Neuronal &Endothelial NO synthase enzymes (nNOS &eNOS) make NO in response to calmodulin (CaM) binding and function broadly in human health and disease. Our long-term goal is to understand the molecular mechanisms that regulate NOS catalysis. NOS contain connected NADPH-FAD (FNR), FMN, and NOSoxy domains that transfer NADPH-derived electrons to the heme and tetrahydrobiopterin (H4B) groups in NOSoxy, thus allowing O2 activation required for NO synthesis. We believe that NOS conformational states and domain motions control its electron transfer reactions. The FMN domain plays a key role by performing separate electron acceptor and donor functions to shuttle electrons through NOS. This creates a three-state, two- equilibrium model that involves separate FMN domain interactions with the FNR (KA) and with the NOSoxy (KB). We hypothesize that structural &conformational features that are common to di-flavin oxidoreductases blend with those unique to NOS enzymes to create novel mechanisms of regulation. How the elements function at the molecular level to regulate NOS conformational behaviors and synchronize electron transfer reactions is a central focus of this proposal. During the current funding period we developed methods to: a) Study KA &KB in nNOS &eNOS b) Simulate their different rates of conformational change &electron flux, and c) Characterize several common &unique structural elements that control their electron transfers. Despite significant progress, connections between NOS conformational states &motions and the electron transfer reactions remain largely unexplored.
Our Aims address this gap through connected biophysical, biochemical, kinetic, and molecular engineering approaches, to achieve a molecular-level understanding of NOS regulation.
Aim 1. Define conformational KA, its control mechanisms, and its role in determining electron flux through nNOSr &eNOSr. We will: Utilize our newly-developed Cys-lite, FRET, &domain locking approaches to: (i) define conformational distributions &populations associated with KA, &test hypotheses regarding control by the flavin reduction state &CaM binding. (ii) Define how the common &unique control elements in NOS orchestrate KA and FMN domain motions to uncover molecular mechanisms regulating electron flux.
Aim 2. What mechanisms govern KB &the associated FMN to NOSoxy electron transfer? We will use our Cys-lite, FRET, &domain locking approaches to: (i) define conformational states and distributions associated with KB that underpin FMN to heme electron transfer, (ii) Determine how conformational properties are influenced by the flavin &heme redox state, CaM binding, and control elements that regulate heme reduction, and (iii) Test whether these same control mechanisms regulate electron transfer to H4B in nNOS. Relevance: By clarifying how NO production is regulated at the enzyme level our work may help develop treatments for human diseases that involve making too much or too little NO, and will illuminate protein structure-function relationships among this important class of redox proteins.

Public Health Relevance

Nitric oxide (NO) is a natural signal molecule made by cells and has many important roles in human health and disease. NO synthase enzymes (NOS) generate NO throughout the body. We are studying NOS enzymes to find out how they control their NO production. By clarifying how their NO production is regulated at the enzyme level, our work may help develop treatments for human diseases that involve making too much or too little NO.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Cleveland Clinic Lerner
Other Basic Sciences
Schools of Medicine
United States
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Rwere, Freeborn; Xia, Chuanwu; Im, Sangchoul et al. (2016) Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone. J Biol Chem 291:14639-61
Haque, Mohammad Mahfuzul; Ray, Sougata Sinha; Stuehr, Dennis J (2016) Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics. J Biol Chem 291:23047-23057
Sarkar, Anindya; Dai, Yue; Haque, Mohammad Mahfuzul et al. (2015) Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMplications for Enzyme Maturation. J Biol Chem 290:21615-28
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He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J et al. (2015) Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics. Proc Natl Acad Sci U S A 112:11835-40
Haque, Mohammad M; Bayachou, Mekki; Tejero, Jesus et al. (2014) Distinct conformational behaviors of four mammalian dual-flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles. FEBS J 281:5325-40
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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
Sabat, Joseph; Egawa, Tsuyoshi; Lu, Changyuan et al. (2013) Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation. J Biol Chem 288:6095-106
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

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