Three unique flavoproteins involved in the metabolism of molecular oxygen in the streptococci have been targeted for this study. The long-term objective of this project is to combine studies of the catalytic roles of flavin and non-flavin oxidation-reduction centers with probes of specific interactions between the flavin cofactor and the protein environment. There are two major questions concerning the mechanism of the FAD-containing NADH peroxidase, which catalyzes the reduction of hydrogen peroxide to water. The possible participation of an active-site protein disulfide in the oxidation-reduction reaction catalyzed by this enzyme will be tested by spectrophotometric reductive titration methods and by chemical modification. Steady-state and stopped-flow kinetic methods will be used to evaluate the catalytic role of the two-electron reduced enzyme. The four-electron transferring NADH oxidase represents a second example of interacting oxidation-reduction sites within a common catalytic center. Static spectral titration methods and atomic absorption spectroscopy will be employed to identify these sites. In addition, correlations of cysteine content and enzyme activity will be combined with analytical ultracentrifugation techniques to define the role of exogenous thiols in the reconstitution of the deflavo oxidase. The FAD-containing glycerol-3-phosphate oxidase is unusual in that the oxidation of this substrate is generally coupled to the reduction of NAD+, not of molecular oxygen. The question of whether the native enzyme exhibits the active-site characteristics of the typical flavoprotein oxidases will be tested by anaerobic photoreductions and by titrations with sulfite. In addition, the apoenzyme will be reconstituted with artificial FAD analogs as probes of the interaction between protein environment and bound flavin. The energy-yielding metabolic processes of the streptococci, which lack cytochromes and hemes, have been largely ignored. The unique flavoproteins targeted in this study represent special adaptations of flavin-mediated oxidation-reduction function which allow these organisms a beneficial respiratory activity.
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