Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping.

Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of (1)O2, and suppressed by the (1)O2 quencher NaN3. Due to the limited mobility of (1)O2 these data demonstrate that exogenous histidine reaches close vicinity of (1)O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that (1)O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied (1)O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518-8527; Cser and Vass 2007, BBA 1767:233-243). We found that the D1-Gln130Glu mutant showed decreased (1)O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both (1)O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which (3)P680 mediated (1)O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.

Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC 7942 cells.

The role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria was analyzed in a Synechococcus sp. PCC 7942 mutant produced by inactivating its cdsA gene presumably encoding cytidine 5'-diphosphate-diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the Synechococcus sp. PCC 7942/DeltacdsA cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a suppression of O(2) evolving activity, and (c) in a modification of Chl fluorescence induction curves. Two-dimensional PAGE showed that in the absence of PG (a) the amount of the PSI monomers increased at the expense of the PSI trimers and (b) PSII dimers were decomposed into monomers. [(35)S]methionine labeling confirmed that PG depletion did not block the de novo synthesis of PSII proteins but slowed down the assembly of the newly synthesized D1 protein into PSII core complexes. Retailoring of PG was observed during PG depletion: the exogenously added artificial dioleoyl PG was transformed into photosynthetically more essential PG derivatives. Concomitantly with a decrease in PG content, SQDG content increased, but it could not restore photosynthetic activity.

Janus-faced charge recombinations in photosystem II photoinhibition.

Light-induced damage of the photosynthetic apparatus in plants is an important phenomenon that primarily affects the photosystem II complex. Here, we propose a new model of photoinhibition in which charge recombination processes have a double-faced role: first, photodamage is induced by singlet oxygen, which is produced via interaction with the triplet reaction center chlorophyll ((3)P(680)) arising from the recombination of the charge-separated state between P(680) and the pheophytin electron acceptor ((3)[P(680)(+)Phe(-)]). Second, photoprotection is provided by competition between (3)[P(680)(+)Phe(-)] formation and direct recombination of the (1)[P(680)(+)Phe(-)] and P(680)(+)Q(A)(-) states. The efficiency of these two pathways is under control of the redox potential of the Phe and Q(A) electron acceptors, which is utilized during adaptation to high light conditions.

Energetics of Photosystem II charge recombination in Acaryochloris marina studied by thermoluminescence and flash-induced chlorophyll fluorescence measurements.

We studied the charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll (Chl) d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Flash-induced chlorophyll fluorescence decay was retarded in the mus and ms time ranges and accelerated in the s time range in Acaryochloris marina relative to that in the Chl a-containing cyanobacterium, Synechocystis PCC 6803. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, which blocks the Q(B) site, the relaxation of fluorescence decay arising from S(2)Q(A)(-) recombination was somewhat faster in Acaryochloris marina than in Synechocystis PCC 6803. Thermoluminescence intensity of the so called B band, arising from the recombination of the S(2)Q(B)(-) charge separated state, was enhanced significantly (2.5 fold) on the basis of equal amounts of PSII in Acaryochloris marina as compared with Synechocystis 6803. Our data show that the energetics of charge recombination is modified in Acaryochloris marina leading to a approximately 15 meV decrease of the free energy gap between the Q(A) and Q(B) acceptors. In addition, the total free energy gap between the ground state and the excited state of the reaction center chlorophyll is at least approximately 25-30 meV smaller in Acaryochloris marina, suggesting that the primary donor species cannot consist entirely of Chl a in Acaryochloris marina, and there is a contribution from Chl d as well.

Molecular mechanisms of light stress of photosynthesis.

Photosynthesis is the basic energy conversion process on Earth, which makes possible the utilization of the energy of sunlight for living organisms. However, light is not only the basic driving force of photosynthesis, but also an important stress factor at the same time. Light-induced decline of photosynthetic activity, generally denoted as photoinhibition, is a general phenomenon in all oxygenic photosynthetic organism under conditions when the metabolic processes cannot keep up with the electron flow produced by the primary photoreactions. Although light-induced damage occurs in all pigmented photosynthetic complexes the primary site of photoinhibition is the photosystem II (PSII) complex, which performs light-driven oxidation of water to protons and oxygen. The main factors, which are responsible for the light sensitivity of photosystem II, are excited pigment molecules, oxygen, manganese, as well as electron donors with high-oxidizing potential. Photosystem II can be efficiently protected from photodamage by the combination of harmless dissipation of absorbed light energy, nonradiative charge recombination, and repair of damaged reaction center complexes, making possible the safe utilization of light, the highly energetic substrate of photosynthesis.

Radiative and non-radiative charge recombination pathways in Photosystem II studied by thermoluminescence and chlorophyll fluorescence in the cyanobacterium Synechocystis 6803.

The mechanism of charge recombination was studied in Photosystem II by using flash induced chlorophyll fluorescence and thermoluminescence measurements. The experiments were performed in intact cells of the cyanobacterium Synechocystis 6803 in which the redox properties of the primary pheophytin electron acceptor, Phe, the primary electron donor, P(680), and the first quinone electron acceptor, Q(A), were modified. In the D1Gln130Glu or D1His198Ala mutants, which shift the free energy of the primary radical pair to more positive values, charge recombination from the S(2)Q(A)(-) and S(2)Q(B)(-) states was accelerated relative to the wild type as shown by the faster decay of chlorophyll fluorescence yield, and the downshifted peak temperature of the thermoluminescence Q and B bands. The opposite effect, i.e. strong stabilization of charge recombination from both the S(2)Q(A)(-) and S(2)Q(B)(-) states was observed in the D1Gln130Leu or D1His198Lys mutants, which shift the free energy level of the primary radical pair to more negative values, as shown by the retarded decay of flash induced chlorophyll fluorescence and upshifted thermoluminescence peak temperatures. Importantly, these mutations caused a drastic change in the intensity of thermoluminescence, manifested by 8- and 22-fold increase in the D1Gln130Leu and D1His198Lys mutants, respectively, as well as by a 4- and 2.5-fold decrease in the D1Gln130Glu and D1His198Ala mutants, relative to the wild type, respectively. In the presence of the electron transport inhibitor bromoxynil, which decreases the redox potential of Q(A)/Q(A)(-) relative to that observed in the presence of DCMU, charge recombination from the S(2)Q(A)(-) state was accelerated in the wild type and all mutant strains. Our data confirm that in PSII the dominant pathway of charge recombination goes through the P(680)(+)Phe(-) radical pair. This indirect recombination is branched into radiative and non-radiative pathways, which proceed via repopulation of P(680)(*) from (1)[P(680)(+)Ph(-)] and direct recombination of the (3)[P(680)(+)Ph(-)] and (1)[P(680)(+)Ph(-)] radical states, respectively. An additional non-radiative pathway involves direct recombination of P(680)(+)Q(A)(-). The yield of these charge recombination pathways is affected by the free energy gaps between the Photosystem II electron transfer components in a complex way: Increase of DeltaG(P(680)(*)<-->P(680)(+)Phe(-)) decreases the yield of the indirect radiative pathway (in the 22-0.2% range). On the other hand, increase of DeltaG(P(680)(+)Phe(-)<-->P(680)(+)Q(A)(-)) increases the yield of the direct pathway (in the 2-50% range) and decreases the yield of the indirect non-radiative pathway (in the 97-37% range).

The role of D1-Ala344 in charge stabilization and recombination in Photosystem II.

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 alpha-carboxylate chain of the D1-Ala344 residue might represent one of the final steps in the assembly of functional catalytic sites for water oxidation.