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

Abstract

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 1O2, and suppressed by the 1O2 quencher NaN3. Due to the limited mobility of 1O2 these data demonstrate that exogenous histidine reaches close vicinity of 1O2 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 1O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied 1O2 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 1O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both 1O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which 3P680 mediated 1O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.