A New Mechanism-Based Inhibitor of Photosynthetic Water Oxidation: Acetone Hydrazone. 1. Equilibrium Reactions

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 Q_A⁻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.