Imre Vass

The Ala344 residue of the D1 protein has been identified as a crucial residue of the catalytic cluster of the water-oxidizing complex, however, its function has not been fully clarified. Here we have used thermoluminescence and flash-induced chlorophyll fluorescence measurements to characterize the effect of the D1-Ala344stop mutation on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutant cannot grow photoautotrophically it shows flash-induced thermoluminescence and chlorophyll fluorescence signals reflecting the stabilization of negative and positive charges on the Q_A and Q_B quinone electron acceptors, and stable Photosystem II donors, respectively. Decay of flash induced chlorophyll fluorescence yield is multiphasic in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with 6 ms, 350 ms, and 26 s time constants. When cells are illuminated with repetitive flashes, fired at 1 ms intervals, the 6 ms phase is gradually decreased with the concomitant increase of the 350 ms phase. After 45 min dark adaptation of mutant cells the 6 ms and 350 ms phases were significantly decreased and a very slow decaying component was formed. Flash induced oscillation of the thermoluminescence B band, which reflects the redox cycling of the water-oxidizing complex in the wild-type cells, was completely abolished in the D1-Ala344stop mutant. The results demonstrate that low efficiency photooxidation of Mn occurs in about 60% of the PSII centers. The photooxidizable Mn is unstable in the dark, and formation of higher S states is inhibited. In addition, the Q_A to Q_B electron transfer step is slowed down as an indirect consequence of the donor side modification. Our data indicate that the stabilization of a Mn ion by the α-carboxylate chain of the D1-Ala344 residue might represent one of the final steps in the assembly of functional catalytic sites for water oxidation.

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S₂ and S₃ states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S₂Q ⁻_B and S₃Q ⁻_B states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.

The His332 residue of the D1 protein has been identified as the likely ligand of the catalytic Mn ions in the water oxidizing complex (Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J. & Iwata, S. (2004) Science 303, 1831–1838). However, its function has not been fully clarified. Here we used thermoluminescence and flash‐induced chlorophyll fluorescence measurements to characterize the effect of the D1‐H333E, D1‐H332D and D1‐H332S mutations on the electron transport of Photosystem II in intact cells of the cyanobacterium Synechocystis 6803. Although the mutants are not photoautotrophic they all show flash‐induced thermoluminescence and chlorophyll fluorescence, which originate from the S₂Q_A^– and S₂Q_B^– recombinations demonstrating that charge stabilization takes place in the water oxidizing complex. However, the conversion of S₂ to higher S states is inhibited and the energetic stability of the S₂Q_A^– charge pair is increased by 75, 50 and 7 mV in the D1‐H332D, D1‐H332E and D1‐H332S mutants, respectively. This is most probably caused by a decrease of E_m(S₂/S₁). Concomitantly, the rate of electron donation from Mn to Tyr‐Z^ḃ during the S₁ to S₂ transition is slowed down, relative to the wild type, 350‐ and 60‐fold in the D1‐H332E and D1‐H332D mutants, respectively, but remains essentially unaffected in D1‐H332S. A further effect of the D1‐H332E and D1‐H332D mutations is the retardation of the Q_A to Q_B electron transfer step as an indirect consequence of the donor side modification. Our data show that although the His residue in the D1‐332 position can be substituted by other metal binding residues for binding photo‐oxidisable Mn it is required for controlling the functional redox energetics of the Mn cluster.

A fundamental discovery of photosynthetis research in the 1950s was the detection of thermally stimulated light emission from preilluminated photosynthetic material [Arnold W and Sherwood H (1957)...

In order to understand the mechanism of photodamage induced by solar radiation under natural conditions, we studied the interaction of visible and ultraviolet-B light in the inactivation and repair...

Inhibition of electron transport and damage to the protein subunits by visible light has been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides....

UV‐B irradiation ofSynechocystis6803 cells inhibits photosystem II activity, which can be restored viade novosynthesis of the D1 (and D2) reaction center subunits. Recently we have shown that of the twopsbAgenes that encode identical D1 proteins inSynechocystis6803, UV‐B preferentially enhances the transcription ofpsbA3compared to that ofpsbA2[Máté, Z., Sass, L., Szekeres, M., Vass, I. and Nagy, F. (1998)J. Biol. Chem.273, 17439–17444]. Here we studied the effect of UV‐B on the synthesis of the D1 protein from thepsbA2andpsbA3genes in the P7 mutant ofSynechocystis6803. In this mutant,psbA2carries the Ala251→Val point mutation, which confers resistance to the photosystem II electron transport inhibitor metribuzin, butpsbA3is the same as in the wild‐type. By applying variable chlorophyll fluorescence measurements to distinguish between metribuzin‐sensitive and metribuzin‐resistant photosystem II centers we quantified the amount of the D1 protein produced from each of thepsbA3andpsbA2genes. When the cells were exposed to UV‐B light, the fraction of D1 protein produced from thepsbA3gene was increased from 15–20 to 32–40% of the total D1. This effect was reversible by transferring the cells to visible light. The rate of D1 production frompsbA3increased with increasing UV‐B intensities, and was a transient phenomenon at low UV‐B levels (0.1 µE·m⁻²·s⁻¹). It is concluded that the enhancement ofpsbA3gene transcription by UV‐B light leads to enhanced D1 protein synthesis from this gene. Our findings demonstrate that the main role ofpsbA3transcription activated by UV‐B is to increase the size of thepsbAmRNA pool available for translation when a rapid repair of the D1 protein is needed under UV‐B stress.

We studied the effect of UV-B radiation (280−320 nm) on the donor- and acceptor-side components of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 by measuring the relaxation of flash-induced variable chlorophyll fluorescence. UV-B irradiation increases the t_1/2 of the decay components assigned to reoxidation of Q_A^- by Q_B from 220 to 330 μs in centers which have the Q_B site occupied, and from 3 to 6 ms in centers with the Q_B site empty. In contrast, the t_1/2 of the slow component arising from recombination of the Q_AQ_B^- state with the S₂ state of the water-oxidizing complex decreases from 13 to 1−2 s. In the presence of DCMU, fluorescence relaxation in nonirradiated cells is dominated by a 0.5−0.6 s component, which reflects Q_A^- recombination with the S₂ state. After UV-B irradiation, this is partially replaced by much faster components (t_1/2 ≈ 800−900 μs and 8−10 ms) arising from recombination of Q_A^- with stabilized intermediate photosystem II donors, P680⁺ and Tyr-Z⁺. Measurement of fluorescence relaxation in the presence of different concentrations of DCMU revealed a 4−6-fold increase in the half-inhibitory concentration for electron transfer from Q_A to Q_B. UV-B irradiation in the presence of DCMU reduces Q_A in the majority (60%) of centers, but does not enhance the extent of UV-B damage beyond the level seen in the absence of DCMU, when Q_A is mostly oxidized. Illumination with white light during UV-B treatment retards the inactivation of PSII. However, this ameliorating effect is not observed if de novo protein synthesis is blocked by lincomycin. We conclude that in intact cyanobacterium cells UV-B light impairs electron transfer from the Mn cluster of water oxidation to Tyr-Z⁺ and P680⁺ in the same way that has been observed in isolated systems. The donor-side damage of PSII is accompanied by a modification of the Q_B site, which affects the binding of plastoquinone and electron transport inhibitors, but is not related to the presence of Q_A^-. White light, at the intensity applied for culturing the cells, provides protection against UV-B-induced damage by enhancing protein synthesis-dependent repair of PSII.