S1-state model of the O2-evolving complex of photosystem II

We introduce a quantum mechanics/molecular mechanics model of the oxygen-evolving complex of photosystem II in the S(1) Mn(4)(IV,III,IV,III) state, where Ca(2+) is bridged to manganese centers by the carboxylate moieties of D170 and A344 on the basis of the new X-ray diffraction (XRD) model recently reported at 1.9 ? resolution. The model is also consistent with high-resolution spectroscopic data, including polarized extended X-ray absorption fine structure data of oriented single crystals. Our results provide refined intermetallic distances within the Mn cluster and suggest that the XRD model most likely corresponds to a mixture of oxidation states, including species more reduced than those observed in the catalytic cycle of water splitting.     

Active and resting states of the O2-evolving complex of photosystem II

During dark adaptation, a change in the O2-evolving complex (OEC) of spinach photosystem II (PSII) occurs that affects both the structure of the Mn site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline EPR signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h at 0 degrees C) and short-term (6 min at 0 degree C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this EPR signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination at 170 K produces an EPR signal with a different hyperfine structure and a wider field range than does illumination at 160 K or below. In contrast, the line shape of the S2-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPR-detected change in the Mn site of the OEC that occurs during dark adaptation is correlated with a change in O2 consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2 following illumination, but only when a functional OEC and excess reductant are present. We assign this slow consumption of O2 to a catalytic reduction of O2 by the OEC in the dark. The rate of O2 consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2 despite the presence of excess reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Calcium depletion modifies the structure of the photosystem II O(2)-evolving complex.

A 5 min exposure of photosystem II to a pH 3 citric acid solution is a simple method for selective removal of Ca(2+) from the O(2)-evolving complex. The resulting preparation retains the 23 and 17 kDa extrinsic polypeptides, but the activity of this material is only 10-20% of that of an untreated control sample. Biochemical characterization of citrate-treated photosystem II reveals that some reaction centers lose the extrinsic proteins during citrate treatment. Furthermore, a comparison of photosystem II preparations treated with citrate, or depleted of 23 and 17 kDa extrinsic polypeptides by high-salt treatment, shows that low concentrations of a small reductant, NH(2)OH, which has little effect on the activity of intact photosystem II, can reduce and inhibit the Mn cluster in both types of preparations. In contrast, a large reductant, hydroquinone, cannot access the majority of O(2)-evolving centers in citrate-treated preparations, while 23 and 17 kDa-depleted material is rapidly inactivated by the reductant. Incubation of the citrate-treated samples in high ( approximately 60 mM) concentrations of CaCl(2) restores 50% of the lost activity; this Ca(2+)-reconstituted activity is chelator-insensitive, indicating that rebinding of Ca(2+) restores the structural integrity of the O(2)-evolving complex. A characterization of Ca(2+) and Cl(-) affinities in steady-state activity assays shows that citrate-treated preparations exhibit a Cl(-) requirement similar to that of polypeptide-depleted photosystem II, while Ca(2+) reactivation of O(2) evolution appears to occur at two structurally distinct sites. One site exhibits a high Ca(2+) affinity, similar to that found in polypeptide-depleted samples, but a second, lower-affinity site also exists, with a K(M) that is approximately 10 times greater than that of the high-affinity site, which is associated with centers that retain the extrinsic polypeptides. These data indicate that citrate-induced Ca(2+) depletion causes release of the 23 and 17 kDa extrinsic polypeptides from some photosystem II reaction centers, and also modifies the structure of the polypeptide-retaining O(2)-evolving centers so that the Mn cluster is exposed to small, but not large, reductants. This change may be due to subtle modifications to the structure of the photosystem II extrinsic proteins that produces a new pathway between the solvent and the Mn cluster or, alternatively, to the opening of an existing channel in the intrinsic lumenal polypeptide domain, between the solvent and the Mn cluster, that is normally occluded by a bound Ca(2+) atom.     

Site-directed mutagenesis of Thermosynechococcus elongatus photosystem II: the O2-evolving enzyme lacking the redox-active tyrosine D

Site-directed mutagenesis in the photosystem II (PSII) oxygen-evolving enzyme was achieved in the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII from this species is the focus of attention because its robustness makes it suitable for enzymological and biophysical studies. PSII, which lacks the redox-active tyrosine Tyr(D), was engineered by substituting a phenylalanine for tyrosine 160 of the D2 protein. An aim of this work was to engineer a mutant for spectroscopy, in particular, for EPR, on the active enzyme. The Tyr(D)(*) EPR signal was monitored in whole cells (i) to control the expression level of the two genes (psbD(1) and psbD(2)) encoding D2 and (ii) to assess the success of the mutagenesis. Both psbD(1) and psbD(2) could be expressed, and recombination occurred between them. The D2-Y160F mutation was introduced into psbD(1) after psbD(2) was deleted and a His-tag was attached to the CP43 protein. The effects of the Y160F mutation were characterized in cells, thylakoids, and isolated PSII. The efficiency of enzyme function under the conditions tested was unaffected. The distribution and lifetime of the redox states (S(n)() states) of the enzyme cycle were modified, with more S(0) in the dark and no rapid decay phase of S(3). Although not previously reported, these effects were expected because Tyr(D)(*) is able to oxidize S(0) and Tyr(D) is able to reduce S(2) and S(3). Slight changes in the difference spectra in the visible and infrared recorded upon the formation and reduction of the chlorophyll cation P(680)(+) and kinetic measurements of P(680)(+) reduction indicated minor structural perturbations, perhaps in the hydrogen-bonding network linking Tyr(D) and P(680), rather than electrostatic changes associated with the loss of a charge from Tyr(D)(*)(H(+)). We show here that this fully active preparation can provide spectra from the Mn(4)CaO(4) complex and associated radical species uncontaminated by Tyr(D)(*).     

The EPR spectrum of tyrosine Z* and its decay kinetics in O2-evolving photosystem II preparations

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.     

Binding and functions of the two chloride ions in the oxygen-evolving center of photosystem II

Photosynthesis Research - Light-driven water oxidation in photosynthesis occurs at the oxygen-evolving center (OEC) of photosystem II (PSII). Chloride ions (Cl?) are essential for oxygen...     

N-terminal sequencing of photosystem II low-molecular-mass proteins. 5 and 4.1 kDa components of the O2-evolving core complex from higher plants

High resolution gel electrophoresis in the low-molecular-mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low-molecular-mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N-terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co-transcribed with them. These two components were associated with the O2-evolving core complex. Sequences of other low-molecular-mass proteins confirmed the previous identification as photosystem II components.     

Computational insights into the O<Subscript>2</Subscript>-evolving complex of photosystem II

Mechanistic investigations of the water-splitting reaction of the oxygen-evolving complex (OEC) of photosystem II (PSII) are fundamentally informed by structural studies. Many physical techniques have provided important insights into the OEC structure and function, including X-ray diffraction (XRD) and extended X-ray absorption fine structure (EXAFS) spectroscopy as well as mass spectrometry (MS), electron paramagnetic resonance (EPR) spectroscopy, and Fourier transform infrared spectroscopy applied in conjunction with mutagenesis studies. However, experimental studies have yet to yield consensus as to the exact configuration of the catalytic metal cluster and its ligation scheme. Computational modeling studies, including density functional (DFT) theory combined with quantum mechanics/molecular mechanics (QM/MM) hybrid methods for explicitly including the influence of the surrounding protein, have proposed chemically satisfactory models of the fully ligated OEC within PSII that are maximally consistent with experimental results. The inorganic core of these models is similar to the crystallographic model upon which they were based, but comprises important modifications due to structural refinement, hydration, and proteinaceous ligation which improve agreement with a wide range of experimental data. The computational models are useful for rationalizing spectroscopic and crystallographic results and for building a complete structure-based mechanism of water-splitting in PSII as described by the intermediate oxidation states of the OEC. This review summarizes these recent advances in QM/MM modeling of PSII within the context of recent experimental studies.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

Probing the structure of photosystem II with amines and phenylhydrazine

Photosynthetic oxygen evolution is catalyzed at the manganese-containing active site of photosystem II (PSII). Amines are analogs of substrate water and inhibitors of oxygen evolution. Recently, the covalent incorporation of (14)C from [(14)C]methylamine and benzylamine into PSII subunits has been demonstrated (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209). To obtain more information concerning these labeling reactions, t-[(14)C]butylamine and phenylhydrazine were employed as probes. Neither compound can be oxidized by a transamination or addition/elimination mechanism, but both can react with activated carbonyl groups, produced as a result of posttranslational modification of amino acid residues, to give amine-derived adducts. (14)C incorporation into the PSII subunits D2/D1 and CP47 was obtained upon treatment of PSII with either t-[(14)C]butylamine or [(14)C]phenylhydrazine. For t-butylamine and methylamine, the amount of labeling increased when PSII was treated with denaturing agents. Labeling of CP47, D2, and D1 with methylamine and phenylhydrazine approached a one-to-one stoichiometry, assuming that D2 and D1 each have one binding site. Evidence was obtained suggesting that reductive stabilization and/or access are modulated by PSII light reactions. These results support the proposal that PSII subunits D2, D1, and CP47 contain quinocofactors and that access to these sites is sterically limited.     

Modification of the properties of S2 state in photosynthetic O2-evolving center by replacement of chloride with other anions

Properties of the S2 state formed in photosystem II membranes in which Cl- had been replaced by various anions were investigated by means of thermoluminescence measurements and low temperature EPR spectroscopy. The Br--substituted membranes showed the normal thermoluminescence B-band arising from S2Q-B charge recombination, whereas the SO2-4-, F--, CH3COO--, and NO-3-substituted membranes showed modified B-bands with variously upshifted peak temperatures. The extent of the peak temperature upshift varied in parallel with the extent of inhibition of O2 evolution depending on the anion species. A normal EPR S2 multiline signal was induced in Br--substituted membranes, but its amplitude was reduced to less than 10% in F--, NO-3-, CH3COO--, and SO2-4-substituted membranes, In contrast, the g = 4.1 signal from S2 was markedly enhanced in F-- and NO-3-substituted membranes, not much affected in CH3COO-- and SO2-4-substituted membranes, and decreased to 70% in Br--substituted membranes. Based on these data, the effect of various types of S2 modification on the O2-evolving activity was discussed. It was suggested that anions have an important role in regulating the interaction between the Mn atoms, and thereby adjust the redox properties of the S2 state to enable further transitions beyond S2.     

Comparative properties of hydroquinone and hydroxylamine reduction of the Ca(2+)-stabilized O2-evolving complex of photosystem II: reductant-dependent Mn2+ formation and activity inhibition.

Calcium binding to photosystem II slows NH2OH inhibition of O2 evolution; Mn2+ is retained by the O2-evolving complex [Mei, R., & Yocum, C. F. (1991) Biochemistry 30, 7836-7842]. This Ca(2+)-induced stability has been further characterized using the large reductant hydroquinone. Salt-washed photosystem II membranes reduced by hydroquinone in the presence of Ca2+ retain 80% of steady-state O2 evolution activity and contain about 2 Mn2+/reaction center that can be detected at room temperature by electron paramagnetic resonance. This Mn2+ produces a weak enhancement of H2O proton spin-lattice relaxation rates, cannot be easily extracted by a chelator, and is reincorporated into the O2-evolving complex upon illumination. A comparison of the properties of Ca(2+)-supplemented photosystem II samples reduced by hydroquinone or NH2OH alone or in sequence reveals the presence of a subpopulation of manganese atoms at the active site of H2O oxidation that is not accessible to facile hydroquinone reduction. At least one of these manganese atoms can be readily reduced by NH2OH following a noninhibitory hydroquinone reduction step. Under these conditions, about 3 Mn2+/reaction center are lost and O2 evolution activity is irreversibly inhibited. We interpret the existence of distinct sites of reductant action on manganese as further evidence that the Ca(2+)-binding site in photosystem II participates in regulation of the organization of manganese-binding ligands and the overall structure of the O2-evolving complex.     

Preparation of O2-evolving Photosystem-II subchloroplasts from spinach

An O₂-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O₂ evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O₂ uptake activity. The preparation showed a chlorophyll $\text{a}\text{b}$ ratio of 1.9 and a $\text{P-700}\text{chlorophyll}$ ratio of $\text{1}\text{2400}$. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O₂-evolving Photosystem II preparation.     

Orientation of the tetranuclear manganese cluster and tyrosine Z in the O(2)-evolving complex of photosystem II: An EPR study of the S(2)Y(Z)(*) state in oriented acetate-inhibited photosystem II membranes.

Inhibitory treatment by acetate, followed by illumination and rapid freezing, is known to trap the S(2)Y(Z)(*) state of the O(2)-evolving complex (OEC) in photosystem II (PS II). An EPR spectrum of this state exhibits broad split signals due to the interaction of the tyrosyl radical, Y(Z)(*), with the S = 1/2 S(2) state of the Mn(4) cluster. We present a novel approach to analyze S(2)Y(Z)(*) spectra of one-dimensionally (1-D) oriented acetate-inhibited PS II membranes to determine the magnitude and relative orientation of the S(2)Y(Z)(*) dipolar vector within the membrane. Although there exists a vast body of EPR data on isolated spins in oriented membrane sheets, the present study is the first of its kind on dipolar-coupled electron spin pairs in such systems. We demonstrate the feasibility of the technique and establish a rigorous treatment to account for the disorder present in partially oriented 1-D membrane preparations. We find that (i) the point-dipole distance between Y(Z)(*) and the Mn(4) cluster is 7.9 +/- 0.2 A, (ii) the angle between the interspin vector and the thylakoid membrane normal is 75 degrees, (iii) the g(z)()-axis of the Mn(4) cluster is 70 degrees away from the membrane normal and 35 degrees away from the interspin vector, and (iv) the exchange interaction between the two spins is -275 x 10(-)(4) cm(-)(1), which is antiferromagnetic. Due to the sensitivity of EPR line shapes of oriented spin-coupled pairs to the interspin distance, the present study imposes a tighter constraint on the Y(Z)-Mn(4) point-dipole distance than obtained from randomly oriented samples. The geometric constraints obtained from the 1-D oriented sample are combined with published models of the structure of Mn-depleted PS II to propose a location of the Mn(4) cluster. A structure in which Y(Z) is hydrogen bonded to a manganese-bound hydroxide ligand is consistent with available data and favors maximal orbital overlap between the two redox center that would facilitate direct electron- and proton-transfer steps.     

Characterization of the manganese O2-evolving complex and the iron-quinone acceptor complex in photosystem II from a thermophilic cyanobacterium by electron paramagnetic resonance and X-ray absorption spectroscopy

The Mn donor complex in the S1 and S2 states and the iron-quinone acceptor complex (Fe2+-Q) in O2-evolving photosystem II (PS II) preparations from a thermophilic cyanobacterium, Synechococcus sp., have been studied with X-ray absorption spectroscopy and electron paramagnetic resonance (EPR). Illumination of these preparations at 220-240 K results in formation of a multiline EPR signal very similar to that assigned to a Mn S2 species observed in spinach PS II, together with g = 1.8 and 1.9 EPR signals similar to the Fe2+-QA- acceptor signals seen in spinach PS II. Illumination at 110-160 K does not produce the g = 1.8 or 1.9 EPR signals, nor the multiline or g = 4.1 EPR signals associated with the S2 state of PS II in spinach; however, a signal which peaks at g = 1.6 appears. The most probable assignment of this signal is an altered configuration of the Fe2+-QA- complex. In addition, no donor signal was seen upon warming the 140 K illuminated sample to 215 K. Following continuous illumination at temperatures between 140 and 215 K, the average X-ray absorption Mn K-edge inflection energy changes from 6550 eV for a dark-adapted (S1) sample to 6551 eV for the illuminated (S2) sample. The shift in edge inflection energy indicates an oxidation of Mn, and the absolute edge inflection energies indicate an average Mn oxidation state higher than Mn(II). Upon illumination a significant change was observed in the shape of the features associated with 1s to 3d transitions. The S1 spectrum resembles those of Mn(III) complexes, and the S2 spectrum resembles those of Mn(IV) complexes. The extended X-ray absorption fine structure (EXAFS) spectrum of the Mn complex is similar in the S1 and S2 states. Simulations indicate O or N ligands at 1.75 +/- 0.05 A, transition metal neighbor(s) at 2.73 +/- 0.05 A, which are assumed to be Mn, and terminal ligands which are probably N and O at a range of distances around 2.2 A. The Mn-O bond length of 1.75 A and the transition metal at 2.7 A indicate the presence of a di-mu-oxo-bridged Mn structure. Simulations indicate that a symmetric tetranuclear cluster is unlikely to be present, while binuclear, trinuclear, or highly distorted tetranuclear structures are possible. The striking similarity of these results to those from spinach PS II suggests that the structure of the Mn complex is largely conserved across evolutionarily diverse O2-evolving photosynthetic species.     

Activating anions that replace Cl- in the O2-evolving complex of photosystem II slow the kinetics of the terminal step in water oxidation and destabilize the S2 and S3 states

Photosystem II, the multisubunit protein complex that oxidizes water to O2, requires the inorganic cofactors Ca2+ and Cl- to exhibit optimal activity. Chloride can be replaced functionally by a small number of anionic cofactors (Br-, NO3-, NO2-, I-), but among these anions, only Br- is capable of restoring rates of oxygen evolution comparable to those observed with Cl-. UV absorption difference spectroscopy was utilized in the experiments described here as a probe to monitor donor side reactions in photosystem II in the presence of Cl- or surrogate anions. The rate of the final step of the water oxidation cycle was found to depend on the activating anion bound at the Cl- site, but the kinetics of this step did not limit the light-saturated rate of oxygen evolution. Instead, the lower oxygen evolution rates supported by surrogate anions appeared to be correlated with an instability of the higher oxidation states of the oxygen-evolving complex that was induced by addition of these anions. Reduction of these states takes place not only with I- but also with NO2- and to a lesser extent even with NO3- and Br- and is not related to the ability of these anions to bind at the Cl- binding site. Rather, it appears that these anions can attack higher oxidation states of the oxygen evolving complex from a second site that is not shielded by the extrinsic 17 and 23 kDa polypeptides and cause a one-electron reduction. The decrease of the oxygen evolution rate may result from accumulated damage to the reaction center protein by the one-electron oxidation product of the anion.