Probing tyrosine Z oxidation in photosystem II core complex isolated from spinach by EPR at liquid helium temperatures

Tyrosine Z (Tyr_Z) oxidation observed at liquid helium temperatures provides new insights into the structure and function of Tyr_Z in active Photosystem II (PSII). However, it has not been reported in PSII core complex from higher plants. Here, we report Tyr_Z oxidation in the S₁ and S₂ states in PSII core complex from spinach for the first time. Moreover, we identified a 500 G-wide symmetric EPR signal (peak position g = 2.18, trough position g = 1.85) together with the g = 2.03 signal induced by visible light at 10 K in the S₁ state in the PSII core complex. These two signals decay with a similar rate in the dark and both disappear in the presence of 6% methanol. We tentatively assign this new feature to the hyperfine structure of the S₁Tyr_Z ^• EPR signal. Furthermore, EPR signals of the S₂ state of the Mn-cluster, the oxidation of the non-heme iron, and the S₁Tyr_Z ^• in PSII core complexes and PSII-enriched membranes from spinach are compared, which clearly indicate that both the donor and acceptor sides of the reaction center are undisturbed after the removal of LHCII. These results suggest that the new spinach PSII core complex is suitable for the electron transfer study of PSII at cryogenic temperatures.     

Effect of bicarbonate on the water-oxidizing complex of photosystem II in the super-reduced S-states

It is shown that the hydrazine-induced transition of the water-oxidizing complex (WOC) to super-reduced S-states depends on the presence of bicarbonate in the medium so that after a 20 min treatment of isolated spinach thylakoids with 3 mM NH₂NH₂ at 20 °C in the CO₂/HCO₃⁻-depleted buffer the S-state populations are: 42% of S₋₃, 42% of S₋₂, 16% of S₋₁ and even formal S₋₄ state is reached, while in the presence of 2 mM NaHCO₃, the same treatment produces 30% of S₋₃, 38% of S₋₂, and 32% of S₋₁ and there is no indication of the S₋₄ state. Bicarbonate requirement for the oxygen-evolving activity, very low in untreated thylakoids, considerably increases upon the transition of the WOC to the super-reduced S-states, and the requirement becomes low again when the WOC returns back to the normal S-states using pre-illumination. The results are discussed as a possible indication of ligation of bicarbonate to manganese ions within the WOC.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Effect of monogalactosyldiacylglycerol and other thylakoid lipids on violaxanthin de-epoxidation in liposomes

In this study we present evidence that one of two reactions of the xanthophyll cycle, violaxanthin de-epoxidation, may occur in unilamellar egg phosphatidylcholine vesicles supplemented with monogalactosyldiacylglycerol (MGDG). Activity of violaxanthin de-epoxidase (VDE) in this system was found to be strongly dependent on the content of MGDG in the membrane; however, only to a level of 30 mol%. Above this concentration the rate of violaxanthin de-epoxidation decreased. The effect of individual thylakoid lipids on VDE-independent violaxanthin transformation was also investigated and unspecific effects of phosphatidylglycerol and sulphoquinovosyldiacyglycerol, probably related to the acidic character of these lipids, were found. The presented results suggest that violaxanthin de-epoxidation most probably takes place inside MGDG-rich domains of the thylakoid membrane. The described activity of the violaxanthin de-epoxidation reaction in liposomes opens new possibilities in the investigation of the xanthophyll cycle and may contribute to a better understanding of this process.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 °C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing “activity gels”. Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being “self-digested”, also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 °C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg²⁺, and inhibited by Zn²⁺, Cd²⁺, EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing “activity gels” or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Phosphorylation and disassembly of the photosystem II core as an early stage of photoinhibition

The presence of heterogeneity in phosphorylated PSII core populations in grana membranes of spinach (Spinacia oleracea L.) was previously demonstrated (Giardi et al., 1991, Biochem. Biophys. Res. Commun. 176, 1298–1304). The effect of photoinhibitory conditions on the distribution of these phosphorylated PSII core populations in thylakoids and PSII particles has been investigated. The sensitivity of the PSII core to strong illumination depended on the phosphorylation state of D₁ and D₂ proteins as well as on the content of the 9-kDa PsbH phosphoprotein. When D₁ and D₂ proteins are under-phosphorylated, the 9-kDa phosphoprotein is tightly bound to the PSII core; thus, a partial protection from photoinhibition is observed. Of the different PSII core populations isolated from membranes photoinhibited for 10 min, the highly phosphorylated populations lack internal antennae CP43 and CP47; perhaps these migrate out to the non-appressed regions of thylakoids. The degradation of the D₁ protein seems to follow the disassembly of the PSII core.     

The effect of outer antenna complexes on the photochemical trapping rate in barley thylakoid Photosystem II

We have investigated the previous suggestions in the literature that the outer antenna of Photosystem II of barley does not influence the effective photosystem primary photochemical trapping rate. It is shown by steady state fluorescence measurements at the F₀ fluorescence level of wild type and the chlorina f2 mutant, using the chlorophyll b fluorescence as a marker, that the outer antenna is thermally equilibrated with the core pigments, at room temperature, under conditions of photochemical trapping. This is in contrast with the conclusions of the earlier studies in which it was suggested that energy was transferred rapidly and irreversibly from the outer antenna to the Photosystem II core. Furthermore, the effective trapping time, determined by single photon counting, time-resolved measurements, was shown to increase from 0.17±0.017 ns in the chlorina Photosystem II core to a value within the range 0.42±0.036-0.47±0.044 ns for the wild-type Photosystem II with the outer antenna system. This 2.5-2.8-fold increase in the effective trapping time is, however, significantly less than that expected for a thermalised system. The data can be explained in terms of the outer antenna increasing the primary charge separation rate by about 50%.     

Highly efficient photoactivation of Mn-depleted photosystem II by imidazole-liganded manganese complexes

The oxygen-evolving complex (OEC) of Mn-depleted photosystem II (PSII) can be reconstituted in the presence of exogenous Mn or a Mn complex under weak illumination, a process called photoactivation. Synthetic Mn complexes could provide a powerful system to analyze the assembly of the OEC. In this work, four mononuclear Mn complexes, [(terpy)₂Mn^II(OOCH₃)]·2H₂O (where terpy is 2,2′:6′,2″-terpyridine), Mn^II(bzimpy)₂, Mn^II(bp)₂(CH₃CH₂OH)₂ [where bzimpy is 2,6-bis(2-benzimidazol-2-yl)pyridine] and [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH (where py is pyridine) were used in photoactivation experiments. Measurements of the photoreduction of 2,6-dichorophenolindophenol and oxygen evolution demonstrate that photoactivation is more efficient when Mn complexes are used instead of MnCl₂ in reconstructed PSII preparations. The most efficient recoveries of oxygen evolution and electron transport activities are obtained from a complex, [Mn^III(HL)(L)(py)(CH₃OH)]CH₃OH, that contains both imidazole and phenol groups. Its recovery of the rate of oxygen evolution is as high as 79% even in the absence of the 33-kDa peptide. The imidazole ligands of the Mn complex probably accelerate P ₆₈₀ ^•+ reduction and consequently facilitate the process of photoactivation. Also, the strong intermolecular hydrogen bond probably facilitates interaction with the Mn-depleted PSII via reorganization of the hydrogen-bonding network, and therefore promotes the recovery of oxygen evolution and electron transport activities.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic binuclear Mn(II) and Mn(IV) complexes: production of hydrogen peroxide

Reconstitution of Mn-depleted PSII particles with synthetic binuclear Mn complexes (one Mn(II)₂ complex and one Mn(IV)₂ complex) was examined. In both cases the electron-transfer rates in the reconstituted systems were found to be up to 75–82% of that measured in native PSII but the oxygen evolution activity remained lower (<5–40%). However, hydrogen peroxide was also produced by the reconstituted samples. These samples therefore represent a new type of reconstituted PSII that generates hydrogen peroxide as the final product in reconstituted PSII centers.     

P680+ reduction in oxygen-evolving Photosystem II core complexes

The kinetics of P680⁺ reduction in oxygen-evolving spinach Photosystem II (PS II) core particles were studied using both repetitive and single-flash 830 nm transient absorption. From measurements on samples in which PS II turnover is blocked, we estimate radical-pair lifetimes of 2 ns and 19 ns. Nanosecond single-flash measurements indicate decay times of 7 ns, 40 ns and 95 ns. Both the longer 40 ns and 95 ns components relate to the normal S-state controlled Y_z P680⁺ electron transfer dynamics. Our analysis indicates the existence of a 7 ns component which provides evidence for an additional process associated with modified interactions involving the water-splitting catalytic site. Corresponding microsecond measurements show decay times of 4 s and 90 s with the possibility of a small component with a decay time of 20–40 s. The precise origin of the 4 s component remains uncertain but appears to be associated with the water-splitting center or its binding site while the 90 s component is assigned to P680⁺-Q_A ^– recombination. An amplitude and kinetic analysis of the flash dependence data gives results that are consistent with the current model of the oxygen-evolving complex.Abbreviations PS II- Photosystem II- - P680- primary donor (Chl-a_II dimer) of PS II - Y_z- Tyr 161 donor to P680 - Q_A- quinone secondary acceptor to P680 - LHC- light-harvesting chlorophyll protein of PS II - BBY- Berthold, Babcock and Yocum PS II membrane fragment preparation - PPBQ- phenyl-p-benzoquinone     

Protein composition of the photosystem II core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803

The presence of four photosystem II proteins, CP47, CP43, D1 and D2, was monitored in mutants of Synechocystis sp. PCC 6803 that have modified or inactivated genes for CP47, CP43, or D2. It was observed that: (1) thylakoids from mutants without a functional gene encoding CP47 are also depleted in D1 and D2; (2) inactivation of the gene for CP43 leads to decreased but significant levels of CP47, D1 and D2; (3) deletion of part of both genes encoding D2, together with deletion of part of the CP43-encoding gene causes a complete loss of CP47 and D1; (4) thylakoids from a site-directed mutant in which the His-214 residue of D2 has been replaced by asparagine do not contain detectable photosystem II core proteins. However, in another site-directed mutant, in which His-197 has been replaced by tyrosine, some CP47 as well as breakdown products of CP43, but no D1 and D2, can be detected. These data could indicate a central function of CP47 and D2 in stable assembly of the photosystem II complex. CP43, however, is somewhat less critical for formation of the core complex, although CP43 is required for a physiologically functional photosystem II unit. A possible model for the assembly of the photosystem II core complex is proposed.     

Quenching in Arabidopsis thaliana mutants lacking monomeric antenna proteins of photosystem II

The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.     

Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: their specificity and evolution

Sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) are lipids with negative charges, distributed among membranes of chloroplasts of plants and their postulated progenitors, cyanobacteria, and also widely among membranes of anoxygenic photosynthetic bacteria. Thus, these acidic lipids are of great interest in terms of their roles in the function and evolution of the photosynthetic membranes. The physiological significance of these lipids in photosynthesis has been examined through characterization of mutants defective in their abilities to synthesize SQDG or PG, and through characterization of isolated thylakoid membranes or photosynthetic particles, the acidic lipid contents of which were manipulated in vitro, for example, on treatment with phospholipase to degrade PG. Responsibility of SQDG or PG has been clarified so far in terms of the structural and/or functional integrity of photosystems I and/or II in cyanobacterial, green algal, and higher plant species. Also implied were distinct levels of the responsibility in the different photosynthetic organisms. Extreme cases involved the indispensability of SQDG for photosynthesis and growth in two prokaryotic, photosynthetic organisms and the contribution of PG to construction of the photosystem-I trimer exclusively in cyanobacteria. Here, roles of these acidic lipids are discussed with a focus on their specificity and the evolution of photosynthetic membranes.Norihiro Sato is the recipient of the Botanical Society Award for Young Scientist, 2003.     

Influence of thylakoid membrane lipids on the structure and function of the plant photosystem II core complex

Main conclusion

MGDG leads to a dimerization of isolated, monomeric PSII core complexes. SQDG and PG induce a detachment of CP43 from the PSII core, thereby disturbing the intrinsic PSII electron transport. The influence of the four thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure and function of isolated monomeric photosystem (PS) II core complexes was investigated. Incubation with the negatively charged lipids SQDG and PG led to a loss of the long-wavelength 77 K fluorescence emission at 693 nm that is associated with the inner antenna proteins. The neutral galactolipids DGDG and MGDG had no or only minor effects on the fluorescence emission spectra of the PSII core complexes, respectively. Pigment analysis, absorption and 77 K fluorescence excitation spectroscopy showed that incubation with SQDG and PG led to an exposure of chlorophyll molecules to the surrounding medium followed by conversion to pheophytin under acidic conditions. Size-exclusion chromatography and polypeptide analysis corroborated the findings of the spectroscopic measurements and pigment analysis. They showed that the negatively charged lipid SQDG led to a dissociation of the inner antenna protein CP43 and the 27- and 25-kDa apoproteins of the light-harvesting complex II, that were also associated with a part of the PSII core complexes used in the present study. Incubation of PSII core complexes with MGDG, on the other hand, induced an almost complete dimerization of the monomeric PSII. Measurements of the fast PSII fluorescence induction demonstrated that MGDG and DGDG only had a minor influence on the reduction kinetics of plastoquinone Q_A and the artificial PSII electron acceptor 2,5-dimethyl-p-benzoquinone (DMBQ). SQDG and, to a lesser extent, PG perturbed the intrinsic PSII electron transport significantly.     

Flash-induced redox changes in oxygen-evolving spinach Photosystem II core particles

Flash-induced redox reactions in spinach PS II core particles were investigated with absorbance difference spectroscopy in the UV-region and EPR spectroscopy. In the absence of artificial electron acceptors, electron transport was limited to a single turnover. Addition of the electron acceptors DCBQ and ferricyanide restored the characteristic period-four oscillation in the UV absorbance associated with the S-state cycle, but not the period-two oscillation indicative of the alternating appearance and disappearance of a semiquinone at the Q_B-site. In contrast to PS II membranes, all active centers were in state S₁ after dark adaptation. The absorbance increase associated with the S-state transitions on the first two flashes, attributed to the Z⁺S₁ZS₂ and Z⁺S₂ZS₃ transitions, respectively, had half-times of 95 and 380 s, similar to those reported for PS II membrane fragments. The decrease due to the Z⁺S₃ZS₀ transition on the third flash had a half-time of 4.5 ms, as in salt-washed PS II membrane fragments. On the fourth flash a small, unresolved, increase of less than 3 s was observed, which might be due to the Z⁺S₀ZS₁ transition. The deactivation of the higher S-states was unusually fast and occurred within a few seconds and so was the oxidation of S₀ to S₁ in the dark, which had a half-time of 2–3 min. The same lifetime was found for tyrosine D⁺, which appeared to be formed within milliseconds after the first flash in about 10% inactive centers and after the third and later flashes by active centers in Z⁺S₃.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - D secondary electron donor of PS II - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S_0–3 redox state of the oxygen-evolving complex - Z secondary electron donor of PS II     

Absorbance difference spectra of the S-state transitions in Photosystem II core particles

Redox changes of the oxygen evolving complex in PS II core particles were investigated by absorbance difference spectroscopy in the UV-region. The oscillation of the absorbance changes induced by a series of saturating flashes could not be explained by the minimal Kok model (Kok et al. 1970) consisting of a 4-step redox cycle, S₀ S₁ S₂ S₃ S₀, although the values of most of the relevant parameters had been determined experimentally. Additional assumptions which allow a consistent fit of all data are a slow equilibration of the S₃ state with an inactive state, perhaps related to Ca²⁺-release, and a low quantum efficiency for the first turnover after dark-adaptation. Difference spectra of the successive S-state transitions were determined. At wavelengths above 370 nm, they were very different due to the different contribution of a Chl bandshift in each spectrum. At shorter wavelengths, the S₁ S₂ transition showed a difference spectrum similar to that reported by Dekker et al. 1984b and attributed to an Mn(III) to Mn(IV) oxidation. The spectrum of absorbance changes associated with the S₂ S₃ transition was similar to that reported by Lavergne 1991 for PS II membranes. The S₀ S₁ transition was associated with a smaller but still substantial absorbance increase in the UV. Differences with the spectra reported by Lavergne 1991 are attributed to electrostatic effects on electron transfer at the acceptor side associated with the S-state dependence of proton release in PS II membranes.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S₀ to S₄ redox state of the oxygen evolving complex - Z secondary electron donor of PS II     

Investigation of the interaction of the water oxidising manganese complex of photosystem II with the aqueous solvent environment

Interaction of the water oxidising manganese complex of photosystem II with the aqueous environment has been investigated using electron paramagnetic resonance spectroscopy and electron spin echo envelope modulation spectroscopy to detect interaction of [2H]methanol with the complex in the S2 state. The experiments show that the classical S2 multiline signal is associated with a manganese environment which is not exposed to the aqueous medium. An electron paramagnetic resonance spectroscopy signal, also induced by 200 K illumination, showing 2H modulation by methanol in the medium and a modified multiline electron paramagnetic resonance spectroscopy signal formed in parallel to it, are suggested to be associated with a second manganese environment exposed to the medium.