Theoretical investigation on peripheral ligands of oxygen-evolving center in photosystem II

The interaction between the Mn-cluster and its peripheral ligands in oxygen-evolving center is still unclear. Theoretical investigation on the coordination of histidine, H2O, and Cl to Mn2O2 units in OEC is conducted. The following conclusions are obtained: (i) both histidine and H2O molecule, bound to the two Mn ions, respectively, are vertical to the Mn2O2 plane, and maintain a large distance; (ii) the two H2O molecules cannot bind to the same Mn2O2 unit. Based on Mn-cluster structure in OEC, we theoretically predict that two H2O molecules bind to the two Mn ions at the "C"-shaped open end in S0 state, while two His residues at the closed end. Cl ion can only terminally ligate at the open end. Individual valence for the four Mn ions in S0 state is assigned.     

Theoretical investigation on peripheral ligands of oxygen-evolving center in photosystem II

The interaction between the Mn-cluster and its peripheral ligands in oxygen-evolving center is still unclear. Theoretical investigation on the coordination of histidine, H2O, and Cl to Mn2O2 units in OEC is conducted. The following conclusions are obtained: (i) both histidine and H2O molecule, bound to the two Mn ions, respectively, are vertical to the Mn2O2 plane, and maintain a large distance; (ii) the two H2O molecules cannot bind to the same Mn2O2 unit. Based on Mn-cluster structure in OEC, we theoretically predict that two H2O molecules bind to the two Mn ions at the "C"-shaped open end in S0 state, while two His residues at the closed end. Cl ion can only terminally ligate at the open end. Individual valence for the four Mn ions in S0 state is assigned.     

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.     

Biosynthetic exchange of bromide for chloride and strontium for calcium in the photosystem II oxygen-evolving enzymes

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.     

Functional linkage between phycobilisome and reaction center in two phycobilisome oxygen-evolving photosystem II preparations isolated from the thermophilic cyanobacterium Synechococcus sp

Photosystem II oxygen-evolving preparations with attached phycobilisomes were isolated from the thermophilic cyanobacterium Synechococcus sp. with beta-octylglucoside or digitonin. Fluorescence emission spectra of the two preparations determined at 77 K largely lacked a far red band which originates from photosystem I. The spectrum of the digitonin preparation was otherwise similar to that of intact cells, whereas the beta-octylglucoside preparation showed a pronounced band at 687 nm, which is considered to be emitted from phycobilisomes. The relative yield of phycobilin fluorescence was similar between the digitonin preparations and the cells but was considerably larger in the beta-octylglucoside preparations at room temperature. The quantum yield of ferricyanide photoreduction determined with light which is absorbed mainly by phycobiliproteins was 0.85 for the digitonin preparation and 0.57 for the beta-octylglucoside preparation. The results indicate that excitation energy is transferred from phycobilisomes to photosystem II reaction centers in the digitonin preparation as efficiently as in intact cells, while a significant portion of light energy harvested by phycobilisomes is not utilized by the primary photochemistry in the beta-octylglucoside preparation. Digitonin and beta-octylglucoside preparations had 65 and 48 chlorophyll a molecules per photosystem II reaction center, respectively. The beta-octylglucoside preparation contained twice as much phycocyanin and allophycocyanin per photosystem II reaction center as the digitonin preparation, which has a phycobiliprotein-to-photosystem II reaction center ratio very similar to that of cells. It is concluded that whereas the beta-octylglucoside preparation contains a considerable amount of free phycobilisomes, all phycobilisomes present in the digitonin preparation are physically and functionally linked to photosystem II reaction center complexes.     

Binding of amines to the O2-evolving center of photosystem II

The binding of several primary amines to the O2-evolving center (OEC) of photosystem II (PSII) has been studied by using low-temperature electron paramagnetic resonance (EPR) spectroscopy of the S2 state. Spinach PSII membranes treated with NH4Cl at pH 7.5 produce a novel S2-state multiline EPR spectrum with a 67.5-G hyperfine line spacing when the S2 state is produced by illumination at 0 degrees C [Beck, W. F., de Paula, J. C., & Brudvig, G. W. (1986) J. Am. Chem. Soc. 108, 4018-4022]. The altered hyperfine line spacing and temperature dependence of the S2-state multiline EPR signal observed in the presence of NH4Cl are direct spectroscopic evidence for coordination of one or more NH3 molecules to the Mn site in the OEC. In contrast, the hyperfine line pattern and temperature dependence of the S2-state multiline EPR spectrum in the presence of tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol, or CH3NH2 at pH 7.5 were the same as those observed in untreated PSII membranes. We conclude that amines other than NH3 do not readily bind to the Mn site in the S2 state because of steric factors. Further, NH3 binds to an additional site on the OEC, not necessarily located on Mn, and alters the stability of the S2-state g = 4.1 EPR signal species. The effects on the intensities of the g = 4.1 and multiline EPR signals as the NH3 concentration was varied indicate that both EPR signals arise from the same paramagnetic site and that binding of NH3 to the OEC affects an equilibrium between two configurations exhibiting the different EPR signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Oxidation of the two beta-carotene molecules in the photosystem II reaction center

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.     

Structure of photosystem II and molecular architecture of the oxygen-evolving centre

Photosynthesis utilizes light energy to oxidize water molecules to molecular oxygen at the oxygen-evolving centre of photosystem II. The structure of photosystem II from the cyanobacterium Thermosynechococcus elongatus has been reported at 3.5A resolution and, for the first time, the complete molecular structure of this 650 kDa complex, including the oxygen-evolving centre, has been revealed.     

Interaction of the high-spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen-evolving PS II reaction center complex and in PS II particles

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q₄₀₀, Fe(III) atom interacting with the PS II primary electron acceptor quinone Q_A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q_B. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipids in oxygen-evolving photosystem II complexes of cyanobacteria and higher plants

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.     

Polypeptides of the oxygen-evolving photosystem II complex. Immunological detection and biogenesis

Oxygen-evolving photosystem II complex was isolated from spinach chloroplasts. The individual polypeptides of the complex were isolated from sodium dodecyl sulfate (SDS)-polyacrylamide gels and antibodies were raised in rabbits against these polypeptides. After washing of the isolation complex by 0.8 M Tris to release the extrinsic proteins, a distinct diffused protein band was revealed at the position of 33 kDa in SDS gels containing 4 M urea. When this band was electroeluted from the gel and subsequently electrophoresed on SDS gels, three distinct protein bands became apparent. Antibodies raised against each one of these polypeptides cross-reacted with the other two polypeptides to varying degrees but not with the other subunits of the complex. The three polypeptides were denoted as "34," "33," and "32" kDa and the 33 being the herbicide-binding protein. Using the antibodies, the relative amounts of the photosystem II polypeptides were followed during greening of etiolated spinach seedlings. While all three extrinsic polypeptides were present in etiolated leaves at relatively high amounts, the other polypeptides could not be detected prior to an approximate 6-h illumination period. Further illumination induced the appearance of all of the rest of the subunits in a relatively similar rate. The oxygen evolution activity was developed parallel to the increase in the amounts of these polypeptides. Therefore, the assembly of the active photosystem II during greening is a two-step process in contrast with the photosystem I reaction center, which is assembled step by step, and the rest of the chloroplast protein complexes, which are assembled by a concerted mechanism.     

Isolation and characterization of oxygen-evolving photosystem II particles and photosystem II core complex from the filamentous cyanobacterium Spirulina platensis

Photosystem (PS) II particles retaining a high rate of O₂ evolution were isolated from the mesophilic filamentous cyanobacterium, Spirulina platensis. To achieve high production of PSII complexes in the cells, irradiance from halogen incandescent lamps was used. Disruption of cells by vibration of glass beads proved to be the most suitable procedure for isolation of thylakoid membranes. The selectivity of detergents for PSII particle preparation rose in the order of Triton X-100 < decyl-β-D-glucopyranoside < dodecyldimethyl-aminooxide < n-heptyl-β-D-thioglucoside < N-dodecyl-N,N-dimethylammonio-3-propane sulphonate < n-octyl-β-thioglycoside < octylglucoside < n-dodecyl-β-D-maltoside. The last four detergents yielded extracts, from which pure PSII particles not contaminated by PSI complexes could be obtained by sucrose-gradient centrifugation (20–45%) at the 43% sucrose level. We assumed both the acceptor and donor sides of the isolated n-dodecyl-β-D-maltoside (DM) particles to be intact due to high oxygen production by DM particles [1,500 meq(e^?) mol^?1 (Chl) s^?1] achieved in the presence of all artificial acceptors tested. The PSII particle fraction from the sucrose gradient was used with immobilized metal (Cu²⁺) affinity chromatography (IMAC) for the preparation of the PSII core complex. By washing the column with a MES buffer containing MgCl₂ and CaCl₂, the phycobiliproteins were stripped off. The PSII core complex was eluted in a buffer containing 1% DM, mannitol, MgCl₂, NaCl, CaCl₂, and ?-aminocaproic acid. SDS-PAGE of the core complex provided pure bands of D1 and D2 proteins and PsbO protein from thylakoid membrane, which were used to raise polyclonal antibodies in rabbits. These antibodies recognized D1 and D2 not only as monomers of 31 and 32 kDa proteins, but also as heterodimers of D1, D2 corresponding to the band of 66 kDa on SDS-PAGE. This was in contrast to antibodies of synthetic determinants, which reacted only with the monomers of D1 and D2 proteins. These negative reactions against heterodimers of D1, D2 supported the hypothesis that dimeric forms of PSII reaction centre proteins have a C-terminal sequence sterically protected against a reaction with specific antibodies.     

FTIR detection of water reactions in the oxygen-evolving centre of photosystem II

Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700-3500 cm(-1) region in FTIR difference spectra during S-state cycling. In the S1-->S2 transition, a band shift from 3588 to 3617 cm(-1) was observed, indicative of a weakened H-bond. Decoupling experiments using D2O:H2O (1:1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S2 formation. In the S2-->S3, S3-->S0 and S0-->S1 transitions, negative bands were observed at 3634, 3621 and 3612 cm(-1), respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600-1300 cm-(1)) as 'fingerprints' of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S2-->S3, S3-->S0 and S0-->S1 transitions was consistent with a view that protons are released in the three transitions other than S1-->S2, while relatively high susceptibility to dehydration in the S2-->S3 and S3-->S0 transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.     

Multiple redox-active chlorophylls in the secondary electron-transfer pathways of oxygen-evolving photosystem II

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680(+). We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and beta-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 and 814 nm and three slow-decaying bands at 810, 825, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl(+)). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b 559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl(+) and Car(+) species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680(+) and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulfoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680(+).     

Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II

The galactolipid digalactosyldiacylglycerol (DGDG) is present in the thylakoid membranes of oxygenic photosynthetic organisms such as higher plants and cyanobacteria. Recent x-ray crystallographic analysis of protein-cofactor supercomplexes in thylakoid membranes revealed that DGDG molecules are present in the photosystem II (PSII) complex (four molecules per monomer), suggesting that DGDG molecules play important roles in folding and assembly of subunits in the PSII complex. However, the specific role of DGDG in PSII has not been fully clarified. In this study, we identified the dgdA gene (slr1508, a ycf82 homolog) of Synechocystis sp. PCC6803 that presumably encodes a DGDG synthase involved in the biosynthesis of DGDG by comparison of genomic sequence data. Disruption of the dgdA gene resulted in a mutant defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew as rapidly as the wild-type cells, indicating that DGDG is not essential for growth in Synechocystis. However, we found that oxygen-evolving activity of PSII was significantly decreased in the mutant. Analyses of the PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO, which stabilize the oxygen-evolving complex, were substantially dissociated from the PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen-evolving activity was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that the PsbU subunit is dissociated from the PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins required for stabilization of the oxygen-evolving complex.     

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.     

Calcium EXAFS establishes the Mn-Ca cluster in the oxygen-evolving complex of photosystem II

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 A, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is approximately 3.5 A distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster.     

Crystallization and the crystal properties of the oxygen-evolving photosystem II from Synechococcus vulcanus

A photosystem II (PSII) complex highly active in oxygen evolution was purified and crystallized from a thermophilic cyanobacterium, Synechococcus vulcanus. The PSII complex in the crystals contained the D1/D2 reaction center subunits, CP47 and CP43 (two chlorophyll-binding core antenna proteins of photosystem II), cytochrome b-559 alpha- and beta-subunits, several low molecular weight subunits, and three extrinsic proteins, that is, 33 and 12 kDa proteins and cytochrome c-550. The PSII complex also retained a high rate of oxygen evolution. The apparent molecular mass of the PSII in the crystals was determined to be 580 kDa by gel filtration chromatography, indicating that the PSII crystallized is a dimer. The crystals diffracted to a maximum resolution of 3.5 A at a cryogenic temperature using X-rays from a synchrotron radiation source, SPring-8. The crystals belonged to an orthorhombic system, and the space group was P2(1)2(1)2(1) with unit cell dimensions of a = 129.7 A, b = 226.5 A, and c = 307.8 A. Each asymmetric unit contained one PSII dimer, which gave rise to a specific volume (V(M)) of 3.6 A(3)/Da based on the calculated molecular mass of 310 kDa for a PSII monomer and an estimated solvent content of 66%. Multiple data sets of native crystals have been collected and processed to 4.0 A, indicating that our crystals are suitable for structure analysis at this resolution.