Calcium exchange and structural changes during the photosynthetic oxygen evolving cycle

PSII catalyzes the oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. PSII contains an oxygen-evolving complex, which is located on the lumenal side of the PSII reaction center and which contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen. This process produces five Sn-states, where n refers to the number of oxidizing equivalents stored. Calcium is required for oxygen production. Strontium is the only divalent cation that replaces calcium and maintains activity. In our previous FT-IR work, we assessed the effect of strontium substitution on substrate-limited PSII preparations, which were inhibited at the S3 to S0 transition. In this work, we report reaction-induced FT-IR studies of hydrated PSII preparations, which undergo the full S-state cycle. The observed difference FT-IR spectra reflect long-lived photoinduced conformational changes in the oxygen-evolving complex; strontium exchange identifies vibrational bands sensitive to substitutions at the calcium site. During the S1' to S2' transition, the data are consistent with an electrostatic or structural perturbation of the calcium site. During the S3' to S0' and S0' to S1' transitions, the data are consistent with a perturbation of a hydrogen bonding network, which contains calcium, water, and peptide carbonyl groups. To explain our data, persistent shifts in divalent cation coordination must occur when strontium is substituted for calcium. A modified S-state model is proposed to explain these results and results in the literature.     

Sucrose and glycerol effects on photosystem II

Photosystem II catalyzes the oxidation of water and the reduction of plastoquinone. The active site cycles among five oxidation states, which are called the S(n) states. PSII purification procedures include the use of the cosolvents, sucrose and/or glycerol, to stabilize water splitting activity and for cryoprotection. In this study, the effects of sucrose and glycerol on PSII were investigated. Sucrose addition was observed to stimulate the steady-state rate of oxygen evolution in the range from 0 to 1.35 M. Glycerol addition was observed to stimulate oxygen evolution in the range from 0 to 30%. Both cosolvents were observed to be inhibitory at higher concentrations. Sucrose addition was shown to have no effect on the rate of Q(A)(-) oxidation or on the K(M) for exogenous acceptor. PSII was then treated to remove extrinsic proteins. In these samples, sucrose addition stimulated activity, but glycerol addition was inhibitory at concentrations higher than approximately 0.5 M. This inhibitory effect of glycerol at relatively low concentrations is attributed to glycerol binding to the active site, when extrinsic subunits are not present. Reaction induced FTIR spectra, associated with the S(1) to S(2) transition of the water-oxidizing complex, exhibited significant differences throughout the 1,800-1,200 cm(-1) region, when glycerol- and sucrose-containing samples were compared. These measurements suggest a cosolvent-induced shift in the pK(A) of an aspartic or glutamic acid side chain, as well as structural changes at the active site. These structural alterations are attributed to a change in preferential hydration of the oxygen-evolving complex.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Functional architecture of higher plant photosystem II supercomplexes

Photosystem II (PSII) is a large multiprotein complex, which catalyses water splitting and plastoquinone reduction necessary to transform sunlight into chemical energy. Detailed functional and structural studies of the complex from higher plants have been hampered by the impossibility to purify it to homogeneity. In this work, homogeneous preparations ranging from a newly identified particle composed by a monomeric core and antenna proteins to the largest C₂S₂M₂ supercomplex were isolated. Characterization by biochemical methods and single particle electron microscopy allowed to relate for the first time the supramolecular organization to the protein content. A projection map of C₂S₂M₂ at 12 Å resolution was obtained, which allowed determining the location and the orientation of the antenna proteins. Comparison of the supercomplexes obtained from WT and Lhcb‐deficient plants reveals the importance of the individual subunits for the supramolecular organization. The functional implications of these findings are discussed and allow redefining previous suggestions on PSII energy transfer, assembly, photoinhibition, state transition and non‐photochemical quenching.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 mol O₂/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl₂ or diphenylcarbazide, or diuron as an inhibitor of electron transfer from Q_A, the primary bound quinone acceptor, to Q_B, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to F_max, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680⁺ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H₂O₂, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O₂ with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680⁺, as well as by oxygen reduction by still unidentified components of PSII.     

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ′, a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ′ subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ′ were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.     

Water oxidation chemistry of photosystem II

Photosystem II (PSII) uses light energy to split water into protons, electrons and O2. In this reaction, nature has solved the difficult chemical problem of efficient four-electron oxidation of water to yield O2 without significant amounts of reactive intermediate species such as superoxide, hydrogen peroxide and hydroxyl radicals. In order to use nature's solution for the design of artificial catalysts that split water, it is important to understand the mechanism of the reaction. The recently published X-ray crystal structures of cyanobacterial PSII complexes provide information on the structure of the Mn and Ca ions, the redox-active tyrosine called YZ and the surrounding amino acids that comprise the O2-evolving complex (OEC). The emerging structure of the OEC provides constraints on the different hypothesized mechanisms for O2 evolution. The water oxidation mechanism of PSII is discussed in the light of biophysical and computational studies, inorganic chemistry and X-ray crystallographic information.     

Photosynthetic generation of oxygen

The oxygen in the atmosphere is derived from light-driven oxidation of water at a catalytic centre contained within a multi-subunit enzyme known as photosystem II (PSII). PSII is located in the photosynthetic membranes of plants, algae and cyanobacteria and its oxygen-evolving centre (OEC) consists of four manganese ions and a calcium ion surrounded by a highly conserved protein environment. Recently, the structure of PSII was elucidated by X-ray crystallography thus revealing details of the molecular architecture of the OEC. This structural information, coupled with an extensive knowledge base derived from a wide range of biophysical, biochemical and molecular biological studies, has provided a framework for understanding the chemistry of photosynthetic oxygen generation as well as opening up debate about its evolutionary origin.     

Kojic Acid Oxidase

Photosystem II (PSII), which catalyzes photosynthetic water oxidation, is composed of more than 20 subunits, including membrane-intrinsic and -extrinsic proteins. The extrinsic proteins of PSII shield the catalytic Mn₄CaO₅ cluster from exogenous reductants and serve to optimize oxygen evolution at physiological ionic conditions. These proteins include PsbO, found in all oxygenic organisms, PsbP and PsbQ, specific to higher plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP in cyanobacteria. Furthermore, red algal PSII has PsbQ′ in addition to PsbO, PsbV, and PsbU, and diatoms have Psb31 in supplement to red algal-type extrinsic proteins, exemplifying the functional divergence of these proteins during evolution. This review provides an updated summary of recent findings on PSII extrinsic proteins and discusses their binding, function, and evolution within various photosynthetic organisms.     

QM/MM computational studies of substrate water binding to the oxygen-evolving centre of photosystem II

This paper reports computational studies of substrate water binding to the oxygen-evolving centre (OEC) of photosystem II (PSII), completely ligated by amino acid residues, water, hydroxide and chloride. The calculations are based on quantum mechanics/molecular mechanics hybrid models of the OEC of PSII, recently developed in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The model OEC involves a cuboidal Mn3CaO4Mn metal cluster with three closely associated manganese ions linked to a single mu4-oxo-ligated Mn ion, often called the 'dangling manganese'. Two water molecules bound to calcium and the dangling manganese are postulated to be substrate molecules, responsible for dioxygen formation. It is found that the energy barriers for the Mn(4)-bound water agree nicely with those of model complexes. However, the barriers for Ca-bound waters are substantially larger. Water binding is not simply correlated to the formal oxidation states of the metal centres but rather to their corresponding electrostatic potential atomic charges as modulated by charge-transfer interactions. The calculations of structural rearrangements during water exchange provide support for the experimental finding that the exchange rates with bulk 18 O-labelled water should be smaller for water molecules coordinated to calcium than for water molecules attached to the dangling manganese. The models also predict that the S1-->S2 transition should produce opposite effects on the two water-exchange rates.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

Dynamics of electron transfer in photosystem II

Photosystem II, being a constituent of light driven photosynthetic apparatus, is a highly organized pigment-protein-lipid complex. The arrangement of PSII active redox cofactors insures efficiency of electron transfer within it. Donation of electrons extracted from water by the oxygen evolving complex to plastoquinones requires an additional activation energy. In this paper we present theoretical discussion of the anharmonic fluctuations of the protein-lipid matrix of PSII and an experimental evidence showing that the fluctuations are responsible for coupling of its donor and acceptor side. We argue that the fast collective motions liberated at temperatures higher that 200 K are crucial for the two final steps of the water splitting cycle and that one can distinguish three different dynamic regimes of PSII action which are controlled by the timescales of forward electron transfer, which vary with temperature. The three regimes of the dynamical behavior are related to different spatial domains of PSII.     

Expression, assembly and auxiliary functions of photosystem II oxygen-evolving proteins in higher plants

The oxygen-evolving complex (OEC) of higher plant photosystem II (PSII) consists of an inorganic Mn₄Ca cluster and three nuclear-encoded proteins, PsbO, PsbP and PsbQ. In this review, we focus on the assembly of these OEC proteins, and especially on the role of the small intrinsic PSII proteins and recently found “novel” PSII proteins in the assembly process. The numerous auxiliary functions suggested during the past few years for the OEC proteins will likewise be discussed. For example, besides being a manganese-stabilizing protein, PsbO has been found to bind calcium and GTP and possess a carbonic anhydrase activity. In addition, specific roles have been suggested for the two isoforms of the PsbO protein in Arabidopsis thaliana. PsbP and PsbQ seem to play an additional role in the formation of PSII supercomplexes and in grana stacking, besides their originally recognized role in providing a proper calcium and chloride ion concentration for water splitting.     

Multifrequency electron spin-echo envelope modulation studies of nitrogen ligation to the manganese cluster of photosystem II

The CalEPR Center at UC-Davis (http://brittepr.ucdavis.edu) is equipped with five research grade electron paramagnetic resonance (EPR) instruments operating at various excitation frequencies between 8 and 130GHz. Of particular note for this RSC meeting are two pulsed EPR spectrometers working at the intermediate microwave frequencies of 31 and 35GHz. Previous lower frequency electron spin-echo envelope modulation (ESEEM) studies indicated that histidine nitrogen is electronically coupled to the Mn cluster in the S2 state of photosystem II (PSII). However, the amplitude and resolution of the spectra were relatively poor at these low frequencies, precluding any in-depth analysis of the electronic structure properties of this closely associated nitrogen nucleus. With the intermediate frequency instruments, we are much closer to the 'exact cancellation' limit, which optimizes ESEEM spectra for hyperfine-coupled nuclei such as 14N and 15N. Herein, we report the results from ESEEM studies of both 14N- and 15N-labelled PSII at these two frequencies. Spectral simulations were constrained by both isotope datasets at both frequencies, with a focus on high-resolution spectral examination of the histidine ligation to the Mn cluster in the S2 state.     

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS1

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O₂ evolution and inhibited the water‐splitting system in PSII. S‐states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSII_X) and PSII_β reaction centers, which increased the fluxes of dissipated energy. JIP test and Q_A^? reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from Q_A^? to Q_B/Q_B^? and accumulation of P₆₈₀⁺. More Q_A^? had to be oxidized through S₂(Q_AQ_B)^? charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.     

Strong-light photoinhibition treatment accelerates the changes of protein secondary structures in triton-treated photosystem I and photosystem II complexes

Changes in the protein secondary structure and electron transport activity of the Triton X-100-treated photosystem I (PSI) and photosystem II (PSII) complexes after strong illumination treatment were studied using Fourier transform-infrared (FT-IR) spectroscopy and an oxygen electrode. Short periods of photoinhibitory treatment led to obvious decreases in the rates of PSI-mediated electron transport activity and PSII-mediated oxygen evolution in the native or Triton-treated PSI and PSII complexes. In the native PSI and PSII complexes, the protein secondary structures had little changes after the photoinhibitory treatment. However, in both Triton-treated PSI and PSII complexes, short photoinhibition times caused significant loss of -helical content and increase of -sheet structure, similar to the conformational changes in samples of Triton-treated PSI and PSII complexes after long periods of dark incubation. Our results demonstrate that strong-light treatment to the Triton-treated PSI and PSII complexes accelerates destruction of the transmembrane structure of proteins in the two photosynthetic membranes.     

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.     

Designing photosystem II: molecular engineering of photo-catalytic proteins

Biological photosynthesis utilizes membrane-bound pigment/protein complexes to convert light into chemical energy through a series of electron-transfer events. In the unique photosystem II (PSII) complex these electron-transfer events result in the oxidation of water to molecular oxygen. PSII is an extremely complex enzyme and in order to exploit its unique ability to convert sunlight into chemical energy it will be necessary to make a minimal model. Here we will briefly describe how PSII functions and identify those aspects that are essential in order to catalyze the oxidation of water into O(2), and review previous attempts to design simple photo-catalytic proteins and summarize our current research exploiting the E. coli bacterioferritin protein as a scaffold into which multiple cofactors can be bound, to oxidize a manganese metal center upon illumination. Through the reverse engineering of PSII and light driven water splitting reactions it may be possible to provide a blueprint for catalysts that can produce clean green fuel for human energy needs.