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 (less than 0.5 s). In thylakoid membranes which have been partially inhibited at low AceH concentrations (less than 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: S1.AceHhv----S2.AceH in equilibrium S1.AceH+hv----S2.AceH+----S1 + AceH2+ 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 (greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)