The metalloenzyme nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its biological activity. NO has a dual role as 1) a diffusible biological messenger for neurotransmission, long-term potentiation, platelet aggregation, and blood pressure regulation and 2) a cytotoxic agent for defense against tumor cells and intracellular parasites. NOS enzymes (NOSs), found in inducible (iNOS), constitutive endothelial (eNOS), and constitutive neuronal (nNOS) isoforms, achieve their important biological function by adopting an intriguing calcium-regulated catalytic mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin (H4B), FMN, FAD and NADPH. NOSs generate NO by expending molecular oxygen and NADPH in each step of a two-step mechanism: first, a monooxygenase-like reaction converts L-arginine to the intermediate N4-hydroxy-L- arginine (NOH-arg), then an unprecedented reaction converts NOH-arg to citrulline and NO. To understand in atomic detail the unique structural metallobiochemistry of NOSs, integrated crystallographic and biochemical studies are proposed for the three major classes of NOS enzymes. Each NOS subunit is divided into two domains joined by a calmodulin-binding hinge region: an oxygenase domain with heme, H4B, and L-arginine binding sites forming the catalytic center for NO production, and a reductase domain with NADPH, FAD, and FMN binding sites supplying electrons to the heme. Electron transfer from the flavins to the heme is controlled by calmodulin (CaM), which fulfills a novel role for a calcium binding protein. The proposed studies first aim to characterize the structural biochemistry for individual oxygenase and reductase domains, which have been overexpressed and crystallized, and then to use these results to determine structures of full-length NOSs and of different analogous isozyme domains. Such parallel structure-function studies will establish common features and variations among these three classes of NOSs. For each NOS domain or isozyme, the proposed work couples expression, purification, and biochemical characterization in the Stuehr laboratory with crystallographic structure determination and analysis in the Getzoff and Tainer laboratories. The results from these coupled molecular biological, spectroscopic, biochemical, and crystallographic experiments will guide the design of site-directed mutants and the selection of appropriate cofactor, substrate, intermediate and inhibitor complexes for further research. This integrated, recursive approach aims to increase understanding of NOS catalysis and ultimately to aid the design of isozyme-selective NOS inhibitors, which will be invaluable tools for discovering isoform functions in vivo and are desirable as therapeutic agents for controlling blood pressure, septic shock, and inflammatory damage.
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