Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

pH dependence of the donor side reactions in Ca2+-depleted photosystem II

We have studied how low pH affects the water-oxidizing complex in Photosystem II when depleted of the essential Ca(2+) ion cofactor. For these samples, it was found that the EPR signal from the Y(Z)(*) radical decays faster at low pH than at high pH. At 20 degrees C, Y(Z)(*) decays with biphasic kinetics. At pH 6.5, the fast phase encompasses about 65% of the amplitude and has a lifetime of approximately 0.8 s, while the slow phase has a lifetime of approximately 22 s. At pH 3.9, the kinetics become totally dominated by the fast phase, with more than 90% of the signal intensity operating with a lifetime of approximately 0.3 s. The kinetic changes occurred with an approximate pK(a) of 4.5. Low pH also affected the induction of the so-called split radical EPR signal from the S(2)Y(Z)(*) state that is induced in Ca(2+)-depleted PSII membranes because of an inability of Y(Z)(*) to oxidize the S(2) state. At pH 4.5, about 50% of the split signal was induced, as compared to the amplitude of the signal that was induced at pH 6.5-7, using similar illumination conditions. Thus, the split-signal induction decreased with an apparent pK(a) of 4.5. In the same samples, the stable multiline signal from the S(2) state, which is modified by the removal of Ca(2+), was decreased by the illumination to the same extent at all pHs. It is proposed that decreased induction of the S(2)Y(Z)(*) state at lower pH was not due to inability to oxidize the modified S(2) state induced by the Ca(2+) depletion. Instead, we propose that the low pH makes Y(Z)(*) able to oxidize the S(2) state, making the S(2) --> S(3) transition available in Ca(2+)-depleted PSII. Implications of these results for the catalytic role of Ca(2+) and the role of proton transfer between the Mn cluster and Y(Z) during oxygen evolution is discussed.     

Structure of donor side components in photosystem II predicted by computer modelling.

Thirty-one and eleven sequences for the photosystem II reaction centre proteins D1 and D2 respectively, were compared to identify conserved single amino acid residues and regions in the sequences. Both proteins are highly conserved. One important difference is that the lumenal parts of the D1 protein are more conserved than the corresponding parts in the D2 protein. The three-dimensional structures around the electron donors tyrosineZ and tyrosineD on the oxidizing side of photosystem II have been predicted by computer modelling using the photosynthetic reaction centre from purple bacteria as a framework. In the model the tyrosines occupy two cavities close to the lumenal surface of the membrane. They are symmetrically arranged around the primary donor P680 and the distances between the centre of the tyrosines and the closest Mg ion in P680 are around 14 A. Both tyrosineZ and tyrosineD are suggested to form a hydrogen bond with histidine 190 from the loop connecting helices C and D in the D1 and D2 proteins, respectively. The Mn cluster in the oxygen evolving complex has been localized by using known and estimated distances from the tyrosine radicals. It is suggested that a binding region for the Mn cluster is constituted by the lumenal ends of helices A and B and the loop connecting them in the D1 protein. This part of the D1 protein contains a large number of strictly conserved carboxylic acid residues and histidines which could participate in the Mn binding. There is little probability that the Mn cluster binds on the lumenal surface of the D2 protein.     

On the inhibitory action of cadmium on the donor side of photosystem II in isolated chloroplasts.

The inhibitory effect of the Cd2+ in the electron transport of the isolated chloroplasts has been observed by measuring the oxygen uptake from the solution and the fluorescence induction. Cd2+ is found to be an inhibitor on the donor side of Photosystem II and its action site, as determined by experiments using hydroxylamine and exogenous Mn, is supposed to be on the water-splitting enzyme itself. Moreover, physicochemical and physiological studies indicate that only the ionic form of Cd is acting at the level of the manganoprotein. It is not possible, from this work, to define precisely in which form Cd is taken up through the thylakoid membranes.     

Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.     

Interaction of molecular oxygen with the donor side of photosystem II after destruction of the water-oxidizing complex

Photosystem II (PSII) is a pigment-protein complex of thylakoid membrane of higher plants, algae, and cyanobacteria where light energy is used for oxidation of water and reduction of plastoquinone. Light-dependent reactions (generation of excited states of pigments, electron transfer, water oxidation) taking place in PSII can lead to the formation of reactive oxygen species. In this review attention is focused on the problem of interaction of molecular oxygen with the donor site of PSII, where after the removal of manganese from the water-oxidizing complex illumination induces formation of long-lived states (P680^+· and TyrZ^·) capable of oxidizing surrounding organic molecules to form radicals.     

Characteristic features of the interaction between Fe(II) cations and the donor side of the manganese-depleted photosystem II

Ferrous iron cations Fe(II) can effectively bind to the donor side of the manganese-depleted photosystem II (PSII(-Mn)) and in this way block electron transfer from diphenylcarbazide (DPC) to the major donor for P680, Y_Z. The present study was focused on the characteristic features of this process. The oxidation and subsequent binding of Fe(II) cations to PSII(-Mn) may proceed in the absence of an artificial electron acceptor, and therefore we investigated the role of O₂ as a putative endogenous acceptor. Oxygen was shown to participate in the blockade of Y_Z by Fe cations, apparently as a structural element of Fe cluster formed at the donor side of PSII(-Mn). The kinetic study of blocking Y_Z by Fe(II) as dependent on light intensity demonstrated that the quantum efficiency of Fe cations binding to the donor side of PSII(-Mn) considerably exceeded that of Mn cations. We also compared the possibilities of extracting the native Mn cluster and reconstructed Fe cations from PSII and an alternative electron transport from DPC to P680⁺ under the conditions of the Y_Z blockade by Fe cations. Neither an alternative donor for P680, Y_D , nor cytochrome b ₅₅₉ participated in the latter process. As a whole, our evidence shows that many features of binding Fe cation to the donor side of PSII(-Mn) are in common with photoassembling the Mn cluster.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 12–20.Original Russian Text Copyright © 2005 by Lovyagina, Davletshina, Kultysheva, Timofeev, Ivanov, Semin.     

Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis

Phosphatidylglycerol (PG) is considered to play an important role in the ordered assembly and structural maintenance of the photosynthetic apparatus in thylakoid membranes. However, its function in photosynthesis remains poorly understood. In this study we have identified a pgsA gene of Synechocystis sp. PCC6803 that encodes a PG phosphate synthase involved in the biosynthesis of PG. A disruption of the pgsA gene allowed us to manipulate the content of PG in thylakoid membranes and to investigate the function of PG in photosynthesis. The obtained pgsA mutant could grow only in the medium containing PG, and the photosynthetic activity of the pgsA mutant dramatically decreased with a concomitant decrease of PG content in thylakoid membranes when the cells grown in the presence of PG were transferred to the medium without PG. This decrease of photosynthetic activity was attributed to the decrease of photosystem (PS)II activity, but not to the decrease in PSI activity. These findings demonstrate that PG is essential for growth of Synechocystis sp. PCC6803 and provide the first direct evidence that PG plays an important role in PSII.     

Effect of phosphatidylglycerol on conformation and micro-environment of tyrosyl residue in photosystem II

The structural aspects in the interaction of phosphatidylglycerol (PG) with photosystem II (PSIl), mainly the effect of PQ on conformation and microenvironment of tyrosine residues of PSIl proteins were studied by Fourier transform infrared (FTIR) spectroscopy. It was found that the binding of PG to PSIl particle induces changes in the conformation and micropolarity of phenol ring in the tyrosine residues. In other words, the PG effect on the PSIl results in blue shift of the stretch vibrational band in the phenol ring from 1620 to 1500 cm-1 with the enhancement of the absorb-ance intensity. Additionally, a new spectrum of hydrogen bond was also observed. The results imply that the hydrogen-bond formation between the OH group of phenol and one of PG might cause changes in the structures of tyrosine residues in PSIl proteins.     

Cytochemical localization of photosystem II donor sites

Summary Tetramethylbenzidene, an artificial donor of electrons to the photosystem (PS) II reaction center, is oxidized to an osmiophilic polymer that allows for the localization of the donor site of PS II. Mesophyll chloroplasts of Sorghum bicolor (L.) Moench. contain 0.1–0.2 m deposits only in the grana stacks and on the lumen side of the thylakoid. Agranal mature bundle sheath plastids (known to be devoid of PS II activity) show little deposition and the products appear randomly distributed along the lamellae. The cytochemical localization of PS II donor sites on the lumen side is consistent with flash photolysis and diaminobenzenesulfonic acid inactivation studies as well as the chemiosmotic theory for the generation of a proton gradient.     

Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 μmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.     

Influence of the disaccharide trehalose on the oxidizing side of photosystem II

Photosynthetica - The steady-state oxygen evolution rate was previously shown to be stimulated by the disaccharide trehalose in PSII suspension. Here we showed a similar increase in the rate of...     

Effect of phosphatidylglycerol on molecular organization of photosystem I

Phosphatidylglycerol (PG) is the only anionic phospholipid in photosynthetic membrane. In this study, photosystem I (PSI) particles obtained from plant spinach were reconstituted into PG liposomes at a relatively high concentration. The results from visible absorption, fluorescence emission, and circular dichroism (CD) spectra reveal an existence of the interactions of PSI with PG. PG effect causes blue-shift and intensity decrease of Chl a peak bands in the absorption and 77 K fluorescence emission. The visible CD spectra indicate that the excitonic interactions for Chl a and Chl b molecules were enhanced upon reconstitution. Furthermore, more or less blue- or red-shift of the peaks characterized by Chl a, Chl b, and carotenoid molecules are also occurred. Simultaneously, an increase in alpha-helix and a decrease particularly in the disordered conformations of protein secondary structures are observed. In addition, the same effect also leads to somewhat more tryptophan (Trp) residues exposed to the polar environment. These results demonstrate that some alteration of molecular organization occurs within both the external antenna LHCI and PSI core complex after PSI reconstitution.     

Reactions of hydroxylamine with the electron-donor side of photosystem II

The reaction of hydroxylamine with the O2-evolving center of photosystem II (PSII) in the S1 state delays the advance of the H2O-oxidation cycle by two charge separations. In this paper, we compare and contrast the reactions of hydroxylamine and N-methyl-substituted analogues with the electron-donor side of PSII in both O2-evolving and inactivated [tris(hydroxymethyl)aminomethane- (Tris-) washed] spinach PSII membrane preparations. We have employed low-temperature electron paramagnetic resonance (EPR) spectroscopy in order to follow the oxidation state of the Mn complex in the O2-evolving center and to detect radical oxidation products of hydroxylamine. When the reaction of hydroxylamine with the S1 state in O2-evolving membranes is allowed to proceed to completion, the S2-state multiline EPR signal is suppressed until after three charge separations have occurred. Chemical removal of hydroxylamine from treated PSII membrane samples prior to illumination fails to reverse the effects of the dark reaction, which argues against an equilibrium coordination of hydroxylamine to a site in the O2-evolving center. Instead, the results indicate that the Mn complex is reduced by two electrons by hydroxylamine, forming the S-1 state. An additional two-electron reduction of the Mn complex to a labile "S-3" state probably occurs by a similar mechanism, accounting for the release of Mn(II) ions upon prolonged dark incubation of O2-evolving membranes with high concentrations of hydroxylamine. In N,N-dimethylhydroxylamine-treated, Tris-washed PSII membranes, which lack O2 evolution activity owing to loss of the Mn complex, a large yield of dimethyl nitroxide radical is produced immediately upon illumination at temperatures above 0 degrees C. The dimethyl nitroxide radical is not observed upon illumination under similar conditions in O2-evolving PSII membranes, suggesting that one-electron photooxidations of hydroxylamine do not occur in centers that retain a functional Mn complex. We suggest that the flash-induced N2 evolution observed in hydroxylamine-treated spinach thylakoid membrane preparations arises from recombination of hydroxylamine radicals formed in inactivated O2-evolving centers.     

Vectorial charge transfer reactions on the donor side of manganese-depleted and reconstituted photosystem 2 core complexes

The light-induced functioning of photosystem 2 (PS 2) is directly linked to the translocation of both electrons and protons across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). Generation of ΔΨ due to S-state transitions of the water oxidation complex was demonstrated for the first time in Mn-depleted and reconstituted PS 2 core complexes incorporated into liposomes. The kinetics and relative amplitudes of the electrogenic reactions in dark-adapted samples during S₁→S₂, S₂→S₃, and S₄→S₀ transitions in response to the first, second and third laser flashes were comparable to those obtained in the intact PS 2 core particles. These results expand current understanding of the nature and mechanisms of electrogenic (vectorial) reactions due to a charge transfer on the donor side of PS 2.     

Peroxynitrite inhibits electron transport on the acceptor side of higher plant photosystem II

Peroxynitrite is a strong oxidant that has been proposed to form in chloroplasts. The interaction between peroxynitrite and photosystem II (PSII) has been investigated to determine whether this oxidant could be a hazard for PSII. Peroxynitrite is shown to inhibit oxygen evolution in PSII membranes in a dose-dependent manner. Analyses by PAM fluorimetry and EPR spectroscopy have demonstrated that the inhibition target of peroxynitrite is on the PSII acceptor side. In the presence of the herbicide DCMU, the chlorophyll (Chl) a fluorescence induction curve is inhibited by peroxynitrite, but the slow phase of the Chl a fluorescence decay does not change. EPR studies demonstrate that the Signal II_slow and Signal II_fast of peroxynitrite-treated Tris-washed PSII membranes are induced at room temperature, implying that the redox active tyrosines Y_Z and Y_D of PSII are not significantly nitrated. A featureless EPR signal with a g value of approximately 2.0043 ± 0.0003 and a line width of 10 ± 1 G is induced under continuous illumination in the presence of peroxynitrite. This new EPR signal corresponds with the semireduced plastoquinone Q_A in the absence of magnetic interaction with the non-heme Fe²⁺. We conclude that peroxynitrite impairs PSII electron transport in the Q_AFe²⁺ niche.