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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM051491-17A1
Application #
8577898
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1994-08-01
Project End
2017-04-30
Budget Start
2013-08-01
Budget End
2014-04-30
Support Year
17
Fiscal Year
2013
Total Cost
$451,292
Indirect Cost
$147,769
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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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; Tejero, Jesus; Bayachou, Mekki et al. (2013) Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis. FEBS J 280:4439-53
Wang, Zhi-Qiang; Tejero, Jesus; Wei, Chin-Chuan et al. (2012) Arg375 tunes tetrahydrobiopterin functions and modulates catalysis by inducible nitric oxide synthase. J Inorg Biochem 108:203-15
Tejero, Jesus; Biswas, Ashis; Haque, Mohammad Mahfuzul et al. (2011) Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase. Biochem J 433:163-74
Haque, Mohammad M; Kenney, Claire; Tejero, Jesus 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
Hannibal, Luciana; Somasundaram, Ramasamy; Tejero, Jesus et al. (2011) Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase. J Biol Chem 286:39224-35

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