Studies of ascorbate-dependent, non-heme iron enzyme ACC oxidase

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DESCRIPTION (provided by applicant): Oxygen-activating enzymes with mononuclear non-heme iron active sites participate in many metabolically important reactions that have tremendous medical, pharmaceutical, and environmental significance, catalyzing a wide range of reactions involving dioxygen (monooxygenation, oxidation, etc.). The present proposal aims to obtain more information about the mechanism by which such iron enzymes activate dioxygen and how cofactors and substrates interact in the active site to allow for catalytic activity. Studies will focus on 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), a unique ascorbate-dependent, non- heme iron enzyme that catalyzes the last step in the biosynthesis of plant hormone ethylene, important in many aspects of plant growth and development, including germination, fruit ripening, and senescence. The specific aims of this proposal are 1) studying the mode of oxygen activation by the iron center in ACCO and the nature of the active oxidant using 18O kinetic isotope effects and pre-steady state kinetics and 2) deciphering the substrate breakdown mechanism by using alternate substrates containing radioactive labels. A range of studies are planned that include the measurement of 18O kinetic isotope effects to determine the order of reductant binding and the role of ascorbate in the reaction, and the characterization of intermediates that accumulate during catalytic turnover by stopped-flow methods. Radioactively labeled substrate analogs will be used in enzyme inactivation studies which could be potentially used for designing mechanism-based inhibitors that could help reduce agricultural waste and sustain healthy nutrition. The ability to control the ethylene synthesis processes in a time-dependent manner may aid in decreasing major losses of agricultural waste due to over-ripening. Potential impact on human healty is significant, particularly where long distance transport of produce is needed or storage occurs in areas that lack sufficient refrigeration.