Dark-stable S1 State Research Articles

Water Networks in Photosystem II Using Crystalline Molecular Dynamics Simulations and Room-Temperature XFEL Serial Crystallography.

Structural dynamics of water and its hydrogen-bonding networks play an important role in enzyme function via the transport of protons, ions, and substrates. To gain insights into these mechanisms in the water oxidation reaction in Photosystem II (PS II), we have performed crystalline molecular dynamics (MD) simulations of the dark-stable S1 state. Our MD model consists of a full unit cell with 8 PS II monomers in explicit solvent (861 894 atoms), enabling us to compute the simulated crystalline electron density and to compare it directly with the experimental density from serial femtosecond X-ray crystallography under physiological temperature collected at X-ray free electron lasers (XFELs). The MD density reproduced the experimental density and water positions with high fidelity. The detailed dynamics in the simulations provided insights into the mobility of water molecules in the channels beyond what can be interpreted from experimental B-factors and electron densities alone. In particular, the simulations revealed fast, coordinated exchange of waters at sites where the density is strong, and water transport across the bottleneck region of the channels where the density is weak. By computing MD hydrogen and oxygen maps separately, we developed a novel Map-based Acceptor-Donor Identification (MADI) technique that yields information which helps to infer hydrogen-bond directionality and strength. The MADI analysis revealed a series of hydrogen-bond wires emanating from the Mn cluster through the Cl1 and O4 channels; such wires might provide pathways for proton transfer during the reaction cycle of PS II. Our simulations provide an atomistic picture of the dynamics of water and hydrogen-bonding networks in PS II, with implications for the specific role of each channel in the water oxidation reaction.

Alteration of the O2-Producing Mn4Ca Cluster in Photosystem II by the Mutation of a Metal Ligand.

The O2-evolving Mn4Ca cluster in photosystem II (PSII) is arranged as a distorted Mn3Ca cube that is linked to a fourth Mn ion (denoted as Mn4) by two oxo bridges. The Mn4 and Ca ions are bridged by residue D1-D170. This is also the only residue known to participate in the high-affinity Mn(II) site that participates in the light-driven assembly of the Mn4Ca cluster. In this study, we use Fourier transform infrared difference spectroscopy to characterize the impact of the D1-D170E mutation. On the basis of analyses of carboxylate and carbonyl stretching modes and the O-H stretching modes of hydrogen-bonded water molecules, we show that this mutation alters the extensive network of hydrogen bonds that surrounds the Mn4Ca cluster in the same manner as that of many other mutations. It also alters the equilibrium between conformers of the Mn4Ca cluster in the dark-stable S1 state so that a high-spin form of the S2 state is produced during the S1-to-S2 transition instead of the low-spin form that gives rise to the S2 state multiline electron paramagnetic resonance signal. The mutation may also change the coordination mode of the carboxylate group at position 170 to unidentate ligation of Mn4. This is the first mutation of a metal ligand in PSII that substantially impacts the spectroscopic signatures of the Mn4Ca cluster without substantially eliminating O2 evolution. The results have significant implications for our understanding of the roles of alternate active/inactive conformers of the Mn4Ca cluster in the mechanism of O2 formation.

Evaluation of new low-valent computational models for the oxygen-evolving complex of photosystem II

The oxidation states of the Mn4CaO5 cluster of the photosynthetic oxygen-evolving complex remain controversial. New quantum chemical models for the dark-stable S1 state suggest that a low-valent Mn(III-IV-III-II) form, opposed to the commonly accepted high-valent Mn(III-IV-IV-III) assignment, agrees with available structural data. We examine the magnetic properties of the models and the consequences for electronic structure and catalytic progression of assuming a neutral second-sphere residue D1-His337. The low-valent model reproduces the experimental diamagnetic ground spin-state. However, the protonation assignment introduces a critical flaw because neutral His337 becomes itself the locus of subsequent oxidation, in contradiction to physiological catalytic progression.

Crystallographic Data Support the Carousel Mechanism of Water Supply to the Oxygen-Evolving Complex of Photosystem II.

Photosystem II (PSII) oxidizes water to produce oxygen through a four-step photocatalytic cycle. Understanding PSII structure–function relations is important for the development of biomimetic photocatalytic systems. The quantum mechanics/molecular mechanics (QM/MM) analysis of substrate water binding to the oxygen-evolving complex (OEC) has suggested a rearrangement of water ligands in a carousel mechanism around a key Mn center. Here, we find that the most recently reported X-ray free-electron laser (XFEL) crystallographic data obtained for the dark-stable S1 state and the doubly flashed S3 state at 2.25 Å resolution support the carousel mechanism. The features in the XFEL data and QM/MM model-simulated difference Fourier maps suggest that water displacement may occur from the so-called “narrow” channel, resulting in binding of a new water molecule to the OEC, and thus provide new insights into the nature of rearrangements of water ligands along the catalytic cycle before O=O bond formation.

Rationalizing the 2.25 Å Resolution Crystal Structure of the Water Oxidising Complex of Photosystem II in the S3 State.

Quantum chemical calculations are described which rationalize the recent X-ray diffraction (XRD) structure at 2.25 Å of the Mn4 Ca water oxidising complex (WOC) of photosystem II (PSII) in the S3 intermediate state. The new S3 XRD structure shows remarkable similarity to earlier atomic resolution (1.9, 1.95 Å) WOC structures in the dark stable S1 state and is inconsistent with most current proposals, from computational chemistry and other sources, regarding the Mn oxidation state levels in the WOC cluster and the nature of water substrate binding, particularly in S3 . This mirrors earlier failures to rationalise the WOC geometry in the 1.9 and 1.95 Å S1 XRD structures, assuming "high" paradigm Mn oxidation models. However, we recently showed that a lower Mn oxidation assumption closely reproduces the S1 XRD structures, computationally. This same "low" Mn oxidation model, now computationally applied in S3 , not only reproduces the latest 2.25 Å XRD structure but also rationalises a number of other important, experimental features of the WOC, including the metal-metal distances inferred from EXAFS studies as well as earlier S3 state XRD structures of lower resolution (4-5 Å). As found previously for S1 , the WOC in the S3 state is computationally revealed to be structurally variable, consistent with some EXAFS and lower-resolution XRD data. This is a direct consequence of at least two MnIII ions being present in all metastable S states.

Two Different Structures of the Oxygen-Evolving Complex in the Same Polypeptide Frameworks of Photosystem II.

The oxygen-evolving complex (OEC) forms the heart of photosystem II (PSII) in photosynthesis. The crystal structure of PSII from Thermosynechococcus vulcanus has been reported at a resolution of 1.9 Å and at an averaged X-ray dose of 0.43 MGy. The OEC structure is suggested to be partially reduced to Mn(II) by EXAFS and DFT computational studies. Recently, the "radiation-damage-free" structures have been published at 1.95 Å resolution using XFEL, but reports continued to appear that the OEC is reduced to the S0-state of the Kok cycle. To elucidate much more precise structure of the OEC, in this study two structures were determined at extremely low X-ray doses of 0.03 and 0.12 MGy using conventional synchrotron radiation source. The results indicated that the X-ray reduction effects on the OEC were very small in the low dose region below 0.12 MGy, that is, a threshold existed for the OEC structural changes caused by X-ray exposure. The OEC structures of the two identical monomers in the crystal were clearly different under the threshold of the radiation dose, although the surrounding polypeptide frameworks of PSII were the same. The assumption that the OECs in the crystal were in the dark-stable S1-state of the Kok cycle should be re-evaluated.

Atomic Structure and Water-splitting Reaction Mechanism of Photosystem II Revealed by X-ray Free Electron Laser

Photosystem II (PSII) is a huge membrane-protein complex which catalyzes light-driven water-oxidation reaction at its catalytic center, the oxygen evolving complex (OEC). The crystal structure of PSII has been determined at 1.9 Å resolution using synchrotron radiation which revealed that the OEC is a Mn4CaO5 cluster. Some of the manganese atoms of the OEC are, however, rapidly reduced by X-ray irradiation which results in slight elongation of the distances between manganese cations. Furthermore, high-resolution 3D structural information is only limited to the dark-stable S1 state and the structures in the other intermediate states are missing. X-ray free electron laser (XFEL) has the potential to address these unsolved problems, and unveil the water-splitting reaction mechanism of PSII. In this review, we will introduce the analyses of the damage-free structure and an intermediate structure of PSII using XFEL, demonstrating the potential to obtain high spatial resolution of biological samples by XFEL.

The O2-Evolving Complex of Photosystem II: Recent Insights from Quantum Mechanics/Molecular Mechanics (QM/MM), Extended X-ray Absorption Fine Structure (EXAFS), and Femtosecond X-ray Crystallography Data.

Efficient photoelectrochemical water oxidation may open a way to produce energy from renewable solar power. In biology, generation of fuel due to water oxidation happens efficiently on an immense scale during the light reactions of photosynthesis. To oxidize water, photosynthetic organisms have evolved a highly conserved protein complex, Photosystem II. Within that complex, water oxidation happens at the CaMn4O5 inorganic catalytic cluster, the so-called oxygen-evolving complex (OEC), which cycles through storage "S" states as it accumulates oxidizing equivalents and produces molecular oxygen. In recent years, there has been significant progress in understanding the OEC as it evolves through the catalytic cycle. Studies have combined conventional and femtosecond X-ray crystallography with extended X-ray absorption fine structure (EXAFS) and quantum mechanics/molecular mechanics (QM/MM) methods and have addressed changes in protonation states of μ-oxo bridges and the coordination of substrate water through the analysis of ammonia binding as a chemical analog of water. These advances are thought to be critical to understanding the catalytic cycle since protonation states regulate the relative stability of different redox states and the geometry of the OEC. Therefore, establishing the mechanism for substrate water binding and the nature of protonation/redox state transitions in the OEC is essential for understanding the catalytic cycle of O2 evolution. The structure of the dark-stable S1 state has been a target for X-ray crystallography for the past 15 years. However, traditional X-ray crystallography has been hampered by radiation-induced reduction of the OEC. Very recently, a revolutionary X-ray free electron laser (XFEL) technique was applied to PSII to reveal atomic positions at 1.95 Å without radiation damage, which brought us closer than ever to establishing the ultimate structure of the OEC in the S1 state. However, the atom positions in this crystal structure are still not consistent with high-resolution EXAFS spectroscopy, partially due to the poorly resolved oxygen positions next to Mn centers and partial reduction due to extended dark adaptation of the sample. These inconsistencies led to the new models of the OEC with an alternative low oxidation state and raised questions on the protonation state of the cluster, especially the O5 μ-oxo bridge. This Account summarizes the most recent models of the OEC that emerged from QM/MM, EXAFS and femtosecond X-ray crystallography methods. When PSII in the S1 state is exposed to light, the S1 state is advanced to the higher oxidation states and eventually binds substrate water molecules. Identifying the substrate waters is of paramount importance for establishing the water-oxidation mechanism but is complicated by a large number of spectroscopically similar waters. Water analogues can, therefore, be helpful because they serve as spectroscopic markers that help to track the motion of the substrate waters. Due to a close structural and electronic similarity to water, ammonia has been of particular interest. We review three competing hypotheses on substrate water/ammonia binding and compile theoretical and experimental evidence to support them. Binding of ammonia as a sixth ligand to Mn4 during the S1 → S2 transition seems to satisfy most of the criteria, especially the most compelling recent EPR data on D1-D61A mutated PSII. Such a binding mode suggests delivery of water from the "narrow" channel through a "carousel" rearrangement of waters around Mn4 upon the S2 → S3 transition. An alternative hypothesis suggests water delivery through the "large" channel on the Ca side. However, both water delivery paths lead to a similar S3 structure, seemingly reaching consensus on the nature of the last detectable S-state intermediate in the Kok cycle before O2 evolution.

Large-scale QM/MM calculations of the CaMn4O5 cluster in the oxygen-evolving complex of photosystem II: Comparisons with EXAFS structures

Large-scale QM/MM calculations including hydrogen-bonding networks in the oxygen evolving complex (OEC) of photosystem II (PSII) were performed to elucidate the geometric structures of the CaMn4O5 cluster in the key catalytic states (Si (i=0–3)). The optimized Mn–Mn, Ca–Mn and Mn–O distances by the large-scale QM/MM starting from the high-resolution XRD structure were consistent with those of the EXAFS experiments in the dark stable S1 state by the Berkeley and Berlin groups. The optimized geometrical parameters for other Si (i=0, 2, 3) states were also consistent with those of EXAFS, indicating the importance of the large-scale QM/MM calculations for the PSII-OEC.

Hydration of the oxygen-evolving complex of photosystem II probed in the dark-stable S1 state using proton NMR dispersion profiles

The hydration of the oxygen-evolving complex (OEC) was characterized in the dark stable S1 state of photosystem II using water R1(ω) NMR dispersion (NMRD) profiles. The R1(ω) NMRD profiles were recorded over a frequency range from 0.01 MHz to 40 MHz for both intact and Mn-depleted photosystem II core complexes from Thermosynechococcus vulcanus (T. vulcanus). The intact-minus-(Mn)-depleted difference NMRD profiles show a characteristic dispersion from approximately 0.03 MHz to 1 MHz, which is interpreted on the basis of the Solomon-Bloembergen-Morgan (SBM) and the slow motion theories as being due to a paramagnetic enhanced relaxation (PRE) of water protons. Both theories are qualitatively consistent with the ST = 1, g = 4.9 paramagnetic state previously described for the S1 state of the OEC; however, an alternative explanation involving the loss of a separate class of long-lived internal waters due to the Mn-depletion procedure can presently not be ruled out. Using a point-dipole approximation the PRE-NMRD effect can be described as being caused by 1-2 water molecules that are located about 10 Å away from the spin center of the Mn4CaO5 cluster in the OEC. The application of the SBM theory to the dispersion observed for PSII in the S1 state is questionable, because the parameters extracted do not fulfil the presupposed perturbation criterion. In contrast, the slow motion theory gives a consistent picture indicating that the water molecules are in fast chemical exchange with the bulk (τw < 1 μs). The modulation of the zero-field splitting (ZFS) interaction suggests a (restricted) reorientation/structural equilibrium of the Mn4CaO5 cluster with a characteristic time constant of τZFS = 0.6-0.9 μs.

Mechanism of Water Splitting by Photosystem II

The light driven water splitting achieved in the oxygen evolving complex (OEC) of Photosystem II is a critical process that sustains our biosphere. Photosynthetic water splitting is fascinating in its complexity, inspiring due to its practical applications in designing artificial photosynthesis, and not yet understood. At the heart of the water splitting process is the Mn4Ca cluster embedded in a fine tuned protein environment.The electronic structure and geometry of this cluster were probed by X-ray spectroscopy at the functional room temperature state.1, 2 Detailed kinetic analysis of the X-ray induced damage was performed and allowed selection of undamaging conditions for experimentation. High-quality extended X-ray absorption fine structure (EXAFS) spectra of the OEC at room temperature will be presented and compared with XRD and DFT derived molecular models of the dark stable S1 state. The determined Debye-Waller factors, sensitive to dynamic processes, support the rigid structure of the Mn4Ca cluster at room temperature.Laser-pump X-ray probe time-resolved X-ray emission measurements (XES) allowed monitoring of changes in the electronic structure of the OEC in real time during the catalysis. Using time-resolved XES we monitored the evolution of the electronic structure of the OEC of Photosystem II during the most critical S3 to S0 transition which results in O2 evolution. Our data show no oxidation but only a gradual reduction of the Mn centers after excitation of the complex past the S3 state. These observations allow us to propose a unique O-O bond formation and water splitting mechanism. Combined with DFT modeling, our analysis reveals the mechanism of catalytic water splitting.1. Davis et al. J. Phys. Chem. B 117, 9161-9169 (2013).2. Davis et al. J. Phys. Chem. Lett. 3, 1858-1864 (2012).

3PT230 光化学系II酸素発生中心の暗所安定状態における電子スピン状態とプロトン化状態の理論的研究(日本生物物理学会第50回年会(2012年度))

3PT230 光化学系II酸素発生中心の暗所安定状態における電子スピン状態とプロトン化状態の理論的研究(日本生物物理学会第50回年会(2012年度))

QM/MM Models of the O2-Evolving Complex of Photosystem II.

This paper introduces structural models of the oxygen-evolving complex of photosystem II (PSII) in the dark-stable S1 state, as well as in the reduced S0 and oxidized S2 states, with complete ligation of the metal-oxo cluster by amino acid residues, water, hydroxide, and chloride. The models are developed according to state-of-the-art quantum mechanics/molecular mechanics (QM/MM) hybrid methods, applied in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus, recently reported at 3.5 Å resolution. Manganese and calcium ions are ligated consistently with standard coordination chemistry assumptions, supported by biochemical and spectroscopic data. Furthermore, the calcium-bound chloride ligand is found to be bound in a position consistent with pulsed electron paramagnetic resonance data obtained from acetate-substituted PSII. The ligation of protein ligands includes monodentate coordination of D1-D342, CP43-E354, and D1-D170 to Mn(1), Mn(3), and Mn(4), respectively; η(2) coordination of D1-E333 to both Mn(3) and Mn(2); and ligation of D1-E189 and D1-H332 to Mn(2). The resulting QM/MM structural models are consistent with available mechanistic data and also are compatible with X-ray diffraction models and extended X-ray absorption fine structure measurements of PSII. It is, therefore, conjectured that the proposed QM/MM models are particularly relevant to the development and validation of catalytic water-oxidation intermediates.

Rapid Loss of Structural Motifs in the Manganese Complex of Oxygenic Photosynthesis by X-ray Irradiation at 10–300 K

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-mu-oxo bridge between manganese atoms, previously separated by approximately 2.7 A, to a mono-mu-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at approximately 200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final Mn(II)4 state the complete loss of inter-manganese distances <3 A was observed, even at 10 K, because of the destruction of mu-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.

The structure of the manganese complex of Photosystem II in its dark-stable S1-state—EXAFS results in relation to recent crystallographic data

Determination of the nuclear geometry of the Mn4Ca complex of photosystem II (PSII) may facilitate an in-depth discussion of the mechanism of photosynthetic water oxidation. The first step of structural analysis by EXAFS (extended X-ray absorption fine-structure) spectroscopy and protein crystallography is the determination of the structure of the dark-stable S1-state of the Mn complex. Approaches for normalization and simulation of EXAFS spectra are critically evaluated, in particular with respect to the accuracy of the determined coordination numbers. It is shown that the number of Mn–Mn interactions with about 2.7 A length is likely to be two in the S1-state. The presence and orientation of the 2.7 A Mn–Mn vectors as well as of the Mn–Ca distances of ∼3.3 A length is predicted by EXAFS analysis and seems to be compatible with the available crystallographic data (Zouni, Witt, J. Kern, P. Fromme, N. Kraus, W. Saenger and P. Orth, Nature, 2001, 409, Kamiya and Shen, Proc. Natl. Acad. Sci. USA, 2003, 100, 98; Ferreira, Iverson, Maghlaoui, Barber and Iwata, Science, 2004, 303, 1831). In the light of XAS results on radiation-induced modifications of the Mn4Ca complex, however, we conclude that in the course of the crystallographic data collection the Mn4Ca complex may be significantly affected by X-ray photoreduction so that the obtained electron density maps do not represent the intact complex in its S1-state. Thus, any detailed structural modelling by combination of EXAFS results and crystallographic data seems to be premature. Two motifs for the connection of Mn ions in the complex, namely either by (μ2-O)2 or (μ2-O, μ3-O) bridges, may account for Mn–Mn distances close to 2.7 A. Three basic possibilities to arrange the Mn ions of the Mn4Ca complex in its S1-state are discussed.

Protein and Ligand Environments of the S2State in Photosynthetic Oxygen Evolution: A Difference FT-IR Study

Oxidation of water in photosynthetic organisms occurs in a chlorophyll containing enzyme, photosystem II. The catalytic manganese cluster of photosystem II cycles among five redox states called the Sn states. There are two forms of the S2 state, which give rise to different EPR signals and which differ in magnetic coupling among the manganese atoms. We have used difference infrared spectroscopy to obtain more information about the environment of the manganese cluster in these two forms of the S2 state. We present the 1600−1200 cm-1 region of the difference spectrum associated with the generation of the multiline state and the g = 4.1 S2 state. The difference spectrum associated with generation of the S2 multiline state from the dark stable S1 state shows broad spectral features in the 1500−1200 cm-1 region at 1490 (negative), 1331 (negative), 1393 (positive), and 1267 (positive) cm-1. Global 15N labeling has little impact on intensities or frequencies in this region of the spectrum. These vibrational features are not observed in manganese-depleted, EDTA-treated photosystem II. Also, these features are not observed in oxygen-evolving photosystem II upon illumination at 80 K, a temperature where the manganese cluster is not oxidized. We conclude that these lines arise from amino acid residues that are close to or ligating to the cluster. From the frequencies and the lack of sensitivity to 15N labeling, we favor the assignment of these lines to the asymmetric and symmetric stretch of one or more glutamate and/or aspartate residue(s). The spectral breadth of the lines is consistent with either an inhomogeneous or a homogeneous broadening mechanism. When illumination conditions are used that generate the g = 4.1 S2 EPR signal, these vibrational lines are not observed. These results are discussed in terms of current models for the catalytic site.

EVIDENCE THAT FORMATE DESTABILIZES THE S1 STATE OF THE OXYGEN-EVOLVING MECHANISM IN PHOTOSYSTEM II

Flash-induced absorption changes at 295 nm and oxygen flash yields were measured in Photosystem II-enriched membranes and thylakoids from spinach. In the presence of formate and reduced 2,6-dichloro-t p-benzoquinone (DCBQ), the normal period-4 oscillation in the oxygen-evolving mechanism is shifted one flash, a maximum appearing on the 4th rather than the 3rd flash. Analysis of the flash-induced sequence shows that in 90–100% of the centers, the oxygen evolving complex is present in the most reduced state (S0), rather than in the singly oxidized state (S1) as is normally the case. An electron donation to the oxidized states S2 or S3 during the flash sequence by reduced tyrosine ‘YD’ cannot account for the data unless the rate of this reaction is dramatically accelerated by formate. Rather, it appears that the normally dark-stable S1 state of the oxygen-evolving mechanism is modified by formate so that it becomes reducible to S0. The reduced form of DCBQ is an efficient mediator to promote the shift to S0. As in other instances where a chemical reduction of S1 is achieved, the ‘formal S0’ state caused by the formate/DCBQ treatment is not spectroscopically identical with the S0 state occurring under illumination. Incubation at high redox potential (550 mV) did not reverse the S-state shift caused by the treatment with formate and DCBQ at lower potential, but induced a distortion of the absorption change sequence that could indicate an enhanced yield of oxygen on the second flash.