The long-term objectives of this study are to understand the mechanisms by which three unique streptococcal flavoproteins catalyze reactions normally ascribed to hemeproteins or to heme- dependent processes. Investigation of the catalytic roles of both flavin and non-flavin redox centers will be essential to the overall evaluation of the key structure-function relationships operative in the three enzymes, which are important in streptococcal oxygen metabolism. Recombinant DNA approaches will be applied to the study of the FAD- containing NADH peroxidase: the specific questions to be addressed include the cysteinyl peptide sequences of the proposed redox- active disulfide and the overall relatedness between the peroxidase and the flavoprotein disulfide reductases. In the context of our mechanistic studies, the roles of two distinct complexes of the reduced peroxidase with pyridine nucleotides in both peroxide reduction and in transhydrogenation will be investigated by static titration and rapid-reaction techniques, as will the specific role of cysteine thiol(s) in peroxide reduction. Studies of the unusual FAD-containing NADH oxidase will focus on the identification of the second non-flavin redox center in this enzyme and on the interaction of this center with the flavin in the overall four-electron reduction of oxygen. The possible role of oxygenated flavin intermediates in the catalytic mechanism will also be investigated by stopped-flow methods and will include studies of the enzyme reconstituted with modified FAD analogs. The possible structural and functional relationship of the NADH oxidase and the NADH peroxidase will be examined by protein chemistry and molecular genetic approaches. There are two major questions concerning the streptococcal alpha- glycerophosphate oxidase. The distinction between hydride- and carbanion-mediated substrate dehydrogenations is an important one, and halogenated analogs of the natural substrate will be used to probe this question in more detail. Secondly, the comparison between this flavoprotein and the FAD-containing anaerobic alpha- glycerophosphate dehydrogenase of E. coli should provide valuable information concerning structural and mechanistic features shared by both.
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