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

Abstract

Extraction of Ca(2+) from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O2 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 Ca(2+)-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 (·-) , as possible products of water oxidation in PSII(-Ca) membranes was examined. During the investigation of O 2 (·-) production in PSII(-Ca) samples, we found that (i) O 2 (·-) is formed on the acceptor side of PSII due to the reduction of O2; (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 O2, thereby increasing the rate of O 2 (·-) 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.

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II

“…S1B). This observation is in agreement with previous reports that chloride-depleted PSII membranes produced more HO • compared with control PSII membranes (33,34). The involvement of chloride in HO • formation is supported by the observation that acetate and DIDS nearly abolished detectable HO • production (Fig.…”

Reactive oxygen species: Reactions and detection from photosynthetic tissues

“…The Cyt c method has been used in vitro to measure O 2 • from PSII-enriched membrane fragments from higher plants [263], from apoplasts of wheat roots [314] and from Chlamydomonas sp. ICE-L suspension [264].…”

“…In low and moderate light intensities (at PPFD 5-200 µmol m -2 s -1 ) Cyt c was mainly reduced by the plastoquinone pool and not by O 2 • [316]. For this reason, comparison of the results obtained with and without added SOD is always required [263]. To enhance the specificity of Cyt c to O 2 • , it is possible to use acetylated Cyt c [318], for example with Chlamydomonas in [264].…”

Visible light promoted photocatalytic water oxidation: proton and electron collection via a reversible redox dye mediator

“…Here the DCPIP plays an equivalent role to NADP + . 14,15 Partially reduced DCPIPH is pink, further reduction gives colourless DCPIPH2 which is equivalent to NADPH+H + produced by light dependant photosynthesis PSII/PSI and can potentially be used as a synthetic proton/electron storage molecule. Ru(bpy)3 2+ and DCPIP can be incorporated into silica sol-gels which facilitates its recovery and re-use in reactions (see ESI for experimental details).…”