Structure/Mechanism of an FMN- and FAD-containing Enzyme

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DESCRIPTION (provided by applicant): NADPH-cytochrome P450 oxidoreductase (CYPOR) and nitric oxide synthase isoforms (NOSs) are mammalian enzymes that contain two flavins, FMN and FAD, and an NADPH-binding site. CYPOR catalyzes the transfer of reducing equivalents from NADPH to cytochromes P450 and is an essential component of the microsomal cytochrome P450 monooxygenase system. The system catalyzes the oxygenation of drugs, xenobiotics, and endogenous substrates, including steroids, lipids, and prostaglandins. Despite intensive studies over four decades to elucidate the mechanism and function of CYPOR and its interactions with P450s, gaps in our understanding still exist. During the last funding period, the principal investigator's laboratory determined the crystal structure of rat CYPOR, solubilized by limited trypsin treatment. Based on this structure, it is proposed to study: (1) mutants of CYPOR to define the role of specific residues in catalysis and (2) holo-CYPOR to determine the role of the membrane-binding domain in the interactions between CYPOR and P450 and (3) to initiate studies, by EPR spectroscopy, of possible structural rearrangement of the CYPOR molecule upon binding to P450s, its physiological electron-transfer partners. Three NOS isoforms, neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) catalyze the NADPH-dependent formation of nitric oxide (NO) and L-citrulline from L-arginine and molecular oxygen. NO is a mediator of neuronal signaling (nNOS), a cytotoxic agent (iNOS), and a vasodilator (eNOS), depending on enzyme source and tissue site of production. Both nNOS and eNOS are constitutively expressed and are activated by Ca++/CaM whereas iNOS is transcriptionally activated by cytokines and is active at normal Ca++ concentrations. Each isoform consists of a heme domain (N-terminus) with characteristics common to P450s, a flavin domain (C-terminus) homologous to CYPOR in both amino acid sequence and function, and a Ca++/CaM-binding region linking the two domains. Despite similar basic chemical mechanisms, overall reaction rates and modes of regulation of NO production differ significantly among the isoforms. To determine the structural basis for these differences, it is proposed to determine the crystal structures of (4) the flavin domains and (5) their variants of the three NOSs, containing their respective Ca++/CaM-binding regions.