Mechanisms of Flavoproteins

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DESCRIPTION (provided by applicant): Flavoproteins are found throughout metabolism, catalyzing a variety of critical steps in the cell. Among the reactions which these enzymes carry out are many in which an unactivated carbon-hydrogen bond is cleaved. This is a fundamental reaction in biological systems, catalyzed by a variety of enzyme classes. The overall goal of the present proposal is to understand the mechanisms by which flavoproteins catalyze carbon-hydrogen bond cleavage reactions and to determine the extent to which different protein folds utilize the same mechanism to catalyze these reactions. The experiments focus on enzymes which oxidize bonds between carbon and either oxygen or nitrogen, transferring a hydride equivalent to the flavin cofactor. Enzymes representative of different classes of substrates have been selected for study: D-amino acid oxidase and tryptophan monooxygenase as D- and L-amino acid oxidases, flavocytochrome b2 as a hydroxy acid oxidizing enzyme, and nitroalkane oxidase, a novel enzyme which represents a unique opportunity to study carbanion formation by a flavoprotein. A variety of mechanistic experiments are proposed to elucidate the details of the carbon-hydrogen bond cleavage step by each enzyme. These are combined with mutational analyses designed to determine the extent to which the protein fold correlates with mechanism. The results of these studies will provide insight into the mechanisms of other flavoproteins, into the roles of the protein in directing the reactivity of the flavin cofactor and into the general problem of cleavage of carbon-hydrogen bonds by biological systems.