Copper monooxygenases play important roles in neurological and endocrine function. Two enzymes, peptidyiglycine alpha-amidating monooxygenase (PHM) and dopamine beta-monooxygenase (DBM), occupy key positions in the biosynthetic pathways to neuropeptide hormones and catecholamine neurotransmitters, respectively. PHM catalyzes the final step in the biosynthesis of amidated peptides which are pivotal to the development and health of organisms from the most primitive to humans. Crystallographic and spectroscopic studies on PHM have suggested two alternative mechanisms which represent new paradigms for dioxygen activation by copper. Both mechanisms are novel, and the differences between them focus to a large extent on the early steps in dioxygen reduction. In this proposal, we have designed a program aimed at distinguishing the two mechanisms and extending our knowledge of this novel chemistry. The experiments fall into five areas: (1) further spectroscopic characterization of resting states of the enzyme and selected mutants aimed at providing a comprehensive tool box of spectral signatures capable of distinguishing each copper in either oxidation state; (2) determining the site of substrate binding by sophisticated spectroscopic approaches; (3) investigating the reactivity of the resting enzyme/mutants towards hydrogen peroxide, superoxide, and nitric oxide, with particular emphasis on determining the site of reactivity; (4) time-resolved measurements of the reaction of reduced enzyme with dioxygen, using rapid spectral detection of intermediates; and (5) comparing and contrasting the PHM results with the homologous DBM enzyme. The extensive structure/function information gained from these studies will assist in assessing these enzymes as therapeutic targets.
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