MECHANISMS INVOLVED IN FLAVIN LINKED OXYGEN METABOLISM

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Presented with the biochemical dilemma of growing in an aerobic environment, but without the capacity to synthesize cytochromes or other hemeproteins, the enterococci depend on several very unusual flavoproteins; these enzymes play key roles both in oxygen-dependent energy metabolism and in the cellular defense against oxidative stress. The NADH oxidase (O2>H2O) and NADH peroxidase (H2 02>2H20) also represent a new type of flavoprotein in which the second non-flavin redox center has been identified as a stable cysteine-sulfenic acid (Cys-SOH). One goal of this study is to complete the crystallographic analysis of the native Cys-SOH and Cys-SH forms of the peroxidase at high resolution; information concerning the modes of Cys-SOH stabilization within the peroxidase can also be applied to a number of redox-regulated DNA-binding proteins, such as OxyR, for which Cys- SOH/Cys-SH redox centers have been proposed. Stopped-flow kinetic analyses of the NADH oxidase, focused on the mechanism of the four- electron transfer involved in the reduction of O2>2H2O, will be pursued; preliminary evidence indicates that a flavin-C(4a)- hydroperoxide species may be formed. Crystallographic studies of the Cys42Ser mutant oxidase will provide detailed information on the structural basis for the distinction between oxidase end peroxidase catalytic cycles in these two homologous proteins. In addition, this structural work will be expanded to include the NADH oxidases from Streptococcus pyogenes and S. pneumoniae, especially in view of recent genetic evidence that this enzyme may represent a promising target for structure-based drug design in these important human pathogens. The uptake and catabolism of glycerol in the enterococci requires the concerted actions of two cytosolic enzymes: glycerol kinase (GlpK) and the FAD-dependent alpha-glycerophosphate oxidase (GlpO). Sequence analysis of GlpO indicates surprisingly that this cytosolic enzyme is not related to other FAD-dependent oxidases (O2>H2O2) but to the membrane-associated alpha-glycerophosphate dehydrogenases (GlpDs); structural and kinetic studies of GlpO are planned which should also provide detailed insight regarding the determinants of both substrate binding and subcellular localization in the homologous GlpDs.