CHEMICAL OXIDATION AND REDUCTION OF THE O-2-EVOLUTION CENTER IN PHOTOSYSTEM-II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O-2-evolution pattern, and in the formation of the S-2-state multiline EPR signal, due to the two-electron reduction of the S-1-state by hydroxylamine to form the S--1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O-2-evolution pattern and S-2-state multiline EPR signal after treatment of PS II with 40 mu M hydroxylamine, in agreement with past work, we find that the two-flash delay in O-2 evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O-2-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 mu M hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S--1-state to the S-1-state. In contrast, adding hypochlorite (OCl-) after treatment with hydroxylamine restored the normal flash-induced O-2-evolution pattern and also restored the formation of the S-2-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S--1-state to the S-1-state. This is the first example of a chemical treatment that advances the delayed flash-induced O-2 evolution pattern.