Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca2+ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O-2 evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as the decoupling effect (Semin et al. Photosynth Res 98:235-249, 2008). Extraction of Cl- from Ca2+-depleted membranes (PSII[-Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H2O2, and O (2) (center dot-) , as possible products of water oxidation in PSII(-Ca) membranes was examined. During the investigation of O (2) (center dot-) production in PSII(-Ca) samples, we found that (i) O (2) (center dot-) is formed on the acceptor side of PSII due to the reduction of O-2; (ii) depletion of Cl- does not inhibit water oxidation, but (iii) Cl- depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl- under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O-2, thereby increasing the rate of O (2) (center dot-) generation. From these observations we conclude that the product of water oxidation is H2O2 and that Cl- anions are not involved in the oxidation of water to H2O2 in decoupled PSII(-Ca) membranes. These results also indicate that Cl- anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H2O molecules to the Mn4CaO5 cluster and/or facilitate the release of H+ ions into the lumenal space.