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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Special Emphasis Panel (ZRG3-BMT (02))
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Scripps Research Institute
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Haque, Mohammad Mahfuzul; Bayachou, Mekki; Fadlalla, Mohammed A et al. (2013) Charge-pairing interactions control the conformational setpoint and motions of the FMN domain in neuronal nitric oxide synthase. Biochem J 450:607-17
Haque, Mohammad Mahfuzul; Tejero, Jesús; Bayachou, Mekki et al. (2013) Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis. FEBS J 280:4439-53
Nagpal, Latika; Haque, Mohammad M; Saha, Amit et al. (2013) Mechanism of inducible nitric-oxide synthase dimerization inhibition by novel pyrimidine imidazoles. J Biol Chem 288:19685-97
Haque, Mohammad Mahfuzul; Fadlalla, Mohammed A; Aulak, Kulwant S et al. (2012) Control of electron transfer and catalysis in neuronal nitric-oxide synthase (nNOS) by a hinge connecting its FMN and FAD-NADPH domains. J Biol Chem 287:30105-16
Haque, Mohammad M; Kenney, Claire; Tejero, Jesús et al. (2011) A kinetic model linking protein conformational motions, interflavin electron transfer and electron flux through a dual-flavin enzyme-simulating the reductase activity of the endothelial and neuronal nitric oxide synthase flavoprotein domains. FEBS J 278:4055-69
Rosenfeld, Robin J; Bonaventura, Joseph; Szymczyna, Blair R et al. (2010) Nitric-oxide synthase forms N-NO-pterin and S-NO-cys: implications for activity, allostery, and regulation. J Biol Chem 285:31581-9
Guan, Zhi-Wen; Haque, Mohammad Mahfuzul; Wei, Chin-Chuan et al. (2010) Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer. J Biol Chem 285:3064-75
Haque, Mohammad Mahfuzul; Fadlalla, Mohammed; Wang, Zhi-Qiang et al. (2009) Neutralizing a surface charge on the FMN subdomain increases the activity of neuronal nitric-oxide synthase by enhancing the oxygen reactivity of the enzyme heme-nitric oxide complex. J Biol Chem 284:19237-47
Garcin, Elsa D; Arvai, Andrew S; Rosenfeld, Robin J et al. (2008) Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nat Chem Biol 4:700-7
Wei, Chin-Chuan; Wang, Zhi-Qiang; Tejero, Jesus et al. (2008) Catalytic reduction of a tetrahydrobiopterin radical within nitric-oxide synthase. J Biol Chem 283:11734-42

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