Oxygen-evolving Complex Of Photosystem II Research Articles

Merging Structural Information from X-ray Crystallography, Quantum Chemistry, and EXAFS Spectra: The Oxygen-Evolving Complex in PSII.

Structural data of the oxygen-evolving complex (OEC) in photosystem II (PSII) determined by X-ray crystallography, quantum chemistry (QC), and extended X-ray absorption fine structure (EXAFS) analyses are presently inconsistent. Therefore, a detailed study of what information can be gained about the OEC through a comparison of QC and crystallographic structure information combined with the information from range-extended EXAFS spectra was undertaken. An analysis for determining the precision of the atomic coordinates of the OEC by QC is carried out. OEC model structures based on crystallographic data that are obtained by QC from different research groups are compared with one another and with structures obtained by high-resolution crystallography. The theory of EXAFS spectra is summarized, and the application of EXAFS spectra to the experimental determination of the structure of the OEC is detailed. We discriminate three types of parameters entering the formula for the EXAFS spectrum: (1) model-independent, predefined, and fixed; (2) model-dependent that can be computed or adjusted; and (3) model-dependent that must be adjusted. The information content of EXAFS spectra is estimated and is related to the precision of atomic coordinates and resolution power to discriminate different atom-pair distances of the OEC. It is demonstrated how a precise adjustment of atomic coordinates can yield a nearly perfect representation of the experimental OEC EXAFS spectrum, but at the expense of overfitting and losing the knowledge of the initial OEC model structure. Introducing a novel type of penalty function, it is shown that moderate adjustment of atomic coordinates to the EXAFS spectrum limited by constraints avoids overfitting and can be used to validate different OEC model structures. This technique is used to identify the OEC model structures whose computed OEC EXAFS spectra agree best with the measured spectrum. In this way, the most likely S-state and protonation pattern of the OEC for the most recent high-resolution crystal structure of PSII are determined. We find that the X-ray free-electron laser (XFEL) structure is indeed not significantly affected by exposure to XFEL pulses and thus results in a radiation-damage-free model of the OEC.

Ecophysiological response of native and invasive Spartina species to extreme temperature events in Mediterranean marshes

The recent IPCC WG2 5th Assessment Report (IPCC 2014), notes an increase in the frequency and duration of extreme climatic events, especially for the Mediterranean region. Together with climate change, the invasion of natural communities by non-indigenous species (NIS) constitutes a serious threat to biodiversity. One of these NIS is the American Spartina patens, now present in Western European marshes. The present study aims to understand the biochemical and photochemical responses of S. patens compared with S. maritima under extreme temperature events. Under normal and extreme heat conditions, S. patens had a higher photosynthetic efficiency (α), compared with cold wave events, where the native S. maritima was far more efficient. This reduced photosynthetic efficiency was mostly due to a decrease in the connectivity between photosystem II (PSII) antennae. This was accompanied by severe damage to the oxygen-evolving complex of PSII. On the other hand, S. patens oxygen evolving complexes (OECs) seem to be temperature insensitive. The light absorption capacity was maintained due to a higher net rate of reaction centre (RC) closure as a counteractive measure of the reduced number of RC, especially in S. maritima. The loss of connectivity between PSII antennae and damage in OECs under heat stress leads to a severe reduction in the maximum yield for photochemistry enhanced by the low probability of each absorbed quanta to produce electronic work. However, while S. maritima presents high energy losses under heat stress, S. patens developed efficient quenching mechanisms under thermal stress, through auroxanthin. In S. patens, cold wave-treated individuals also displayed a very active line of enzymatic defences for reactive oxygen species scavenging. In fact, only cold treated individuals of this species presented higher activities of anti-oxidant enzymes, revealing some degree of adaptation to this new environment. In contrast, in S. maritima the exposure to extreme heat periods led, in most cases, to a decrease in the enzymatic defences, leaving the cell prone to oxidative damage. In summary, S. patens appears to have a higher fitness for the incoming climatic scenarios, being more tolerant to heat stress, while S. maritima will have its photobiological fitness decreased. This will impose a shift in the salt marsh biodiversity, favouring the non-indigenous S. patens expansion.

A supramolecular ruthenium macrocycle with high catalytic activity for water oxidation that mechanistically mimics photosystem II.

Mimicking the ingenuity of nature and exploiting the billions of years over which natural selection has developed numerous effective biochemical conversions is one of the most successful strategies in a chemist's toolbox. However, an inability to replicate the elegance and efficiency of the oxygen-evolving complex of photosystem II (OEC-PSII) in its oxidation of water into O2 is a significant bottleneck in the development of a closed-loop sustainable energy cycle. Here, we present an artificial metallosupramolecular macrocycle that gathers three Ru(bda) centres (bda = 2,2'-bipyridine-6,6'-dicarboxylic acid) that catalyses water oxidation. The macrocyclic architecture accelerates the rate of water oxidation via a water nucleophilic attack mechanism, similar to the mechanism exhibited by OEC-PSII, and reaches remarkable catalytic turnover frequencies >100 s(-1). Photo-driven water oxidation yields outstanding activity, even in the nM concentration regime, with a turnover number of >1,255 and turnover frequency of >13.1 s(-1).

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.

Theory of chemical bonds in metalloenzymes XX: magneto-structural correlations in the CaMn4O5 cluster in oxygen-evolving complex of photosystem II

ABSTRACTMagneto-structural correlations in oxygen-evolving complex (OEC) of photosystem II (PSII) have been elucidated on the basis of theoretical and computational results in combination with available electron paramagnetic resonance (EPR) experimental results, and extended x-ray absorption fine structure (EXAFS) and x-ray diffraction (XRD) results. To this end, the computational methods based on broken-symmetry (BS) UB3LYP solutions have been developed to elucidate magnetic interactions in the active manganese catalyst for water oxidation by sunlight. The effective exchange interactions J for the CaMn(III)Mn(IV)3O5(H2O)3Y(Y = H2O or OH−) cluster (1) model of OEC of PSII have been calculated by the generalised approximate spin projection (GAP) method that eliminates the spin contamination errors of the BS UB3LYP solution. Full geometry optimisations followed by the zero-point energy (ZPE) correction have been performed for all the spin configurations of 1 to improve the J values that are compared with accumulated EPR in the S2 state of Kok cycle and magnetic susceptibility results of Christou model complex Ca2Mn(IV)3O4 (2). Using the calculated J values, exact diagonalisation of the spin Hamiltonian matrix has been carried out to obtain excitation energies and spin densities of the ground and lower excited states of 1. The calculated excitation energies are consistent with the available experimental results. The calculated spin densities (projection factors) are also compatible with those of the EPR results. The calculated spin densities have been used to calculate the isotropic hyperfine (Aiso) constants of 55Mn ions revealed by the EPR experiments. Implications of the computational results are discussed in relation to the structural symmetry breaking (SSB) in the S1, S2 and S3 states, spin crossover phenomenon induced by the near-infrared excitation and the right- and left-handed scenarios for the O–O bond formation for water oxidation.

The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex.

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b₆/f (Cyt b₆/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b₆/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700(+) indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b₆/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.

Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser.

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question.

Theoretical modelling of biomolecular systems I. Large-scale QM/MM calculations of hydrogen-bonding networks of the oxygen evolving complex of photosystem II

Quantum mechanical (QM)/molecular mechanics (MM) calculations by the use of a large-scale QM model (QM Model V) have been performed to elucidate hydrogen-bonding networks and proton wires for proton release pathways (PRP) of water oxidation reaction in the oxygen evolving complex (OEC) of photosystem II (PSII). Full geometry optimisations of PRP by the QM/MM model have been carried out starting from the geometry of heavy atoms determined by the recent high-resolution X-ray diffraction (XRD) experiment of PSII refined to 1.9 Å resolution. Computational results by the QM/MM calculations have elucidated the hydrogen-bonding O···O(N) and O···H distances and O(N)–H···O angles in PRP, together with the Cl–O(N) and Cl···H distances and O(N)–H···Cl angles for chloride anions. The optimised hydrogen-bonding networks are well consistent with the XRD results and available experiments such as extended X-ray absorption fine structure, showing the reliability of channel structures of OEC of PSII revealed by the XRD experiment. The QM/MM computations have elucidated possible roles of chloride anions in the OEC of PSII. The QM/MM computational results have provided useful information for understanding and explanation of accumulated mutation experiments of key amino acid residues in the OEC of PSII. Implications of the present results are discussed in relation to three steps for theoretical modelling of water oxidation in the OEC of PSII and bio-inspired working hypotheses for developments of artificial water oxidation systems by use of 3d transition-metal complexes.

Polyoxometalate-Based Cobalt–Phosphate Molecular Catalysts for Visible Light-Driven Water Oxidation

A series of all-inorganic, abundant-metal-based, high-nuclearity cobalt-phosphate (Co-Pi) molecular catalysts [{Co4(OH)3(PO4)}4(SiW9O34)4](32-) (1), [{Co4(OH)3(PO4)}4(GeW9O34)4](32-) (2), [{Co4(OH)3(PO4)}4(PW9O34)4](28-) (3), and [{Co4(OH)3(PO4)}4(AsW9O34)4](28-) (4) were synthesized and shown to be highly effective at photocatalytic water oxidation. The {Co16(PO4)4} cluster contains a Co4O4 cubane which is structurally analogous to the [Mn3CaO4] core of the oxygen-evolving complex (OEC) in photosystem II (PSII). Compounds 1-4 were shown to be the first POM-based Co-Pi-cluster molecular catalysts for visible light-driven water oxidation, thus serving as a functional model of the OEC in PSII. The systematic synthesis of four isostructural analogues allowed for investigating the influence of different heteroatoms in the POM ligands on the photocatalytic activities of these Co-Pi cluster WOCs. Further, the POM-based photocatalysts readily recrystallized from the photocatalytic reaction systems with the polyoxoanion structures unchanged, which together with the laser flash photolysis, dynamic light-scattering, (31)P NMR, UV-vis absorption, POM extraction, and ICP-MS analysis results collectively confirmed that compounds 1-4 maintain their structural integrity under the photocatalytic conditions. This study provides not only a valuable molecular model of the "Co-Pi" catalysts with a well-defined structure but also an unprecedented opportunity to fine-tune high-nuclearity POM clusters for visible light-driven water splitting.

2P257 光合成酸素発生中心(PSII-OEC)の立体構造と電荷状態変化についての理論的研究(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

2P257 光合成酸素発生中心(PSII-OEC)の立体構造と電荷状態変化についての理論的研究(18B. 光生物:光合成,ポスター,第52回日本生物物理学会年会(2014年度))

Theory of chemical bonds in metalloenzymes XIX: labile manganese oxygen bonds of the CaMn4O5 cluster in oxygen evolving complex of photosystem II

Spin polarisation effects of labile manganese–oxygen bonds in the X-ray diffraction structure of the oxygen-evolving complex (OEC) of photosystem II (PSII) at 1.9 Å resolution have been investigated by the UB3LYP computations on the basis of three different theoretical models with and without hydrogen bonds: quantum-mechanical (QM) Model I, QM(Model II)/MM and QM Model III. The spin densities on the manganese and oxygen atoms of the CaMn4O5 cluster revealed by these computations have elucidated internal, semi-internal and external reductions of high-valent manganese ions in the CaMn4O5 cluster in OEC of PSII. The internal reduction of Mn(IV) ions by the back charge transfer from oxygen dianions is remarkable in the small QM Model I, whereas it is significantly reduced in the case of more realistic QM Model III including hydrogen bonding stabilisations of oxygen dianions. However, semi-internal reduction of the CaMn4O5 cluster with remote amino acid residues such as Asp61 anion occurs even in QM Model III, indicating the necessity of large QM parts for redox-active systems such as OEC of PSII. The computational results have clearly demonstrated important roles of confinement effects of the CaMn4O5 cluster with labile Mn–O bonds with protein. These computational results have been applied to molecular design of artificial robust catalysts for water oxidation by use of sunlight.

The nature of chemical bonds of the CaMn4O5 cluster in oxygen evolving complex of photosystem II: Jahn‐Teller distortion and its suppression by Ca doping in cubane structures

AbstractFull geometry optimizations of mixed‐valence (MV) cubane Mn(III)4‐ωMn(IV)ωO4 cluster (ω = 0–4) by UB3LYP have been performed to elucidate Jahn‐Teller (JT) effects for the Mn(III) ions. The optimized geometries have elucidated acute triangle for Mn(III)2 Mn(X) (X = III,IV) core in the cubane, indicating the JT effect. The JT effect is not remakable for Mn(IV)2Mn(III) cores, showing obtuse triangle and it diminishes in Mn(IV)3 core with equilateral triangle. The replacement of one of Mn ions with Ca (or Sr) ion in the cubane structure has also been examined to elucidate multiple roles of Ca(II) ion. The Ca‐doping suppresses the JT effect even for Mn(III)2Mn(X) (X = III,IV) core because acute CaMnMn triangles with longer Ca‐Mn distances than the Mn‐Mn distance are newly formed. The suppression of the JT effect for Mn(IV)2 Mn(III) by Ca doping is also found because of the same reason. The Ca‐doped cubane in the CaMn4O5 cluster (1) of oxygen evolving complex (OEC) of photosystem II (PSII) exhibits two acute CaMnMn triangles and one almost equilateral CaMnMn triangle because of the elongation of the Mn1(d)‐Mn3(b) distance with the coordination of the extra Mn4(a) ion to the cubane. The Mn1(d)Mn2(c)Mn3(b) triangle becomes obtuse because of the suppression of the JT effect, indicating the Mn1(d)(III)Mn2(c)(IV)Mn3(b)(IV) valence state in the S1 state of 1. The JT distortion for Mn(III) ions modified by the Ca(or Sr) doping is coupled with the labile structural deformation for intracluster electron transfers via double exchange mechanism in 1. The structural anomalies of 1 revealed by the new XRD experiment are also crucial for derivation of several guiding principles for theoretical modeling of water splitting reaction at OEC of PSII. The hybrid DFT computational results for 1 have also supported these guiding principles that provide possible orbital and spin correlation diagrams for water splitting reactions. The Huckel‐Hubbard‐Hund (HHH) model has been used for qualitative understanding of these diagrams. Implications of the present computational results are discussed in relation to electronic and spin states of redox active Ca‐doped Mott insulators constructed with magnetic transition metals such as Mn, Fe, Co, Ni, etc. Strongly correlated electron systems (SCES) modified by doping of Ca, Sr, Zn, etc. have been elucidated as possible candidates of bio‐inspired artificial catalysts for water oxidation. © 2012 Wiley Periodicals, Inc.

The Effect of Backbone Constraints: The Case of Water Oxidation by the Oxygen‐Evolving Complex in PSII

The procedure for fixing atoms of amino acid residues in cluster model calculations on enzymes is reviewed. Examples from recent calculations on photosystem II (PSII) and Mo,Cu-dependent CO dehydrogenase are given. In this context, the cluster model work on finding a mechanism for O-O bond formation and a structure of the oxygen-evolving complex in PSII is also reviewed. In that work, fixing certain atoms played an important role. The main part of the present study concerns the mechanism in PSII using models based on the new high-resolution (1.9 Å) X-ray structure, which is compared to that using the old, theoretically suggested, structure. It is concluded that the mechanism remains the same, with a similar barrier height. Finally, a connection between the OEC structure and Mn,Ca-containing minerals is also briefly discussed.

Possible mechanisms of water splitting reaction based on proton and electron release pathways revealed for CaMn4O5 cluster of PSII refined to 1.9 Å X‐ray resolution

AbstractRecently, Umena et al. have revealed the X‐ray diffraction structure of the CaMn4O5 cluster in the oxygen evolving complex (OEC) of photosystem II (PSII) refined to 1.9 Å resolution. Their X‐ray structure has first elucidated hydrogen‐bonding networks and proton release pathways at OEC of PSII. Here, several working hypotheses (heuristic principles) for water splitting reaction are derived from their X‐ray structure for theoretical modeling. These hypotheses suggest how water can be oxidized at OEC of PSII: namely possible reaction mechanisms for the reaction. To confirm them, we have also performed broken‐symmetry (BS) UB3LYP calculations for active site models based on their XRD structure. The bond lengths of formal Mn(V)O with labile dπ‐pπ bonds are optimized to clarify possible roles of the species that are often introduced as a key intermediate in the catalytic (Kok) cycle for water splitting reaction at OEC of PSII. Location of the transition structure for the oxygen‐oxygen (OO) bond formation is also performed by the energy optimization technique. The natural orbital (NO) analysis of the UB3LYP solutions has been performed to obtain the natural molecular orbitals and their occupation numbers that have been useful for classification of localized d‐electrons, labile chemical bonds and closed‐shell (valence) orbitals. The localized d‐electrons characterized by the NO analysis are the origins for the magnetism revealed by ENDOR and other magnetic experiments. On the other hand, the nature of labile (soft) dπ‐pπ bonds responsible for the OO bond formation has been investigated on the basis of chemical indices such as effective bond order (b), diradical character (y), and spin density (Q) indices that are calculated using the orbital overlap between broken‐symmetry orbitals. These chemical indices have been calculated for the transition structure of the OO bond formation at OEC of PSII. Implications of present computational results are discussed in relation to the derived hypotheses and available accumulated experimental results. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2012

Characterization of Proton Coupled Electron Transfer in a Biomimetic Oxomanganese Complex: Evaluation of the DFT B3LYP Level of Theory

The capabilities and limitations of the Becke-3-Lee-Yang-Parr (B3LYP) density functional theory (DFT) for modeling proton coupled electron transfer (PCET) in the mixed-valence oxomanganese complex 1 [(bpy)(2)Mn(III)(mu-O)(2)Mn(IV)(bpy)(2)](3+) (bpy = 2,2'-bipyridyl) are analyzed. Complex 1 serves as a prototypical synthetic model for studies of redox processes analogous to those responsible for water oxidation in the oxygen-evolving complex (OEC) of photosystem II (PSII). DFT B3LYP free energy calculations of redox potentials and pKa's are obtained according to the thermodynamic cycle formalism applied in conjunction with a continuum solvation model. We find that the pKa's of the oxo-ligands depend strongly on the oxidation states of the complex, changing by approximately 10 pH units (i.e., from pH~2 to pH~12) upon III,IV-->III,III reduction of complex 1. These computational results are consistent with the experimental pKa's determined by solution magnetic susceptibility and near-IR spectroscopy as well as with the pH dependence of the redox potential reported by cyclic voltammogram measurements, suggesting that the III,IV-->III,III reduction of complex 1 is coupled to protonation of the di-mu-oxo bridge as follows: [(bpy)(2)Mn(III)(mu-O)(2) Mn(IV)(bpy)(2)](3+)+H(+)+e(-)-->[(bpy)(2)Mn(III)(mu-O)(mu-OH)Mn(III)(bpy)(2)](3+). It is thus natural to expect that analogous redox processes might strongly modulate the pKa's of oxo and hydroxo/water ligands in the OEC of PSII, leading to deprotonation of the OEC upon oxidation state transitions.

The Ligand Environment of the S2 State of Photosystem Ii: A Study of the Hyperfine Interactions of the Tetranuclear Manganese Cluster by 2D Hyscore Spectroscopy.‡

The solar water-splitting protein complex, photosystem II (PSII), catalyzes the light-driven oxidation of water to dioxygen in Nature. The four-electron water oxidation reaction occurs at the tetranuclear manganese-calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The mechanism of light-driven water oxidation has been a subject of intense interest and the OEC of PSII has been studied extensively by structural methods. While the recent X-ray crystal structures, single crystal EXAFS and EPR spectroscopy investigations provide a model for the geometry of the catalytic Mn4Ca-oxo cluster, there is limited knowledge of the protein environment that surrounds the catalytic site. It is suggested that the binding and activation of the substrate water molecules at the Mn4Ca-oxo cluster in the OEC of PSII are facilitated by key amino acid residues that could be ligated to the catalytic cluster. In this study, we demonstrate the application of two-dimensional (2D) hyperfine sublevel correlation spectroscopy to determine the magnetic couplings of the S2 state of PSII. We utilize 2D difference spectroscopy to facilitate unambiguous assignments of the spectral features arising from the substrate molecules and surrounding amino acid residues in the S2 state of PSII. The results presented here, for the first time, identify previously unknown ligands to the catalytic cluster and provide avenues for the assignment of residues by site-directed mutagenesis and the refinement of computational and mechanistic models of PSII.®€ This study is supported by the Office of Basic Energy Sciences, United States Department of Energy (DE-FG02-0ER06-15).

Ammonia Triggers Photodamage of Photosystem II in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.

Investigation of substrate water interactions at the high-affinity Mn site in the photosystem II oxygen-evolving complex

18 O isotope exchange measurements of photosystem II (PSII) in thylakoids from wild-type and mutant Synechocystis have been performed to investigate binding of substrate water to the high-affinity Mn4 site in the oxygen-evolving complex (OEC). The mutants investigated were D1-D170H, a mutation of a direct ligand to the Mn4 ion, and D1-D61N, a mutation in the second coordination sphere. The substrate water 18 O exchange rates for D61N were found to be 0.16+/-0.02 s(-1) and 3.03+/-0.32 s(-1) for the slow and fast phases of exchange, respectively, compared with 0.47+/-0.04 s(-1) and 19.7+/-1.3 s(-1) for the wild-type. The D1-D170H rates were found to be 0.70+/-0.16 s(-1) and 24.4+/-4.6 s(-1) and thus are almost within the error limits for the wild-type rates. The results from the D1-D170H mutant indicate that the high-affinity Mn4 site does not directly bind to the substrate water molecule in slow exchange, but the binding of non-substrate water to this Mn ion cannot be excluded. The results from the D61N mutation show an interaction with both substrate water molecules, which could be an indication that D61 is involved in a hydrogen bonding network with the substrate water. Our results provide limitations as to where the two substrate water molecules bind in the OEC of PSII.

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.

Time-resolved vibrational spectroscopy detects protein-based intermediates in the photosynthetic oxygen-evolving cycle.

Photosynthetic oxygen production by photosystem II (PSII) is responsible for the maintenance of aerobic life on earth. The production of oxygen occurs at the PSII oxygen-evolving complex (OEC), which contains a tetranuclear manganese (Mn) cluster. Photo-induced electron transfer events in the reaction center lead to the accumulation of oxidizing equivalents on the OEC. Four sequential photooxidation reactions are required for oxygen production. The oxidizing complex cycles among five oxidation states, called the S(n) states, where n refers to the number of oxidizing equivalents stored. Oxygen release occurs during the S(3)-to-S(0) transition from an unstable intermediate, known as the S(4) state. In this report, we present data providing evidence for the production of an intermediate during each S state transition. These protein-derived intermediates are produced on the microsecond to millisecond time scale and are detected by time-resolved vibrational spectroscopy on the microsecond time scale. Our results suggest that a protein-derived conformational change or proton transfer reaction precedes Mn redox reactions during the S(2)-to-S(3) and S(3)-to-S(0) transitions.