Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

Does aspartate 170 of the D1 polypeptide ligate the manganese cluster in photosystem II? An EPR and ESEEM Study

Aspartate 170 of the D1 polypeptide provides part of the high-affinity binding site for the first Mn(II) ion that is photooxidized during the light-driven assembly of the (Mn)(4) cluster in photosystem II [Campbell, K. A., Force, D. A., Nixon, P. J., Dole, F., Diner, B. A., and Britt, R. D. (2000) J. Am. Chem. Soc. 122, 3754-3761]. However, despite a wealth of data on D1-Asp170 mutants accumulated over the past decade, there is no consensus about whether this residue ligates the assembled (Mn)(4) cluster. To address this issue, we have conducted an EPR and ESEEM (electron spin-echo envelope modulation) study of D1-D170H PSII particles purified from the cyanobacterium Synechocystis sp. PCC 6803. The line shapes of the S(1) and S(2) state multiline EPR signals of D1-D170H PSII particles are unchanged from those of wild-type PSII particles, and the signal amplitudes correlate approximately with the lower O(2) evolving activity of the mutant PSII particles (40-60% compared to that of the wild type). These data provide further evidence that the assembled (Mn)(4) clusters in D1-D170H cells function normally, even though the assembly of the (Mn)(4) cluster is inefficient in this mutant. In the two-pulse frequency domain ESEEM spectrum of the 9.2 GHz S(2) state multiline EPR signal of D1-D170H PSII particles, the histidyl nitrogen modulation observed at 4-5 MHz is unchanged from that of wild-type PSII particles and no significant new modulation is observed. Three scenarios are presented to explain this result. (1) D1-Asp170 ligates the assembled (Mn)(4) cluster, but the hyperfine couplings to the ligating histidyl nitrogen of D1-His170 are too large or anisotropic to be detected by ESEEM analyses conducted at 9.2 GHz. (2) D1-Asp170 ligates the assembled (Mn)(4) cluster, but D1-His170 does not. (3) D1-Asp170 does not ligate the assembled (Mn)(4) cluster.     

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

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

Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms

The pulsed electron paramagnetic resonance (EPR) methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the structure of the Photosystem II oxygen-evolving complex (OEC), including the paramagnetic manganese cluster and its immediate surroundings. Recent unpublished results from the pulsed EPR laboratory at UC-Davis are discussed, along with aspects of recent publications, with a focus on substrate and cofactor interactions. New data on the proximity of exchangeable deuterons around the Mn cluster poised in the S(0)-state are presented and interpreted. These pulsed EPR results are used in an evaluation of several recently proposed mechanisms for PSII water oxidation. We strongly favor mechanistic models where the substrate waters bind within the OEC early in the S-state cycle. Models in which the O-O bond is formed by a nucleophilic attack by a Ca(2+)-bound water on a strong S(4)-state electrophile provide a good match to the pulsed EPR data.     

D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy

We report both mid-frequency (1800-1200 cm(-)(1)) and low-frequency (670-350 cm(-)(1)) S(2)/S(1) FTIR difference spectra of photosystem II (PSII) particles isolated from wild-type and D1-D170H mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Both mid- and low-frequency S(2)/S(1) spectra of the Synechocystis wild-type PSII particles closely resemble those from spinach PSII samples, which confirms an earlier result by Noguchi and co-workers [Noguchi, T., Inoue, Y., and Tang, X.-S. (1997) Biochemistry 36, 14705-14711] and indicates that the coordination environment of the oxygen evolving complex (OEC) in Synechocystis is very similar to that in spinach. We also found that there is no appreciable difference between the mid-frequency S(2)/S(1) spectra of wild-type and of D1-D170H mutant PSII particles, from which we conclude that D1-Asp170 does not undergo a significant structural change during the S(1) to S(2) transition. This result also suggests that, if D1-Asp170 ligates Mn, it does not ligate the Mn ion that is oxidized during the S(1) to S(2) state transition. Finally, we found that a mode at 606 cm(-)(1) in the low-frequency wild-type S(2)/S(1) spectrum shifts to 612 cm(-)(1) in the D1-D170H mutant spectrum. Because this 606 cm(-)(1) mode has been previously assigned to an Mn-O-Mn cluster mode of the OEC [Chu, H.-A., Sackett, H., and Babcock, G. T. (2000) Biochemistry 39, 14371-14376], we conclude that D1-Asp170 is structurally coupled to the Mn-O-Mn cluster structure that gives rise to this band. Our results suggest that D1-Asp170 either directly ligates Mn or Ca(2+) or participates in a hydrogen bond to the Mn(4)Ca(2+) cluster. Our results demonstrate that combining FTIR difference spectroscopy with site-directed mutagenesis has the potential to provide insights into structural changes in Mn and Ca(2+) coordination environments in the different S states of the OEC.     

Participation of the C-terminal region of the D1-polypeptide in the first steps in the assembly of the Mn4Ca cluster of photosystem II

Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.     

D1-S169A substitution of photosystem II reveals a novel S2-state structure

In photosystem II (PSII), photosynthetic water oxidation occurs at the O₂-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S₀–S₄) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S₂ to S₁ transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7–2.7× slower rate of charge recombination with Q_A⁻ relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an E_a of 5.87 ± 1.15 kcal mol⁻¹ for decay back to the S₁ state, compared to 0.80 ± 0.13 kcal mol⁻¹ for the wild-type S₂ state. In addition, we find that ammonia does not affect the S₂-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S₂ state of D1-S169A PSII. The altered S₂ state of D1-S169A PSII provides insight into the S₂➔S₃ state transition.     

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

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

Release and reactive-oxygen-mediated damage of the oxygen-evolving complex subunits of PSII during photoinhibition

Under photoinhibitory illumination of spinach PSII membranes, the oxygen-evolving complex subunits, OEC33, 24 and 18, were released from PSII. The liberated OEC33 and also OEC24 to a lesser extent were subsequently damaged and then exhibited smeared bands in SDS/urea-PAGE. Once deteriorated, OEC33 could not bind to PSII. The effects of scavengers and chelating reagents on the damage indicated that hydroxyl radicals generated from superoxide in the presence of metal ions were responsible for the damage. These results suggest that, like the D1 protein of the PSII reaction center complex, OEC subunits suffer oxidative damage and turnover under illumination.     

Evidence for changes in the nucleotide conformation in the active site of H(+)-ATPase as determined by pulsed EPR spectroscopy

The conformation of di- and triphosphate nucleosides in the active site of ATPsynthase (H(+)-ATPase) from thermophilic Bacillus PS3 (TF1) and their interaction with Mg(2+)/Mn(2+) cations have been investigated using EPR, ESEEM, and HYSCORE spectroscopies. For a ternary complex formed by a stoichiometric mixture of TF1, Mn(2+), and ADP, the ESEEM and HYSCORE data reveal a (31)P hyperfine interaction with Mn(2+) (|A((31)P)| approximately 5.20 MHz), significantly larger than that measured for the complex formed by Mn(2+) and ADP in solution (|A((31)P)| approximately 4.50 MHz). The Q-band EPR spectrum of the Mn.TF1.ADP complex indicates that the Mn(2+) binds in a slightly distorted environment with |D| approximately 180 x 10(-4) cm(-1) and |E| approximately 50 x 10(-4) cm(-1). The increased hyperfine coupling with (31)P in the presence of TF1 reflects the specific interaction between the central Mn(2+) and the ADP beta-phosphate, illustrating the role of the enzyme active site in positioning the phosphate chain of the substrate for efficient catalysis. Results with the ternary Mn.TF1.ATP and Mn.TF1.AMP-PNP complexes are interpreted in a similar way with two hyperfine couplings being resolved for each complex (|A((31)P(beta))| approximately 4.60 MHz and |A((31)P(gamma))| approximately 5.90 MHz with ATP, and |A((31)P(beta))| approximately 4.20 MHz and |A((31)P(gamma))| approximately 5.40 MHz with AMP-PNP). In these complexes, the increased hyperfine coupling with (31)P(gamma) compared with (31)P(beta) reflects the smaller Mn.P distance with the gamma-phosphate compared with the beta-phosphate as found in the crystal structure of the analogous enzyme from mitochondria [3.53 vs 3.70 A (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628)] and the different binding modes of the two phosphate groups. The ESEEM and HYSCORE data of a complex formed with Mn(2+), ATP, and the isolated beta subunit show that the (31)P hyperfine coupling is close to that measured in the absence of the protein, indicating a poorly structured nucleotide site in the isolated beta subunit in the presence of ATP. The inhibition data obtained for TF1 incubated in the presence of Mg(2+), ADP, Al(NO(3))(3), and NaF indicate the formation of the inhibited complex with the transition state analogue namely Mg.TF1.ADP.AlF(x) with the equilibrium dissociation constant K(D) = 350 microM and rate constant k = 0.02 min(-1). The ESEEM and HYSCORE data obtained for an inhibited TF1 sample, Mn.TF1.ADP.AlF(x), confirm the formation of the transition state analogue with distinct spectroscopic footprints that can be assigned to Mn.(19)F and Mn.(27)Al hyperfine interactions. The (31)P(beta) hyperfine coupling that is measured in the inhibited complex with the transition state analogue (|A((31)P(beta))| approximately 5.10 MHz) is intermediate between those measured in the presence of ADP and ATP and suggests an increase in the bond between Mn and the P(beta) from ADP upon formation of the transition state.     

Structural characterization of photosystem II complex from red alga Porphyridium cruentum retaining extrinsic subunits of the oxygen-evolving complex.

The structure of photosystem II (PSII) complex isolated from thylakoid membranes of the red alga Porphyridium cruentum was investigated using electron microscopy followed by single particle image analysis. The dimeric complexes observed contain all major PSII subunits (CP47, CP43, D1 and D2 proteins) as well as the extrinsic proteins (33 kDa, 12 kDa and the cytochrome c(550)) of the oxygen-evolving complex (OEC) of PSII, encoded by the psbO, psbU and psbV genes, respectively. The single particle analysis of the top-view projections revealed the PSII complex to have maximal dimensions of 22 x 15 nm. The analysis of the side-view projections shows a maximal thickness of the PSII complex of about 9 nm including the densities on the lumenal surface that has been attributed to the proteins of the OEC complex. These results clearly demonstrate that the red algal PSII complex is structurally very similar to that of cyanobacteria and to the PSII core complex of higher plants. In addition, the arrangement of the OEC proteins on the lumenal surface of the PSII complex is consistent to that obtained by X-ray crystallography of cyanobacterial PSII.     

Evidence that the C-terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: an isotope-edited FTIR study

Isotope-edited FTIR difference spectroscopy was employed to determine if the C-terminal alpha-COO(-) group of the D1 polypeptide ligates the (Mn)(4) cluster in photosystem II (PSII) and, if so, if it ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated photoautotrophically in the presence of L-[1-(13)C]alanine or unlabeled ((12)C) L-alanine. In wild-type cells, both the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 and all alanine-derived peptide carbonyl groups will be labeled. In D1-A344G and D1-A344S mutant cells, the C-terminal alpha-COO(-) group of the D1 polypeptide will not be labeled because this group is no longer provided by alanine. The resultant S(2)-minus-S(1) FTIR difference spectra of purified wild-type and mutant PSII particles showed that one symmetric carboxylate stretching mode that is altered during the S(1) --> S(2) transition is sensitive to L-[1-(13)C]alanine-labeling in wild-type PSII particles but not in D1-A344G and D1-A344S PSII particles. Because the only carboxylate group that can be labeled in the wild-type PSII particles but not in the mutant PSII particles is the C-terminal alpha-COO(-) group of the D1 polypeptide, we assign the L-[1-(13)C]alanine-sensitive symmetric carboxylate stretching mode to the alpha-COO(-) group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at approximately 1356 cm(-1) in the S(1) state and at approximately 1339 or approximately 1320 cm(-1) in the S(2) state. These frequencies are consistent with unidentate ligation of the (Mn)(4) cluster by the alpha-COO(-) group of D1-Ala344 in both the S(1) and S(2) states. The apparent 17-36 cm(-1) downshift in frequency in response to the S(1) --> S(2) transition is consistent with the alpha-COO(-) group of D1-Ala344 ligating a Mn ion whose charge increases during the S(1) --> S(2) transition. Accordingly, we propose that the alpha-COO(-) group of D1-Ala344 ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Control experiments were conducted with Mn-depleted wild-type PSII particles. These experiments showed that tyrosine Y(D) may be structurally coupled to the carbonyl oxygen of an alanine-derived peptide carbonyl group.     

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.     

No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.     

Correlation between pH dependence of O2 evolution and sensitivity of Mn cations in the oxygen-evolving complex to exogenous reductants

Photosynthesis Research - Effects of pH, Ca2+, and Cl− ions on the extraction of Mn cations from oxygen-evolving complex (OEC) in Ca-depleted photosystem II (PSII(-Ca)) by exogenous...     

Structural models of the manganese complex of photosystem II and mechanistic implications

Photosynthetic water oxidation and O? formation are catalyzed by a Mn?Ca complex bound to the proteins of photosystem II (PSII). The catalytic site, including the inorganic Mn?CaO(n)H(x) core and its protein environment, is denoted as oxygen-evolving complex (OEC). Earlier and recent progress in the endeavor to elucidate the structure of the OEC is reviewed, with focus on recent results obtained by (i) X?ray spectroscopy (specifically by EXAFS analyses), and (ii) X-ray diffraction (XRD, protein crystallography). Very recently, an impressive resolution of 1.9? has been achieved by XRD. Most likely however, all XRD data on the Mn?CaO(n)H(x) core of the OEC are affected by X-ray induced modifications (radiation damage). Therefore and to address (important) details of the geometric and electronic structure of the OEC, a combined analysis of XRD and XAS data has been approached by several research groups. These efforts are reviewed and extended using an especially comprehensive approach. Taking into account XRD results on the protein environment of the inorganic core of the Mn complex, 12 alternative OEC models are considered and evaluated by quantitative comparison to (i) extended-range EXAFS data, (ii) polarized EXAFS of partially oriented PSII membrane particles, and (iii) polarized EXAFS of PSII crystals. We conclude that there is a class of OEC models that is in good agreement with both the recent crystallographic models and the XAS data. On these grounds, mechanistic implications for the O?O bond formation chemistry are discussed. This article is part of a Special Issue entitled: Photosystem II.     

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.     

Oxomanganese complexes for natural and artificial photosynthesis

The oxygen-evolving complex (OEC) of Photosystem II (PSII) is an oxomanganese complex that catalyzes water-splitting into O2, protons and electrons. Recent breakthroughs in X-ray crystallography have resolved the cuboidal OEC structure at 1.9 ? resolution, stimulating significant interest in studies of structure/function relations. This article summarizes recent advances on studies of the OEC along with studies of synthetic oxomanganese complexes for artificial photosynthesis. Quantum mechanics/molecular mechanics hybrid methods have enabled modeling the S1 state of the OEC, including the ligation proposed by the most recent X-ray data where D170 is bridging Ca and the Mn center outside the CaMn3 core. Molecular dynamics and Monte Carlo simulations have explored the structural/functional roles of chloride, suggesting that it regulates the electrostatic interactions between D61 and K317 that might be critical for proton abstraction. Furthermore, structural studies of synthetic oxomanganese complexes, including the [H2O(terpy)MnIII(μ-O)2MnIV(terpy)OH2]3+ (1, terpy=2,2':6',2″-terpyridine) complex, provided valuable insights on the mechanistic influence of carboxylate moieties in close contact with the Mn catalyst during oxygen evolution. Covalent attachment of 1 to TiO2 has been achieved via direct deposition and by using organic chromophoric linkers. The (III,IV) oxidation state of 1 attached to TiO2 can be advanced to (IV,IV) by visible-light photoexcitation, leading to photoinduced interfacial electron transfer. These studies are particularly relevant to the development of artificial photosynthetic devices based on inexpensive materials.     

Evidence from biosynthetically incorporated strontium and FTIR difference spectroscopy that the C-terminus of the D1 polypeptide of photosystem II does not ligate calcium

Recent FTIR studies have provided evidence that the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 is a unidentate ligand of a Mn ion in photosystem II [Chu, H.-A., Hiller, W., and Debus, R. J. (2004) Biochemistry 43, 3152-3166; Kimura, Y., Mizusawa, N., Yamanari, T., Ishii, A., and Ono, T.-A. (2005) J. Biol. Chem. 280, 2078-2083]. However, the FTIR data could not exclude Ca ligation. Furthermore, the recent approximately 3.5 A X-ray crystallographic structural model positions the alpha-COO(-) group of D1-Ala344 near a Ca ion [Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838]. Therefore, to conclusively establish whether the alpha-COO(-) group of D1-Ala344 ligates Mn or Ca, the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344 was identified in the S(2)-minus-S(1) FTIR difference spectrum of PSII particles having Sr substituted for Ca. Cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated in media having Sr substituted for Ca and containing either l-[1-(13)C]alanine or unlabeled ((12)C) alanine. The S(2)-minus-S(1) FTIR difference spectra of the purified PSII particles show that substituting Sr for Ca alters several carboxylate stretching modes, including some that may correspond to one or more metal ligands, but importantly does not alter the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344. In unlabeled PSII particles, this mode appears at approximately 1356 cm(-)(1) in the S(1) state and at either approximately 1337 or approximately 1320 cm(-)(1) in the S(2) state, irrespective of whether the PSII particles contain Ca or Sr. These data are inconsistent with Ca ligation and show, therefore, that the C-terminal alpha-COO(-) group of the D1 polypeptide ligates a Mn ion. These data also show that substituting Ca with the larger Sr ion perturbs other unidentified carboxylate groups, at least one of which may ligate the Mn(4) cluster.     

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.