with the one-electron photochemistry occurring at the Photosystem II reaction center. We have used X-ray absorption spectroscopy to study the local structure of the Mn complex and have proposed a model for it, based on studies of the Mn K-edges and the extended X-ray absorption fine structure of the S1 and S2 states. The proposed model consists of two di-?-oxo-bridged binuclear Mn units with Mn-Mn distances of ~2.7 Å that are linked to each other by a mono-?-oxo bridge with a Mn-Mn separation of ~3.3 Å. The Mn-Mn distances are invariant in the native S1 and S2 states. This report describes the application of X-ray absorption spectroscopy to S3 samples created under physiological conditions with saturating flash illumination. Significant changes are observed in the Mn-Mn distances in the S3 state compared to the S1 and the S2 states. The two 2.7 Å Mn-Mn distances that characterize the di-?-oxo centers in the S1 and S2 states are lengthened to ~2.8 and 3.0 Å in the S3 state, respectively. The 3.3 Å Mn-Mn and Mn-Ca distances also increase by 0.04-0.2 Å. These changes in Mn-Mn distances are interpreted as consequences of the onset of substrate/water oxidation in the S3 state. Mn-centered oxidation is evident during the S0?S1 and S1?S2 transitions. We propose that the changes in Mn-Mn distances during the S2?S3 transition are the result of ligand or water oxidation, leading to the formation of an oxyl radical intermediate formed at a bridging or terminal position. The reaction of the oxyl radical with OH(-), H2O, or an oxo group during the subsequent S state conversion is proposed to lead to the formation of the O-O bond. Models that can account for changes in the Mn-Mn distances in the S3 state and the implications for the mechanism of water oxidation are discussed.
Project description:Calcium is an abundant, nontoxic metal that finds many roles in synthetic and biological systems including the oxygen-evolving complex (OEC) of photosystem II. Characterization methods for calcium centers, however, are underdeveloped compared to those available for transition metals. Valence-to-core X-ray emission spectroscopy (VtC XES) selectively probes the electronic structure of an element's chemical environment, providing insight that complements the geometric information available from other techniques. Here, the utility of calcium VtC XES is established using an in-house dispersive spectrometer in combination with density functional theory. Spectral trends are rationalized within a molecular orbital framework, and K?2,5 transitions, derived from molecular orbitals with primarily ligand p character, are found to be a promising probe of the calcium coordination environment. In particular, it is shown that calcium VtC XES is sensitive to the electronic structure changes that accompany oxo protonation in Mn3CaO4-based molecular mimics of the OEC. Through correlation to calculations, the potential of calcium VtC XES to address unresolved questions regarding the mechanism of biological water oxidation is highlighted.
Project description:The atmospheric dioxygen (O(2)) is produced at a tetramanganese complex bound to the proteins of photosystem II (PSII). To investigate product inhibition at elevated oxygen partial pressure (pO(2) ranging from 0.2 to 16 bar), we monitored specifically the redox reactions of the Mn complex in its catalytic S-state cycle by rapid-scan and time-resolved X-ray absorption near-edge spectroscopy (XANES) at the Mn K-edge. By using a pressure cell for X-ray measurements after laser-flash excitation of PSII particles, we found a clear pO(2) influence on the redox reactions of the Mn complex, with a similar half-effect pressure as determined (2-3 bar). However, XANES spectra and the time courses of the X-ray fluorescence collected with microsecond resolution suggested that the O(2) evolution transition itself (S(3)-->S(0)+O(2)) was not affected. Additional (nonstandard) oxidation of the Mn complex at high pO(2) explains our experimental findings more readily. Our results suggest that photosynthesis at ambient conditions is not limited by product inhibition of the O(2) formation step.
Project description:The S1 ? S2 transition of the oxygen-evolving complex (OEC) of photosystem II does not involve the transfer of a proton to the lumen and occurs at cryogenic temperatures. Therefore, it is commonly thought to involve only Mn oxidation without any significant change in the structure of the OEC. Here, we analyze structural changes upon the S1 ? S2 transition, as revealed by quantum mechanics/molecular mechanics methods and the isomorphous difference Fourier method applied to serial femtosecond X-ray diffraction data. We find that the main structural change in the OEC is in the position of the dangling Mn and its coordination environment.
Project description:Two structurally homologous Mn compounds in different oxidation states were studied to investigate the relative influence of oxidation state and ligand environment on Mn K-edge X-ray absorption near-edge structure (XANES) and Mn Kbeta X-ray emission spectroscopy (Kbeta XES). The two manganese compounds are the di-mu-oxo compound [L'2Mn(III)O2Mn(IV)L'2](ClO4)3, where L' is 1,10-phenanthroline (Cooper, S. R.; Calvin, M. J. Am. Chem. Soc. 1977, 99, 6623-6630) and the linear mono-mu-oxo compound [LMn(III)OMn(III)L](ClO4)2, where L- is the monoanionic N,N-bis(2-pyridylmethyl)-N'-salicylidene-1,2-diaminoethane ligand (Horner, O.; Anxolabéhère-Mallart, E.; Charlot, M. F.; Tchertanov, L.; Guilhem, J.; Mattioli, T. A.; Boussac, A.; Girerd, J.-J. Inorg. Chem. 1999, 38, 1222-1232). Preparative bulk electrolysis in acetonitrile was used to obtain higher oxidation states of the compounds: the Mn(IV)Mn(IV) species for the di-mu-oxo compound and the Mn(III)Mn(IV) and Mn(IV)Mn(IV) species for the mono-mu-oxo compound. IR, UV/vis, EPR, and EXAFS spectra were used to determine the purity and integrity of the various sample solutions. The Mn K-edge XANES spectra shift to higher energy upon oxidation when the ligand environment remains similar. However, shifts in energy are also observed when only the ligand environment is altered. This is achieved by comparing the di-mu-oxo and linear mono-mu-oxo Mn-Mn moieties in equivalent oxidation states, which represent major structural changes. The magnitude of an energy shift due to major changes in ligand environment can be as large as that of an oxidation-state change. Therefore, care must be exercised when correlating the Mn K-edge energies to manganese oxidation states without taking into account the nature of the ligand environment and the overall structure of the compound. In contrast to Mn K-edge XANES, Kbeta XES spectra show less dependence on ligand environment. The Kbeta1,3 peak energies are comparable for the di-mu-oxo and mono-mu-oxo compounds in equivalent oxidation states. The energy shifts observed due to oxidation are also similar for the two different compounds. The study of the different behavior of the XANES pre-edge and main-edge features in conjunction with Kbeta XES provides significant information about the oxidation state and character of the ligand environment of manganese atoms.
Project description:Manganese K-edge X-ray absorption (XAS) and K? emission (XES) spectroscopies were used to investigate the factors contributing to O-O bond activation in a small-molecule system. The recent structural characterization of a metastable peroxo-bridged dimeric Mn(III)2 complex derived from dioxygen has provided the first opportunity to obtain X-ray spectroscopic data on this type of species. Ground state and time-dependent density functional theory calculations have provided further insight into the nature of the transitions in XAS pre-edge and valence-to-core (VtC) XES spectral regions. An experimentally validated electronic structure description has also enabled the determination of structural and electronic factors that govern peroxo bond activation, and have allowed us to propose both a rationale for the metastability of this unique compound, as well as potential future ligand designs which may further promote or inhibit O-O bond scission. Finally, we have explored the potential of VtC XES as an element-selective probe of both the coordination mode and degree of activation of peroxomanganese adducts. The comparison of these results to a recent VtC XES study of iron-mediated dintrogen activation helps to illustrate the factors that may determine the success of this spectroscopic method for future studies of small-molecule activation at transition metal sites.
Project description:Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn-Mn and Ca(Sr)-Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn-Mn and Ca(Sr)-Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn-Mn and Mn-Ca(Sr) atoms in the O-O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.
Project description:Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95?Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
Project description:The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2-flash (2F) and 3-flash (3F) photosystem II samples, and of a transient 3F' state (250??s after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn reduction, does not yet occur within 250??s after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5?Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.