CI-NMR measurement of chloride binding to the oxygen-evolving complex of spinach Photosystem II

The mechanism by which Cl⁻ activates the oxygen-evolving complex (OEC) of Photosystem II (PS II) in spinach was studied by ³⁵Cl-NMR spectroscopy and steady-state measurements of oxygen evolution. Measurements of the excess ³⁵Cl-NMR linewidth in dark-adapted, Cl⁻-depleted thylakoid and Photosystem II membranes show an overall hyperbolic decrease which is interrupted by sharp increases in linewidth (linewidth maxima) at approx. 0.3 mM, 0.75 mM, 3.25 mM (2.0 mM in PS II membranes), and 7.0 mM Cl⁻. The rate of the Hill reaction (H₂O → 2,6-dichlorophenolindophenol) at low light intensities (5% of saturation) as a function of [Cl⁻] in thylakoids shows three intermediary plateaus in the concentration range between 0.1 and 10 mM Cl⁻ indicating kinetic cooperativity with respect to Cl⁻. The presence of linewidth maxima in the ³⁵Cl-NMR binding curve indicates that Cl⁻ addition exposes four types of Cl⁻ binding site that were previously inaccessible to exchange with Cl⁻ in the bulk solution. These results are best explained by proposing that Cl⁻ binds to four sequestered (salt-bridged) domains within the oxygen-evolving complex. Binding of Cl⁻ is facilitated by the presence of H⁺ and vice versa. The pH dependence of the excess ³⁵Cl-NMR linewidth at 0.75 mM Cl⁻ shows that Cl⁻ binding has a maximum at pH 6.0 and two smaller maxima at pH 5.4 and 6.5 which may suggest that as many as three groups (perhaps histidine) with pK_a values in the region may control the binding.     

Reactivation by chloride of hill activity in heat- and tris-treated thylakoid membranes from Beta vulgaris

Hill activity (photoreduction of 2,6,dichlorophenol indophenol) of heat inactivated (40°C, 3 min) and Tris-washed (0.8M, pH 8.3) thylakoids of Beta vulgaris (beet-spinach) was partially restored if they were incubated with 150 mM MgCl₂ prior to the assay. Mg(NO₃)₂ or MgSO₄ were unable to restore this activity. The extent of this reactivation was dependent upon the degree of inactivation by heating and upon the composition of the isolation and the resuspension buffer used during the heat treatment. Washing of heat-treated thylakoids with phosphate-EDTA buffer prior to incubation with MgCl₂ did not affect the extent of this reactivation. Chloride ions seem to be required for the reactivation of Hill activity damaged either by heat or by Tris.Most commonly used chloroplast isolation and resuspension media, except for Tris-HCl as resuspension medium, were suitable for restoration of Hill activity in heat-damaged thylakoids by preincubation with 150 mM MgCl₂ prior to the assay. Pretreatment with MgCl₂ stimulated Hill activity in Tris-treated and heat-damage thylakoids if phosphate buffer was used for their resuspension. However, the same pretreatment inhibited Hill activity in unheated thylakoids isolated in Tris medium and resuspended in the same medium. On the other hand, MgCl₂ pretreatment induced restoration of the Hill activity of the heated thylakoids when Tricine or Hepes was used as the resuspension medium. It appears that the presence of Tris somehow hampers the Cl^– induced reactivation. The stimulation of Hill activity by MgCl₂ treatment in unheated (control) thylakoids is possibly induced by Mg²⁺ ions and not by Cl^– ions.Abbreviations Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1. 1-dimethyl-urea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2 hydroxyethyl piperazine-N, 2 ethano-sulfonic acid - HT heat-treated - PS II photosystem II - Tricine N-tri (hydroxymethyl) methyl glycine - Tris Tris-(hydroxymethyl) amino-methane     

Multiple anion effects on photosystem II in chloroplast membranes

We investigated the activity of several anions at various sites on photosystem II, in particular those associated with the Cl^- effect (anion binding-site I) and the HCO₃ ^- effect (anion binding-site II). Chlorophyll a fluorescence changes were used to monitor partial photosystem II reactions either in the oxygen-evolving mechanism or involving endogenous quinone electron acceptors. We find that anions such as NO₃ ^-, HCO₃ ^-, HCO₂ ^-, F^-, NO₂ ^-, and acetate can, depending on conditions, bind to either anion binding-site I, anion binding-site II, or both sites simultaneously. The anions N₃ ^- and Au(CN)₂ ^- are exceptions. In their presence, oxygen-consumption reactions are enhanced. The results demonstrate that an exclusive site or mode of action of an anion on photosystem II cannot be determined by measuring the Hill reaction alone. Anion interactions with photosystem II are shown to be very complex and, therefore, caution is advisable in interpreting related experiments. Carbonic anhydrase associated with photosystem II was also investigated as a possible target for some anion effects. In Cl^--depleted thylakoids, NO₃ ^-, stimulated both electron transport and carbonic anhydrase activity at low concentrations, while higher concentrations inhibited both. However, carbonic anhydrase was more sensitive to inhibition by NO₃ ^- than was electron flow. Possible interpretations are discussed; the electron transport and carbonic anhydrase activity appear not to be functionally linked.Abbreviations ABSI Anion binding-site(s) I associated with the oxygen-evolving mechanism - ABSII Anion binding-site(s) II, which controls quinone-related reactions on the electron-acceptor side of photosystem II - OAc^- Acetate - Chl Chlorophyll - DCMU 3—(3,4-dichlorophenyl)-1,1-dimethyl urea - DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - Mes 2-[N-morpholino]ethanesulphonic acid - Mops 3-[N-morpholino]propanesulphonic acid - Tes N-Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid - Tricine N-Tris[hydroxymethyl]methylglycine     

Studies of the slowly exchanging chloride in Photosystem II of higher plants

³⁶Cl^- was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of ³⁶Cl^-, saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10^-4 s^-1 at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on ³⁶Cl^-, which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pK_a of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br^- and NO₃ ^-. The slowly exchanging Cl^- did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S₂ state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S₂ state EPR signals.Abbreviations Chl chlorophyll - EPR electron paramagnetic resonance - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - MWCO molecular weight cut off - PPBQ phenyl-p-benzoquinone - PS II Photosystem II     

Chloride binding proteins: mechanistic implications for the oxygen-evolving complex of Photosystem II

Chloride plays a key role in activating the photosynethetic oxygen-evolving complex (OEC) of Photosystem II, but the OEC is only one of many enzymes affected by this anion. Some of the mechanistic features of Cl^– involvement in water-splitting resemble those of other proteins whose structure and chemistry are known in detail. An overview of the similarities and differences between these Cl^–-binding systems is presented.The literature survey for this Minireview was, for the most part, completed in 1987.     

A model for the mechanism of chloride activation of oxygen evolution in photosystem II

A hypothesis is proposed to explain the function of Cl^- in activating the oxygenevolving complex (OEC) of photosystem II (PS II), based on the results of recent ³⁵Cl-NMR studies. The putative mechanism involves Cl^- binding to two types of sites. An intrinsic site is suggested to be composed of three histidyl residues (His 332 and His 337 from D1 and His 337 D2). It is proposed that Cl^- binding to this site accelerates the abstraction of H⁺ from water by raising the pK_a's of the histidine imidazole groups. Cl^- binding also stimulates the transfer of H⁺ from this intrinsic site to a set of extrinsic sites on the 33 kD extrinsic polypeptide. The extrinsic Cl^- binding sites are suggested to involve four protein domains that are linked together by salt-bridge contacts. Chloride and H⁺ donated from the intrinsic site attack these intramolecular salt-bridges in a defined sequence, thereby exposing previously inaccessible Cl^- and H⁺ binding sites and stimulating the oxidation of water. This hypothesis also proposes a possible structure for the Mn active site within the D1/D2 complex. Specific amino-acid residues that are likely to participate as Mn lignads are identified on the lumenal portions of the D1 and D2 proteins that are different from those in the L and M subunits of photosynthetic bacteria; the choice of these residues is based on the metal coordination chemistry of these residues, their location within the polypeptide chain, the regularity of their spacing, and their conservation through evolution. The catalytic Mn-binding residues are suggested to be D-61, E-65, E-92, E-98, D-103; D-308, E-329, E-342 and E-333 in D1, and H-62, E-70, H-88, E-97, D-101; E-313, D-334, E-338 and E-345 in D2. Finally, this hypothesis identifies sites on both D2 and the 33 kD extrinsic polypeptide that might be involved in high- and low-affinity Ca²⁺ binding.To whom correspondence should be addressed     

Spectroscopic studies of the manganese complex of Photosystem II

Our recent EPR and EXAFS experiments investigating the structure of the oxygen-evolving complex of PS II are discussed. PS II treatments which affect the cofactors calcium and chloride have been used to poise samples in modified forms of the S-states, S₁, S₂ and S₃. X-ray absorption studies indicate a similar overall structure for the manganese complex between treated and native samples although the influence of the treatments and cofactors is observed. Manganese oxidation (or oxidation of a ligand to the manganese cluster) is indicated to occur on each of the transitions S₁ S₂ and S₂ S₃ in these modified samples. The cluster appears to contain at least two inequivalent Mn-Mn pairs. In the native samples the Mn-Mn distance is 2.7 Å, but in samples where the calcium site is affected, one of the pairs has a 3.0 Å Mn-Mn distance. The intensity of the 3.3/3.6 Å interaction is reduced on sodium chloride treatment (calcium depletion) perhaps indicating calcium binding close to the manganese cluster. From EPR data we also propose that treatments which affect calcium and chloride binding cause a modification of the native S₂ state, slow the reduction of Y_z and allow an S3 EPR signal to be observed following illumination. The origin of the S3 EPR signal, a modified S₃ or S₂ X where X is an organic radical of unknown charge, is discussed in relation to the results from the EXAFS studies.Abbreviations EPR electron paramagnetic resonance spectroscopy - EXAFS extended X-ray absorption fine structure - HTG n-heptyl -d-thioglucoside - MES 2(N-morpholino)ethanesulfonic acid - OEC oxygen evolving complex - PPBQ phenyl-1,4-benzoquinone - PS II Photosystem II - Y_z redox active tyrosine     

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.     

The donor side of Photosystem II as the copper-inhibitory binding site

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H₂OSiMo (in the presence and absence of DCMU) and H₂OPPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H₂O SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenilcarbazide - F_o initial non-variable fluorescence - F_I intermediate fluorescence yield - F_m maximum fluorescence yield - F_v variable fluorescence yield - Mes 2,-(N-morpholino)ethanosulfonic acid - OEC oxygen-evolving complex - P680 Primary electron donor chlorophyll - Pheo pheophytin - PPBQ phenyl-p-benzo-quinone - PS II Photosystem II - SiMo Silicomolybdate - Q_B secondary quinone acceptor - Q_A primary quinone aceptor - Tris N-tris(hydroxymethyl)amino ethane - Tyr_z electron carrier functioning between P680 and the Mn cluster This article is dedicated to Prof. Dr. Harmut Lichtenthaler on the occasion of his 60th birthday.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

The state of manganese in the photosynthetic apparatus: 5. The chloride effect in photosynthetic oxygen evolution. Is halide coordinated to the EPR-active manganese in the O2-evolving complex? Studies of the substructure of the low-temperature multiline EPR signal

The role of chloride in the manganese-containing oxygen-evolving complex of Photosystem II has been studied by observing the amplitude of the multiline EPR signal as a function of Cl⁻ concentration or when Cl⁻ is replaced by Br⁻ or F⁻. The correlation of the multiline EPR signal intensity and O₂ activity with the concentration of Cl⁻ shows that chloride is involved in oxygen evolution at the S₂ or earlier S states, and that it is necessary for the production of an EPR-detectable S₂ state. We have developed a new method for the preparation of subchloroplast PS II particles containing Br⁻ and F⁻) and have used these particles for studying the EPR fine structure at high resolution. The fine structure shows a multiplet of 4–6 lines with 10–15 G spacing; at the resolution of our experiment there are no significant differences between the Cl⁻-and Br⁻-containing samples, suggesting that the halide is not a ligand of the EPR-active Mn. Various structural possibilities for the Mn complex, which would account for the observed fine structure of the multiline EPR spectrum are discussed.     

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors

Structural roles of functional Ca²⁺ and Cl⁻ ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640–350 cm⁻¹) and mid- (1800–1200 cm⁻¹) frequency S₂/S₁ Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca²⁺-depleted and Ca²⁺-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with ⁴⁴Ca isotope. In contrast, Sr²⁺-substituted particles showed unique spectral changes in the low-frequency Mn–O–Mn mode at 606 cm⁻¹ as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl⁻-depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl⁻-reconstituted OEC. However, Cl⁻-depletion did not exert significant effects on the low-frequency spectrum.     

Manganese K-edge X-ray absorption spectra of the cyclic S-states in the photosynthetic oxygen-evolving system

A set of Mn K-edge XANES spectra due to the redox states S₀–S₃ of the OEC were determined by constructing a highly-sensitive X-ray detection system for use with physiologically native PS II membranes capable of cycling under a series of saturating laser-flashes. The spectra showed almost parallel upshifts with relatively high K-edge half-height energies given by 6550.9±0.2 eV, 6551.7±0.2 eV, 6552.5±0.2 eV and 6553.6±0.2 eV for the S₀, S₁, S₂ and S₃ states, respectively. The successive difference spectra between S₀ and S₁, S₁ and S₂, and S₂ and S₃ states were found to exhibit a similar peak around 6552–6553 eV, indicating that one Mn(III) ion or its direct ligand is univalently oxidized upon each individual S-state transition from S₀ to S₃. The present data, together with other observations of EPR and pre-edge XANES spectroscopy, suggest that the oxidation state of the Mn cluster undergoes a periodic change; S₀: Mn(III,III,III,IV) S₁: Mn(III,IV,III,IV) S₂: Mn(III,IV,IV,IV) S₃: Mn(IV,IV,IV,IV) or Mn(III,IV,IV,IV)·L⁺ with L being a direct ligand of a Mn(III) ion.Abbreviations Chl chlorophyll - D tyrosine 160 on the D2 protein, an accessory electron donor in PS II - D⁺ the oxidized form of D - EDTA ethylene-diaminetetraacetic acid - EPR electron paramagnetic resonance - EXAFS extended X-ray absorption fine structure - HL py-2,6-bis[bis(2-pyridylmethyl)aminomethyl]-4-methylphenol - Mes 2-(N-morpholino)ethanesulfonic acid - N4 py-tris(2-pyridylmethyl)amine - OEC oxygen evolving complex - P680 primary electron donor of PS II - PS II Photosystem II - Q₄₀₀ a high spin Fe³⁺ of the iron-quinone acceptor complex in PS II - SSD solid state detector - XAFS X-ray absorption fine structure - XANES X-ray absorption near edge structure     

Chloride ligation in inorganic manganese model compounds relevant to Photosystem II studied using X-ray absorption spectroscopy

Chloride ions are essential for proper function of the photosynthetic oxygen-evolving complex (OEC) of Photosystem II (PS II). Although proposed to be directly ligated to the Mn cluster of the OEC, the specific structural and mechanistic roles of chloride remain unresolved. This study utilizes X-ray absorption spectroscopy (XAS) to characterize the Mn–Cl interaction in inorganic compounds that contain structural motifs similar to those proposed for the OEC. Three sets of model compounds are examined; they possess core structures Mn^IV₃O₄X (X=Cl, F, or OH) that contain a di--oxo and two mono--oxo bridges or Mn^IV₂O₂X (X=Cl, F, OH, OAc) that contain a di--oxo bridge. Each set of compounds is examined for changes in the XAS spectra that are attributable to the replacement of a terminal OH or F ligand, or bridging OAc ligand, by a terminal Cl ligand. The X-ray absorption near edge structure (XANES) shows changes in the spectra on replacement of OH, OAc, or F by Cl ligands that are indicative of the overall charge of the metal atom and are consistent with the electronegativity of the ligand atom. Fourier transforms (FTs) of the extended X-ray absorption fine structure (EXAFS) spectra reveal a feature that is present only in compounds where chloride is directly ligated to Mn. These FT features were simulated using various calculated Mn–X interactions (X=O, N, Cl, F), and the best fits were found when a Mn–Cl interaction at a 2.2–2.3 Å bond distance was included. There are very few high-valent Mn halide complexes that have been synthesized, and it is important to make such a comparative study of the XANES and EXAFS spectra because they have the potential for providing information about the possible presence or absence of halide ligation to the Mn cluster in PS II.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0520-1Abbreviations bpea N,N-bis(2-pyridylmethyl)ethylamine - EXAFS extended X-ray absorption fine structure - FT Fourier transform - IPE inflection point energy - OEC oxygen evolving complex - PS II Photosystem II - tacn 1,4,7-triazacyclononane - XANES X-ray absorption near edge structure - XAS X-ray absorption spectroscopy - XRD X-ray diffraction     

Regeneration of the high-affinity manganese-binding site in the reaction center of an oxygen-evolution deficient mutant of Scenedesmus by protease action

The O₂-evolution deficient mutant (LF-1) of Scenedesmus obliquus inserts an unprocessed D1 protein into the thylakoid membrane and binds less than half the wild type (WT) level of Mn. LF-1 photosystem II (PS II) membrane fragments lack that part of the high-affinity Mn²⁺-binding site found in WT membranes which may be associated with histidine residues on the D1 protein (Seibert et al. 1989 Biochim Biophys Acta 974: 185–191). Hsu et al. (1987 Biochim Biophys Acta 890: 89–96) purport that the high-affinity site (characterized by competitive inhibition of DPC-supported DCIP photoreduction by M concentrations of Mn²⁺) in Mn-extracted PS II membranes is also the binding site for Mn functional in O₂ evolution. Proteases (papain, subtilisin, and carboxypeptidase A) can be used to regenerate the high-affinity Mn²⁺-binding site in LF-1 PS II membranes but not in thylakoids. Experiments with the histidine modifier, DEPC, suggest that the regenerated high-affinity Mn²⁺-binding sites produced by either subtilisin or carboxypeptidase A treatments were the same sites observed in WT membranes. However, none of the protease treatments produced LF-1 PS II membranes that could be photoactivated. Reassessment of the processing studies of Taylor et al. (1988 FEBS Lett 237: 229–233) lead us to believe that their procedure also does not result in substantial photoactivation of LF-1 PS II membranes. We conclude that (1) the unprocessed carboxyl end of the D1 protein in LF-1 is located on the lumenal side of the PS II membrane, (2) the unprocessed fragment physically obstructs or perturbs that part of the high-affinity Mn²⁺-binding site undetectable in LF-1, and (3) the D1 protein must be processed at the time of insertion into the membrane for normal O₂-evolution function to result.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-1,4-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DEPC diethylpryocarbonate - DPC 1,5-diphenylcarbazide - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - LDS-PAGE lithium dodecylsulfate polyacrylamide gel electrophoresis - LF-1 a low-fluorescent mutant of Scenedesmus obliquus - MES 4-morpholineethanesulfonic acid - PS II photosystem II - PMSF phenylmethylsulfonyl fluoride - RC photosystem II reaction center - Tris tris(hydroxymethyl)aminomethane - WT wild type Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC-02-83CH10093.     

The role of calcium in the pH-dependent control of Photosystem II

pH-dependent inactivation of Photosystem (PS) II and related quenching of chlorophyll-a-fluorescence have been investigated in isolated thylakoids and PS II-particles and related to calcium release at the donor side of PS II. The capacity of oxygen evolution (measured under light saturation) decreases when the pH is high and the pH in the thylakoid lumen decreases below 5.5. Oxygen evolution recovers upon uncoupling. The pH-response of inactivation can be described by a 1 H⁺-transition with an apparent pK-value of about 4.7. The yield of variable fluorescence decreases in parallel to the inactivation of oxygen evolution. pH-dependent quenching requires light and can be inhibited by DCMU. In PS II-particles, inactivation is accompanied by a reversible release of Ca²⁺-ions (one Ca²⁺ released per 200 Chl). In isolated thylakoids, where a pH was created by ATP-hydrolysis, both inactivation of oxygen evolution (and related fluorescence quenching) by internal acidification and the recovery of that inactivation can be suppressed by calcium-channel blockers. In the presence of the Ca²⁺-ionophore A23187, recovery of Chl-fluorescence (after relaxation of the pH) is stimulated by external Ca²⁺ and retarded by EGTA. As shown previously (Krieger and Weis 1993), inactivation of oxygen evolution at low pH is accompanied by an upward shift of the midpoint redox-potential, E_m, of Q_A. Here, we show that in isolated PS II particles the pH-dependent redox-shift (about 160 mV, as measured from redox titration of Chl-fluorescence) is suppressed by Ca²⁺-channel blockers and DCMU. When a redox potential of –80 to –120mV was established in a suspension of isolated thylakoids, the primary quinone acceptor, Q_A, was largely reduced in presence of a pH (created by ATP-hydrolysis) but oxidized in presence of an uncoupler. Ca²⁺-binding at the lumen side seems to control redox processes at the lumen- and stroma-side of PS II. We discuss Ca²⁺-release to be involved in the physiological process of high energy quenching.     

Total recovery of O2 evolution and nanosecond reduction kinetics of chlorophyll-a II + (P-680+) after inhibition of water cleavage with acetate

Oxygen evolution and reduction kinetics of the photooxidized Chl-a_II ⁺ have been measured in oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp.

1. Incubation of PS II particles with acetate resulted in an inhibition of oxygen evolution and a retardation of the Chl-a_II ⁺=reduction kinetics from the nanosecond range to the microsecond range, indicating a modification of the donor side of photosystem II (PS II).
2. After the first two flashes given to a dark-adapted, acetate treated sample, Chl-a_II ⁺ was re-reduced with a half-life time of 160 μs by a component of the donor side of PS II. Under repetitive excitation Chl-a_II ⁺ was re-reduced in 500 μs by electron back reaction from the primary acceptor Q_A ^- (X-320^-). Obviously, in the presence of acetate only two electrons are available from the donor side.
3. Both oxygen evolution and nanosecond reduction kinetics of Chl-a_II ⁺ were restored to the control level when acetate was removed.
4. The results indicate a tight coupling between O₂ evolution and nanosecond reduction kinetics of Chl-a_II ⁺.
5. The reversible inhibition is probably due to a replacement of Cl^- by acetate within the water splitting enzyme.
6. Due to its strongly retarded kinetics, the reversibly modified system may facilitate investigations of the mechanism of the donor side.

     

The roles of the extrinsic subunits in Photosystem II as revealed by EPR

The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z⁺, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z⁺ still occurs as evidenced by the near absence of Signal II_f. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S₂ state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl₂, Signal II_f is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z⁺. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.     

Light-dependent inactivation of photosynthetic oxygen evolution during NaCl treatment of photosystem II particles: The role of the 24-kDa protein

Photosystem (PS) II particles prepared from spinach thylakoids with Triton X-100 were treated with 1.5 M NaCl either in the light or dark. Under both conditions, the 24-kDa and 18-kDa proteins were released from the particles, but rebound to them when the NaCl concentration was reduced to 34 mM by dilution. Oxygen evolution measured after the dilution was inactivated following NaCl treatment in the light, but not following treatment in the dark. The inactivation in the light was suppressed when 5 mM CaCl₂ was added during or after the NaCl treatment. Based on these observations, a scheme is proposed for the mechanism of light-dependent inactivation of oxygen evolution during NaCl treatment of PS II particles and for the function of the 24-kDa protein in regulating the conformation of a supposed Ca²⁺-binding intrinsic protein.Abbreviations Chl chlorophyll - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 4-morpholineethanesulphonic acid - PS photosystem - SDS sodium dodecylsulphate