Imre Vass

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.

Sequence Analysis of the D1 and D2 Reaction Center Proteins of Photosystem II

Abstract A compilation of 38 sequences for the D1 and 15 sequences for the D 2 reaction center proteins of photosystem II is presented. The sequences have been compared and a similarity index that takes into account the degree of conservation and the quality of the changes in each position has been calculated. The similarity index is used to identify and describe functionally important domains in the D1 / D2 heterodimer. Comparative hydropathy plot are presented for the aminoacid sidechains that constitute the binding domain of the tyrosine radicals, TyrZ and TyrD, in photosystem II. The structure around TyrZ is more hydrophilic than the structure around TyrD. The hydrophilic residues are clustered in the part of the binding pocket for TyrZ that is turned towards the lumenal side of the thylakoid membrane. Most prominent is the presence of two conserved carboxylic am inoacids, D1-Asp 170 and D1-Glu 189. Their respective carboxyl-groups come close in space and are proposed to constitute a metal binding site together with D1-Gln 165. The distance between the proposed metal binding site and the center of the ring of TyrZ is approximately 7Å . The cavity that constitutes the binding site for TyrD is composed of residues from the D 2 protein. Its character is more hydrophobic than the TyrZ site and the environment around TyrD lacks the cluster of putative metal binding side-chains.

Ca2+ depletion modifies the electron transfer on both donor and acceptor sides in Photosystem II from spinach

Ca2+ depletion of Photosystem II from spinach results in reversible retardation of electron transfer on both donor and acceptor sides. On the donor side, a decrease of the electron transfer rate from TyrZ results in an enhanced charge recombination between the oxidized primary donor, P680+, and the reduced acceptor quinone, QA−, which in turn leads to a decrease in the amplitude of the fluorescence yield. In addition, slow electron transfer from the manganese cluster in the dark-stable S2 state results in the appearance of a transient EPR signal from TyrZox which decays with half-times of 600 ms and 5 s. On the acceptor side, the disappearance of the 400 μs decay transient in the fluorescence yield indicates that the electron transfer from QA− to QB has been severely inhibited. These results suggests that removal of a Ca2+ ion from the donor side in PS II, which results in the inhibition of oxygen evolution and in the appearance of an EPR signal in the S′3 state leads to structural changes which are transmitted to the acceptor side. The strikingly similar behavior after depletion of Ca2+ of the TyrZox EPR signal and the split radical signal from the S′3 state suggests that both signals involves the same oxidized amino acid residue, TyrZox. The absence of large effects on the EPR properties of the non-heme iron suggests that the structural changes on the acceptor side are subtle in nature. Chemical modification of histidine results in inhibition of QA− to QB electron transfer and to changes in the magnetic properties of the oxidized non-heme iron but only to minor perturbations of the donor-side. This suggests that histidine, susceptible to chemical modification, is located mainly on the acceptor side of PS II.

The accessory electron donor tyrosine-D of Photosystem II is slowly reduced in the dark during low-temperature storage of isolated thylakoids

Storage of thylakoids for several months at 203 K in the dark changes the flash pattern of oxygen evolution by gradually shifting the first oxygen maximum from the third flash, where it is usually observed, to the fourth flash. This effect is accompanied with the increase of a fast phase (t12 = 1.5 s) in the decay of the S2 and S3 states of the water-oxidizing complex in Photosystem II. In parallel to the changes in the oxygen flash pattern the EPR signal from the stable tyrosine-D+ radical (Signal IIslow) completely disappears with a half-time of approx. 13 weeks. The normal oxygen yield sequence, showing the first maximum at the third flash, as well as the original amplitude of Signal IIslow can be restored by a single flash or by continuous illumination at room temperature. These data show that tyrosine-D+ is reduced by an endogenous redox component in Photosystem II during dark storage of thylakoids at 203 K. In parallel with the reduction of tyrosine-D+ we observed the oxidation of high potential cytochrome b-559, and it is proposed that at low temperature an electron can be transferred from high-potential cytochrome b-559 to tyrosine-D+ in a slow reaction in most of the centers.

Redox interaction of tyrosine-D with the S-states of the water-oxidizing complex in intact and chloride-depleted Photosystem II

The light-induced oxidation of Tyrosine-D in Photosystem II has been studied by time-resolved measurements of the EPR Signal IIslow at room temperature. When induced with single turnover flashes, the oxidation of Tyrosine-D undergoes a period-four oscillation as a function of flash number, showing Tyrosine-D+ formation in the S2 and S3 oxidation states of the water-oxidizing complex. The kinetics of Tyrosine-D oxidation by the S2 and S3 states are almost identical in the pH range of 4.5 to 7.8, and show the same pH dependence for the S3 state as has previously been observed for the S2 state (Vass and Styring (1991) Biochemistry 30, 830–839). It is concluded from the pH-dependent oxidation kinetics that a proton binding with a pK around 7.0–7.2 retards electron transfer from Tyrosine-D to the water-oxidizing complex both in the S2 and in the S3 states. In addition, our results imply that the S2/S1 and S3/S2 redox couples have about the same redox potential relative to that of the Tyrosine-D+/Tyrosine-D couple. Removal of chloride from Photosystem II induced an approximately 10-times slowdown in the Tyrosine-D oxidation kinetics by the S2 state. This result indicates that Tyrosine-D can interact with the S2 state in the absence of chloride. The retarded oxidation kinetics observed under these conditions are consistent with the previously demonstrated stabilization of the chloride-free S2 state. We also observed the flash-induced oxidation of Tyrosine-Z in a large fraction of the chloride depleted Photosystem II centers. In this system Tyr-Z+ was abnormally stable and decayed biphasically with 500 ms and 12–15 s half-times.

Mutation of a putative ligand to the non-heme iron in Photosystem II: implications for QA reactivity, electron transfer, and herbicide binding

Abstract In Photosystem II (PS II), a non-heme iron is present near the electron-accepting quinones Q A and Q B . A putative ligand to this non-heme iron, His-268 in the D2 protein, was mutated to Gln in the cyanobacterium Synechocystis sp. PCC 6803. The resulting mutant H268Q has lost photoautotrophic capacity and shows large alterations in the properties both of Q A and of the Q B pocket. In the mutant, the quantum efficiency of Q A reduction is decreased by approximately 50-fold, electron transfer from Q A to the plastoquinone pool is blocked, Q A apparently can be displaced by exogenous quinones, and the stability of reduced Q A is increased by more than an order of magnitude. Also the affinity of the PS II-directed herbicide diuron to the PS II complex is decreased to undetectable levels. We suggest that these mutation-induced changes in the properties of the acceptor side of PS II are caused by a functional loss of the non-heme iron. This would imply that the non-heme iron in PS II plays a functionally more important role than observed in reaction centers from purple bacteria, and has drastic effects on the properties of Q A . Moreover, the results obtained on the D2 mutant H268Q illustrate that the D2 protein can have a pronounced influence on the properties of the Q B /herbicide-binding environment, which is associated mostly with the D1 protein. Thus, the non-heme iron in PS II appears to both affect Q A reactivity and alter the properties of the Q B pocket.

Molecular mechanisms behind light-induced inhibition of photosystem II electron transport and degradation of reaction centre polypeptides

Excessively high light intensities are damaging to the photosynthetic apparatus. Photosynthetic organisms are therefore faced with the problem of maintaining sufficient excitation power under limiting light conditions whilst avoiding photodamage under high light conditions. The primary target for this photodamage is Photosystem II (PSII), which undergoes inhibition of its electron transport followed by irreversible damage to and degradation of the D~ and D 2 reaction centre polypeptides. This degradation is the initial event in a repair cycle of photodamaged complexes and is followed by protein synthesis and reassembly of functional PSII. This report will deal with recent information relevant to the molecular mechanisms of PSII electron transport impairment and of protein degradation, particularly the D I protein, and the chemical link between these two events. Data will be presented in support of the following sequence of reactions (Fig. 1), which take place at PSII under high-light stress: (i) overreduction of the acceptor side, leading to the formation of stably reduced QA species; (ii) these events facilitate the formation of chlorophyll triplets which react with molecular oxygen to form singlet oxygen; (iii) this highly reactive and damaging species will oxidize pigments and/or amino-acid residues leading to irreversible damage to the reaction centre, mainly to the D l protein; (iv) the oxidative damage induces a conformational change in the D~ protein which triggers it for

UV-B and UV-A Radiation Effects on Photosynthesis at the Molecular Level

Ultraviolet radiation is a well known damaging factor of plant photosynthesis. Here we studied the mechanism of damage induced by the UV-B and UV-A spectral regions to the light energy converting Photosystem II (PSII) complex, which is the origin of electron flow for the whole photosynthetic process. Our results show that the primary UV damage occurs at the catalytic Mn cluster of water oxidation, which is most likely sensitized by the UV absorption of Mn(III) and Mn(IV) ions ligated by organic residues. The presence of visible light enhances the photodamage of PSII, but has no synergistic interaction with UV radiation. UV-induced damage of PSII can be repaired via de novo synthesis of the D1 and D2 reaction center protein subunits. This process is facilitated by low intensity visible light, which thereby can protect against UV-induced damage. However, the photodamage induced by visible light at high intensity (above 1000 µEm−2s−1) cancels the protective effect. The protein repair of PSII is also retarded by the lack of DNA repair as shown in a photolyase deficient cyanobacterial mutant.

Cytochrome b-559 Is Important for Modulating Electron Transfer on the Acceptor Side of Photosystem II and for Photoprotection During Assembly of the Mn4Ca Complex

To investigate the physiological role of cytochrome b-559 (Cyt b-559) we have constructed a PsbE-H23C mutant of C. reinhardtii in which the His-ligand to the haem provided by the the α subunit has been replaced by Cys. The mutant assembles PSII at reduced levels. To investigate the co-ordination of a haem group by the protein, H23C mutants were generated in a cytochrome b 6 f-less (Cyt b 6 f) background. Analysis by absorbance spectroscopy of pure thylakoids confirmed the lack of a haem group in vivo. Reduction of QB is impaired in the mutant. The mutant is also more sensitive to photoinhibition (PI) than WT cells. Under conditions when the Mn cluster is being photoactivated H23C cells are also more susceptible to PI than WT. Our results indicate that Cyt b-559 plays a role in PSII electron transfer, PSII photoprotection and PSII repair.

Non-photochemical-quenching Mechanisms in the Cyanobacterium Thermosynechococcus elongatus

In the cyanobacterium Synechocystis PCC 6803 grown under iron replete or iron deplete conditions, blue light induces a photoprotective Non-Photochemical-Quenching (NPQ) mechanism. This energy dissipation mechanism involves the phycobilisomes and the Orange Carotenoid Protein (OCP) encoded by the slr1963 gene. In the thermophilic cyanobacterium Thermosynechococcus elongatus there are no OCP-like genes, but instead two adjacent genes encode for the N-terminal and C-terminal domains of an OCP-like gene. The goal of our study was to elucidate the possible role of these genes in the blue-light induced NPQ mechanism. Blue light (at any intensity) was not able to induce any quenching of fluorescence in T. elongatus cells grown in the presence or absence of Fe. In contrast, normal state transitions were observed. The blue-light induced NPQ was also absent in Synechococcus elongatus cells in which the OCP gene is lacking, but it was present in Arthrospira maxima and Anabaena variabilis cells possessing the OCP protein.