S(-3) state of the water oxidase in photosystem II.

The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.