Evidence for a direct manganese-oxygen ligand in water binding to the S2 state of the photosynthetic water oxidation complex

The interaction of water with the water oxidizing Mn complex of photosystem II has been investigated using electron spin-echo envelope modulation spectroscopy in the presence of H(2)(17)O. The spectra show interaction of the (17)O with the preparation in the S(2) state induced by 200 K illumination. The modulation is observed only in the center of the multiline spectrum. The inferred hyperfine coupling terms are compatible with water (not hydroxyl) oxygen bound to a particular quasi-axial Mn(III) center in a coupled Mn cluster.     

Electron transfer from the water oxidizing complex at cryogenic temperatures: the S1 to S2 step

We report the detection of a "split" electron paramagnetic resonance (EPR) signal during illumination of dark-adapted (S(1) state) oxygen-evolving photosystem II (PSII) membranes at <20 K. The characteristics of this signal indicate that it arises from an interaction between an organic radical and the Mn cluster of PSII. The broad radical signal decays in the dark following illumination either by back-reaction with Qa*- or by forward electron transfer from the Mn cluster. The forward electron transfer (either from illumination at 11 K followed by incubation in the dark at 77 K or by illumination at 77 K) results in the formation of a multiline signal similar to, but distinct from, other well-characterized multiline forms found in the S0 and S2 states. The relative yield of the "S1 split signal", which we provisionally assign to S1X*, where X could be YZ* or Car*+, and that of the 77 K multiline signal indicate a relationship between the two states. An approximate quantitation of the yield of these signals indicates that up to 40-50% of PSII centers can form the S1 split signal. Ethanol addition removes the ability to observe the S1 split signal, but the multiline signal is still formed at 77 K. The multiline forms with <700 nm light and is not affected by near-infrared (IR) light, showing that we are detecting electron transfer in centers not responsive to IR illumination. The results provide important new information about the mechanism of electron abstraction from the water oxidizing complex (WOC).     

Comparison of the structure of the manganese complex in the S1 and S2 states of the photosynthetic O2-evolving complex: an x-ray absorption spectroscopy study

A Mn-containing enzyme complex is involved in the oxidation of H2O to O2 in algae and higher plants. X-ray absorption spectroscopy is well suited for studying the structure and function of Mn in this enzyme complex. Results of X-ray K-edge and extended X-ray absorption fine structure (EXAFS) studies of Mn in the S1 and S2 states of the photosynthetic O2-evolving complex in photosystem II preparations from spinach are presented in this paper. The S2 state was prepared by illumination at 190 K or by illumination at 277 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU); these are protocols that limit the photosystem II reaction center to one turnover. Both methods produce an S2 state characterized by a multiline electron paramagnetic resonance (EPR) signal. An additional protocol, illumination at 140 K, produces as a state characterized by the g = 4.1 EPR signal. We have previously observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark-adapted S1 state to the S2 state produced by illumination at 190 K [Goodin, D. B., Yachandra, V. K., Britt, R. D., Sauer, K., & Klein, M. P. (1984) Biochim. Biophys. Acta 767, 209-216]. The Mn K-edge spectrum of the 277 K illuminated sample is similar to that produced at 190 K, indicating that the S2 state is similar when produced at 190 or 277 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermediates of the S(3) state of the oxygen-evolving complex of photosystem II

The S(3) state of the water-oxidizing complex (WOC) of photosystem II (PSII) is the last state that can be trapped before oxygen evolution occurs at the transient S(4) state. A number of EPR-detectable intermediates are associated with this critical state. The preceding paper examined mainly the decay of S(3) at cryogenic temperatures leading to the formation of a proton-deficient configuration of S(2) termed S(2)'. This second paper examines all intermediates formed by the near-IR light (NIR) excitation of the S(3) state and compares these with the light-excitation products of the S(2)' state. The rather complex set of observations is organized in a comprehensive flowchart, the central part of which is the S(3)...Q(A)(-) state. This state can be converted to various intermediates via two main pathways: (A) Excitation of S(3) by NIR light at temperatures below 77 K results presumably in the formation of an excited S(3) state, S(3), which decays via either of two pathways. Slowly at liquid helium temperatures but much faster at 77 K, S(3) decays to an EPR-silent state, denoted S(3)' ', which by raising the temperature to ca. 190 K converts to a spin configuration of the Mn cluster, characterized by g = 21, 3.7 in perpendicular and g = 23 in parallel mode EPR, denoted S(3)'. Upon further warming to 220 K, S(3)' relaxes to the untreated S(3) state. Below about 77 K and more favorably at liquid helium temperatures, an alternative pathway of S(3) decay via the metallo-radical intermediate S(2)'Z*...Q(A)(-) can be traced. This leads to the metastable state S(2)'Z...Q(A) via charge recombination. S(2)'Z* is characterized by a split-radical signal at g = 2, while all S(2)' transients are characterized by the same g = 5/2.9 (S = (7)/(2)) configuration of the Mn cluster with small modifications, reflecting an influence of the tyr Z oxidation state on the crystal-field symmetry at the Mn cluster. (B) S(2)'...Q(A) can be reached alternatively by the slow charge recombination of S(3) and Q(A)(-) at 77 K. White-light illumination of S(2)'.Q(A) below about 20 K results in charge separation, reforming the intermediate S(2)'Z*...Q(A)(-). Thermally activated branches to the main pathways are also described, e.g., at elevated temperatures tyr Z* reoxidizes S(2)' to the S(3) state. The above observations are discussed in terms of a molecular model of the S(3) state of the OEC. Main aspects of the model are the following. Intermediates, isoelectronic to S(3), are attributed to the NIR-induced translocation of the positive hole to different Mn ligands, or to tyr Z. On the basis of a comparison of the electron-donating efficiency of tyr Z and tyr D at cryogenic temperatures, it is inferred that the Mn cluster acts as the main proton acceptor from tyr Z. Water associated with the Mn cluster is assumed to be in hydrogen-bonding equilibrium with tyr Z, and an array comprising this water and adjacent water (or OH or O) ligands to Mn followed by a sequence of proton acceptors is proposed to act as an efficient proton translocation pathway. Oxidation of the tyrosine by P(680)(+) repels protons to and out from the Mn cluster. This proposed role of tyr Z in the water-splitting process is described as a proton repeller/electron abstractor.     

Mechanism of photosynthetic water oxidation: combining biophysical studies of photosystem II with inorganic model chemistry

A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.     

ESEEM studies of substrate water and small alcohol binding to the oxygen-evolving complex of photosystem II during functional turnover

We report the first examination of exchangeable proton and MeOH interactions with the Mn catalytic cluster in photosystem II, under functional flash turnover conditions, using 2H ESEEM spectroscopy on the S2 and S0 multiline states. Deuterium-labeled water (D2O) and methyl d3-labeled methanol (DMeOH) are employed. It was discovered that a hyperfine resolved multiline S0 signal could be seen in the presence of D2O, the hyperfine structure of which depended on the presence or absence of methanol (MeOH). In the presence of DMeOH, significant dipolar coupling of the three methyl deuterons to the multiline centers in the S2 and S0 states was seen (S2, 0.65, 0.39(2) MHz; and S0, 0.60, 0.37(2) MHz). These are consistent with direct binding of the methoxy fragment to Mn. Assuming terminal Mn-OMe ligation, the couplings indicated a spin projection coefficient (rho) magnitude of approximately 2 for the ligating Mn in both the S2 and S0 states, with inferred Mn-O distances of approximately 1.9-2.0 A. In the presence of D2O, four classes of exchangeable deuterons were identified by ESEEM in S2 and S0. Three of these classes (1, 2, and 4) exhibited populations and coupling strengths that were essentially constant under various conditions of sample preparation, illumination turnover, and small alcohol addition. Class 3 could be modeled with constant coupling but a highly variable deuteron population (n3 approximately 0-10) depending in part on the preparation used. For all classes, the coupling parameters were very similar in S2 and S0. The favored interpretation is that the two strongest coupling classes (1 and 2) represent close binding of one water molecule to a single Mn which has an oxidation state of II in S0 and III in S2, and rho approximately 2 in both cases. This water is not displaced by MeOH, but either the water or MeOH is singly deprotonated upon MeOH binding. Class 4 represents approximately 2 water molecules which are not closely bound to Mn (Mn-deuteron distances of approximately 3.7-4.7 A). Class 3 probably represents protein matrix protons within approximately 4 A of the Mn in the cluster, which can be variably exchanged in different preparations.     

Water-sensitive low-frequency vibrations of reaction intermediates during S-state cycling in photosynthetic water oxidation

In photosynthetic water oxidation, two water molecules are converted to an oxygen molecule through five reaction intermediates, designated S(n) (n = 0-4), at the catalytic Mn cluster of photosystem II. To understand the mechanism of water oxidation, changes in the chemical nature of the substrate water as well as the Mn cluster need to be defined during S-state cycling. Here, we report for the first time a complete set of Fourier transform infrared difference spectra during S-state cycling in the low-frequency (670-350 cm(-1)) region, in which interactions between the Mn cluster and its ligands can be detected directly, in PS II core particles from Thermosynechococcus elongatus. Furthermore, vibrations from oxygen and/or hydrogen derived from the substrate water and changes in them during S-state cycling were identified using multiplex isotope-labeled water, including H2(18)O, D2(16)O, and D2(18)O. Each water isotope affected the low-frequency S-state cycling spectra, characteristically. The bands sensitive only to (16)O/(18)O exchange were assigned to the modes from structures involving Mn and oxygen having no interactions with hydrogen, while the bands sensitive only to H/D exchange were assigned to modes from amino acid side chains and/or polypeptide backbones that associate with water hydrogen. The bands sensitive to both (16)O/(18)O and H/D exchanges were attributed to the structure involving Mn and oxygen structurally coupled with hydrogen in a direct or an indirect manner through hydrogen bonds. These bands include the changes of intermediate species derived from substrate water during the process of photosynthetic water oxidation.     

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.     

Structure of an active water molecule in the water-oxidizing complex of photosystem II as studied by FTIR spectroscopy

The vibrations of a water molecule in the water-oxidizing complex (WOC) of photosystem II were detected for the first time using Fourier transform infrared (FTIR) spectroscopy. In a flash-induced FTIR difference spectrum upon the S(1)-to-S(2) transition, a pair of positive and negative bands was observed at 3618 and 3585 cm(-1), respectively, and both bands exhibited downshifts by 12 cm(-1) upon replacement of H(2)(16)O by H(2)(18)O. Upon D(2)O substitution, the bands largely shifted down to 2681 and 2652 cm(-1). These observations indicate that the bands at 3618 and 3585 cm(-1) arise from the O-H stretching vibrations of a water molecule, probably substrate water, coupled to the Mn cluster in the S(2) and S(1) states, respectively. The band frequencies indicate that the O-H group forms a weak H-bond and this H-bonding becomes weaker upon S(2) formation. Intramolecular coupling with the other O-H vibration of this water molecule was studied by a decoupling experiment using a H(2)O/D(2)O (1:1) mixture. The downshifts by decoupling were estimated to be 4 and 12 cm(-1) for the 3618 (S(2)) and 3585 cm(-1) (S(1)) bands, both of which were much smaller than 52 cm(-1) of water in vapor, indicating that the observed water has a considerably asymmetric structure; i.e., one of the O-H groups is weakly and the other is strongly H-bonded. The smaller coupling in the S(2) than the S(1) state means that this H-bonding asymmetry becomes more prominent upon S(2) formation. Such a structural change may facilitate the proton release reaction that takes place in the later step by lowering the potential barrier. The present study showed that FTIR detection of the O-H vibrations is a useful and promising method to directly monitor the chemical reactions of substrate water and clarify the molecular mechanism of photosynthetic water oxidation.     

A calcium-specific site influences the structure and activity of the manganese cluster responsible for photosynthetic water oxidation

EPR studies have revealed that removal of calcium using citric acid from the site in spinach photosystem II which is coupled to the photosynthetic O2-evolving process produces a structural change in the manganese cluster responsible for water oxidation. If done in the dark, this yields a modified S1' oxidation state which can be photooxidized above 250 K to form a structurally altered S2' state, as seen by formation of a "modified" multiline EPR signal. Compared to the "normal" S2 state, this new S2'-state EPR signal has more lines (at least 25) and 25% narrower 55Mn hyperfine splittings, indicative of disruption of the ligands to manganese. The calcium-depleted S2' oxidation state is greatly stabilized compared to the native S2 oxidation state, as seen by a large increase in the lifetime of the S2' EPR signal. Calcium reconstitution results in the reduction of the oxidized tyrosine residue 161YD+ (Em approximately 0.7-0.8 V, NHE) within the reaction center D1 protein in both the S1' and S2' states, as monitored by its EPR signal intensity. We attribute this to reduction by Mn. Thus a possible structural role which calcium plays is to bring YD+ into redox equilibrium with the Mn cluster. Photooxidation of S2' above 250 K produces a higher S state (S3 or S4) having a new EPR signal at g = 2.004 +/- 0.003 and a symmetric line width of 163 +/- 3 G, suggestive of oxidation of an organic donor, possibly an amino acid, in magnetic contact with the Mn cluster. This EPR signal forms in a stoichiometry of 1-2 relative to YD+.(ABSTRACT TRUNCATED AT 250 WORDS)     

No evidence from FTIR difference spectroscopy that aspartate-170 of the D1 polypeptide ligates a manganese ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.     

Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z.

The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).     

Mutation of arginine 357 of the CP43 protein of photosystem II severely impairs the catalytic S-state cycle of the H2O oxidation complex

Basic amino acid side chains situated in active sites may mediate critical proton transfers during an enzymatic catalytic cycle. In the case of photosynthetic water oxidation, a strong base is postulated to facilitate the deprotonation of the active site Mn4-Ca cluster, thereby allowing the otherwise thermodynamically constrained transfer of an electron away from the Mn4-Ca cluster to the oxidized redox active tyrosine radical, YZ*, generated by photosynthetic charge separation. Arginine 357 of the CP43 polypeptide may be located in the second coordination shell of the O2-evolving Mn4-Ca cluster of photosystem II (PSII) according to current structural models. An ostensibly conservative substitution mutation, CP43-357K, was investigated using polarographic and fluorescence techniques in evaluating its potential impact on S-state cycling. Cells containing the CP43-357K mutation lost their capacity for autotrophic growth and exhibited a drastic reduction in O2 evolving activity ( approximately 15% of that of the wild type) despite the fact that mutant cells contained more than 80% of the concentration of charge-separating PSII reaction centers and more than half of these contained photooxidizable Mn. Fluorescence kinetics indicated that acceptor side electron transfer, dominated by the transfer of electrons from QA- to QB, was unaffected, but the fraction of centers containing Mn clusters capable of forming the S2 state was reduced to approximately 40% of that of the wild type. Analysis of O2 yields using a bare platinum electrode indicated a severe defect in the S-state cycling properties of the mutant H2O oxidation complexes. Although O2 evolution was delayed to the third flash during a train of single-turnover saturating flashes, the pattern of O2 emission did not exhibit a discernible periodicity indicating a very high miss factor, which was estimated to be approximately 45% compared to the wild-type value of approximately 10%. On the other hand, the multiflash fluorescence measurements indicate that the yield of formation of the S2 state from S1 is diminished by approximately 20%, although this latter estimate is complicated by the presence of damaged PSII centers. Taken together, the experiments indicate that the high miss factor observed during S-state cycling is likely due to a defect in the higher S-state transitions. These results are discussed in relation to the idea that CP43-R357 may serve as a ligand to bicarbonate or as the catalytic base proposed to mediate proton-coupled electron transfer (PCET) in the higher S states of the catalytic cycle of H2O oxidation.     

Trapping of the S2 to S3 state intermediate of the oxygen-evolving complex of photosystem II

Photosystem II preparations poised in the S(2)...Q(A) state produce no detectable intermediate during straightforward illumination at liquid helium temperatures. However, upon flash illumination in the range of 77-190 K, they produce a transient state which at -10 degrees C advances to S(3) or after rapid cooling to 10 K gives rise to a 116 G wide metalloradical EPR signal. The latter decays with half-times on the order of a few minutes, presumably by charge recombination, and can be regenerated repeatedly by illumination at 10 K. The constraints for Tyr Z oxidation are attributed to the presence of excess positive charge in S(2). Elevated temperatures are required presumably to overcome a thermal barrier in the deprotonation of Tyr Z(+) or most likely to allow secondary proton transfer away from the base partner of Tyr Z. Treatment with 5% (v/v) MeOH appears to remove the constraints for Tyr Z oxidation, and a 160 G wide metalloradical EPR signal is produced by illumination at 10 K, which decays with a half-time of ca. 80 s. Formation of the metalloradical signals is accompanied by reversible changes in the Mn multiline signal. The intermediates are assigned to Tyr Z(*) magnetically interacting with the Mn cluster in S(2), S(2)Y(Z)(*). A molecular model which extends an earlier suggestion and provides a plausible explanation of a number of observations, including the binding of small molecules to the Mn cluster, is presented.     

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.     

Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1Y(Z*)

Near-IR (NIR) excitation at liquid He temperatures of photosystem II (PSII) membranes from the cyanobacterium Synechococcus vulcanus or from spinach poised in the S2 state results in the production of a g = 2.035 EPR resonance, reminiscent of metalloradical signals. The signal is smaller in the spinach preparations, but it is significantly enhanced by the addition of exogenous quinones. Ethanol (2-3%, v/v) eliminates the ability to trap the signal. The g = 2.035 signal is identical to the one recently obtained by Nugent et al. by visible-light illumination of the S1 state, and preferably assigned to S1Y(Z*) [Nugent, J. H. A., Muhiuddin, I. P., and Evans, M. C. W. (2002) Biochemistry 41, 4117-4126]. The production of the g = 2.035 signal by liquid He temperature NIR excitation of the S2 state is paralleled by a significant reduction (typically 40-45% in S. vulcanus) of the S2 state multiline signal. This is in part due to the conversion of the Mn cluster to higher spin states, an effect documented by Boussac et al. [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007], and in part due to the conversion to the g = 2.035 configuration. Following the decay of the g = 2.035 signal at liquid helium temperatures (decay halftimes in the time range of a few to tens of minutes depending on the preparation), annealing at elevated temperatures (-80 degrees C) results in only partial restoration of the S2 state multiline signal. The full size of the signal can be restored by visible-light illumination at -80 degrees C, implying that during the near-IR excitation and subsequent storage at liquid helium temperatures recombination with Q(A-) (and therefore decay of the S2 state to the S1 state) occurred in a fraction of centers. In support of this conclusion, the g = 2.035 signal remains stable for several hours (at 11 K) in centers poised in the S2...Q(A) configuration before the NIR excitation. The extended stability of the signal under these conditions has allowed the measurement of the microwave power saturation and the temperature dependence in the temperature range of 3.8-11 K. The signal intensity follows Curie law temperature dependence, which suggests that it arises from a ground spin state, or a very low-lying excited spin state. The P1/2 (microwave power at half-saturation) value is 1.7 mW at 3.8 K and increases to 96 mW at 11 K. The large width of the g = 2.035 signal and its relatively fast relaxation support the assignment to a radical species in the proximity of the Mn cluster. The whole phenomenology of the g = 2.035 signal production is analogous to the effects of NIR excitation on the S3 state [Ioannidis, N., Nugent, J. H. A., and Petrouleas, V. (2002) Biochemistry 41, 9589-9600] producing an S2'Y(Z*) intermediate. In the present case, the intermediate is assigned to S1Y(Z*). The NIR-induced increase in the oxidative capability of the Mn cluster is discussed in relation to the photochemical properties of a Mn(III) ion that exists in both S2 and S3 states. The EPR properties of the S1Y(Z*) intermediate cannot be reconciled easily with our current understanding of the magnetic properties of the S1 state. It is suggested that oxidation of tyr Z alters the magnetic properties of the Mn cluster via exchange of a proton.     

An evaluation of structural models for the photosynthetic water-oxidizing complex derived from spectroscopic and X-ray diffraction signatures

Four of the five intermediate oxidation states (S-states) in the catalytic cycle of water oxidation used by O2-evolving photoautotrophs have been previously characterized by EPR and/or ENDOR spectroscopy, with the first reports for the S0, S1, and S3 states available in just the last three years. The first electron density map of the Mn cluster derived from X-ray diffraction measurements of single crystals of photosystem II at 3.8-4.2 A resolution has also appeared this year. This wealth of new information has provided significant insight into the structure of the inorganic core (Mn4OxCa1Cl1-2), the Mn oxidation states, and the location and function of the essential Ca2+ cofactor within the water-oxidizing complex (WOC). We summarize these advances and provide a unified interpretation of debated structural proposals and Mn oxidation states, based on an integrated analysis of the published data, particularly from Mn X-ray absorption spectroscopy (XAS) and EPR/ENDOR data. Only three magnetic spin-exchange models for the inter-manganese interactions are possible from consideration of the EPR data for the S0, S1, S2 and S(-N) (NO-reduced) states. These models fall into one of three types denoted butterfly, funnel, or tetrahedron. A revised set of eight allowed chemical structures for the Mn4Ox core can be deduced that are shown to be consistent with both EPR and XAS. The popular "dimer-of-dimers" structural model is not compatible with the possible structural candidates. EPR data have identified two inter-manganese couplings that are sensitive to the S-state, suggesting two possible bridging sites for substrate water molecules. Spin densities derived from 55Mn hyperfine data together with Mn K-edge energies from Ca-depleted samples provide an internally consistent assignment for the Mn oxidation states of Mn4(3III,IV) for the S2 state. EPR and XAS data also provide a consistent picture, locating Ca2+ as an integral part of the inorganic core, probably via shared bridging ligands with Mn (aqua/hydroxo/carboxylato/chloro). XAS data reveal that the Ca2+ cofactor increases the Mn(1s-->4p) transition energy by 0.6-1 eV with minimal structural perturbation versus the Ca-depleted WOC. Thus, calcium binding appears to increase the Mn-ligand covalency by increasing electron transfer from shared ligands to Mn, suggesting a direct role for Ca2+ in substrate water oxidation. Consideration of both the XAS and the EPR data, together with reactivity studies on two model complexes that evolve O2, suggest two favored structure types as feasible models for the reactive S4 state that is precursor to the O2 evolution step. These are a calcium-capped "cuboidal" core and a calcium-capped "funnel" core.     

Structural and oxidation state changes of the photosystem II manganese complex in four transitions of the water oxidation cycle (S0 --> S1, S1 --> S2, S2 --> S3, and S3,4 --> S0) characterized by X-ray absorption spectroscopy at 20 K and room temperature

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine(Z) (Tyr(Z)) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr(Z) with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr(Z) and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr(Z) oxidation at cryogenic temperature in the S(0) and S(1) states was around neutral pH and was essentially pH-independent. The yield of Tyr(Z) oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr(Z)(.) in the S(0) and S(1) states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr(Z) oxidation and Tyr(Z)(.) reduction at cryogenic temperature in the S(0) and S(1) states of the active PSII. Theoretical calculations indicate that Tyr(Z) becomes more difficult to oxidize when a H(2)O molecule interacts directly with it. It is suggested that Tyr(Z) is probably located in a hydrophobic environment with no direct interaction with the substrate H(2)O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S(0) and S(2) states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D(2)O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S(0) and S(2) state multiline disappeared after 3 h of D(2)O incubation in the S(0) and S(1) states, respectively. The signal with 4.0 MHz hyperfine constants in S(0) state multiline disappeared after 3 h of D(2)O incubation in the S(0) state, while the similar signal in S(2) state multiline disappeared only after 24 h of D(2)O incubation in the S(1) state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 A, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.