This proposal emanates from the interests of the Principal Investigator in flavoproteins containing both FAD and FMN as prosthetic groups, extending from her early studies on NADPI-I-cytochrome P450 reductase (CYPOR) to the three nitric oxide synthases (NOS). Since both enzymes employ these flavin cofactors as conduits of electron transfer from NADPH to the heme moiety responsible for the specific oxygenation reaction being catalyzed, it is hypothesized that they utilize similar chemical but different regulatory mechanisms to achieve diverse biological outcomes. In cytochrome P450 systems, the separate flavin (CYPOR) and heme- (cytochrome P450) containing proteins are membrane-bound but in NOS isoforms, FAD, FMN, heme, and tetrahydrobiopterin are bound to the same polypeptide chain. It is proposed that CYPOR can serve as a model for NOS flavoprotein catalysis. In seeking to understand more fully the influence of structure, determined for CYPOR and the FAD/NADPH-binding domain of nNOS from studies with Dr. Jung-Ja Kim (Medical College of Wisconsin), on the function of these diverse enzymes, three Specific Aims are planned: 1) Dissection of Electron transport activities catalyzed by either holoenzymes or functional domains of CYPOR and NOS isoforms to determine the necessary sequences/structures for various functions; 2) Determination of changes in oxidation-reduction behavior within the proteins as a function of activation or regulation by intramolecular and intermolecular interactions; and 3) Probing of the flavoprotein domains of CYPOR and the NOS isoforms using chemical derivatives of diphenyleneiodonium designed to act as competitive, allosteric, or covalently-linked inhibitors. With the various inhibitors, as well as the different modular or chimeric constructs, the quest for structural information will continue in collaboration with Dr. Jung-Ja P. Kim, Medical College of Wisconsin. Techniques will include state and stopped-flow spectrophotometry, surface plasmon resonance, x-ray crystallography, redox potential studies, and NMR to examine mechanism. Upon determination of the unique structural features in the flavoprotein domains of constitutive isoforms in comparison with inducible NOS and CYPOR, a basis for drug design could develop.
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