The process of photosynthetic water oxidation has been investigated by using a new type of water oxidation inhibitor, the alkyl hydrazones. Acetone hydrazone (AceH), (CH3)2CNNH2, inhibits water oxidation by a mechanism that is analogous to that of NH2OH. This involves binding to the water-oxidizing complex (WOC), followed by photoreversible reduction of manganese (loss of the S1 → S2 reaction). At higher AceH concentrations the S1 state is reduced in the dark and Mn is released, albeit to a lesser extent than with NH2OH. Following extraction of Mn, AceH is able to donate electrons rapidly to the reaction center tyrosine radical Z+ (161Tyr-D1 protein), more slowly to a reaction center radical C+, and not at all to the dark-stable tyrosine radical D+ (160Tyr-D2 protein) which must be sequestered in an inaccessible site. Manganese, Z+, and C+ thus appear to be located in a common protein domain, with Mn being the first accessible donor, followed by Z+ and then C+. Photooxidation of Cyt b-559 is suppressed by AceH, indicating either reduction or competition for donation to P680+. Unexpectedly, Cl− was found not to interfere or compete with AceH for binding to the WOC in the S1 state, in contrast to the reported rate of binding of N,N-dimethylhydroxylamine, (CH3)2NOH [Beck, W., & Brudvig, G. (1988) J. Am. Chem. Soc. 110, 1517-1523]. We interpret the latter behavior as due to ionic screening of the thylakoid membrane, rather than a specific Cl− site involved in water oxidation. AceH appears not to bind to the acceptor side of PSII as evidenced by normal EPR signals both for QA−Fe(II), the primary electron acceptor, and for the oxidized Fe(III) acceptor (Q400 species), in contrast to that observed with the NH2OH. AceH can be oxidized in solution by a variety of oxidants including Mn(III) to form a reactive diazo intermediate, (CH3)2CNN, which reacts with carbonyl compounds. Oxidation to this diazo intermediate is postulated to be responsible for inhibition of the WOC.
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