The mechanism of photosynthetic water oxidation in spinach was investigated with a newly developed inhibitor of the water-oxidizing complex, acetone hydrazone (AceH), (CH3)2CNNH2 [Tso, J., Petrouleas, V., & Dismukes, G. C. (1990) Biochemistry (preceding paper in this issue)], by using fluorescence induction and single-turnover flashes to monitor O2 yield and thermoluminescence intensity. AceH binds slowly (1-3 min) in the dark to the S1 (resting) oxidation state of the water-oxidizing complex in thylakoids and PSII membranes. Once bound, it causes a two-flash delay in the pattern of O2 release seen in a train of flashes. This is initiated by reduction of manganese in the S2 oxidation state of the complex in a fast reaction (<0.5 s). In thylakoid membranes which have been partially inhibited at low AceH concentrations (<2 mM) the inhibition can be reversed by a single flash and a subsequent dark period. This behavior can be explained by two sequential one-electron oxidation steps: Dissociation of the unobserved radical intermediate, AceH+, from S1 is proposed to account for the recovery from inhibition after one flash. In contrast, recovery from inhibition after a single flash is not observed in detergent-isolated PSII membranes or in intact thylakoid membranes at higher AceH concentrations (>2 mM), where the two-flash delay in O2 release is seen. This suggests either a concerted two-electron process, S2 → S0, or tight binding of AceH+ to S1. Fluorescence induction shows that AceH inhibition does not affect the electron-transfer reactions within the photoreaction center protein. Thermoluminescence shows no evidence for abnormal activation barriers to recombination from both (S2 + S3)QA− and (S2 + S3)QB−, indicative of a lack of observable structural alteration of these states by AceH. The binding of AceH lowers the binding affinity for DCMU, a herbicide that binds to the QB acceptor site. A small yield of O2 (<5%) is observed on the first flash in AceH-inhibited PSII membranes, in contrast to untreated membranes where no O2 forms. This suggests that O2 may bind to S−1 centers which form in <5% of the S1 centers that undergo two-electron reduction in the dark. These centers could then release O2 after forming the S0 state with a single flash.
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