Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. Our long-term goal is to determine the molecular mechanism of NOS enzyme catalysis, including how the interdomain interactions modulate the catalytically relevant IET processes and the NOS enzyme function. In this project we will focus on investigating important roles of the FMN domain docking to the heme domain in regulating the NOS isoform function. An exciting recent development in the NOS enzymes is the discovery of importance of the interdomain FMN-heme interactions for modulating reactivity and structure of the catalytic heme active site. The recent findings raise two new important mechanistic questions that must be addressed to understand NOS regulation mechanism: 1) what specific interdomain interactions govern and facilitate the productive docking of the FMN domain to the heme domain? 2) how can the FMN domain docking, in addition to controlling the catalytically essential FMN-heme IET, modulate reactivity of the heme active site? Our two synergistic and complementary Aims are designed to directly address these important questions. We will employ an integrated program of pulsed electron paramagnetic resonance, laser flash photolysis and mutational studies. In the absence of a structure of full-length mammalian NOS, the combined approach is highly appropriate for the proposed study. This project seeks to address fundamental questions relating to the NOS enzymes. Our study will identify key structural determinants in controlling NOS isoform activity by modulating the alignment of the FMN and heme domains. The new information could facilitate bio-rational development of new selective activators or inhibitors for these clinically important enzymes, in order to provide better therapeutic interventions. In accordance with the guideline of the R15 program we will continue to train our 21st century biomedical scientists by this multi-disciplinary project.

Public Health Relevance

The NOS family is a key target for development of new pharmaceuticals for a wide range of diseases that currently lack effective treatments, including stroke and cancer. However, due to an incomplete understanding of the molecular mechanisms of NOS regulation, NOS inhibitors have not yet been available for clinical treatments. The proposed studies will significantly improve the fundamental understanding of NOS isoform regulation, and could facilitate bio-rational development of new selective mechanism-based drug entities to target these clinically important enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM081811-02A1
Application #
8232160
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2007-08-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2012
Total Cost
$332,200
Indirect Cost
$112,200
Name
University of New Mexico Health Sciences Center
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
829868723
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Astashkin, Andrei V; Chen, Li; Zhou, Xixi et al. (2014) Pulsed electron paramagnetic resonance study of domain docking in neuronal nitric oxide synthase: the calmodulin and output state perspective. J Phys Chem A 118:6864-72
Feng, Changjian; Chen, Li; Li, Wenbing et al. (2014) Dissecting regulation mechanism of the FMN to heme interdomain electron transfer in nitric oxide synthases. J Inorg Biochem 130:130-40
Li, Wenbing; Chen, Li; Lu, Changyuan et al. (2013) Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase. Inorg Chem 52:4795-801
Li, Wenbing; Fan, Weihong; Chen, Li et al. (2012) Role of an isoform-specific serine residue in FMN-heme electron transfer in inducible nitric oxide synthase. J Biol Inorg Chem 17:675-85
Feng, Changjian (2012) Mechanism of Nitric Oxide Synthase Regulation: Electron Transfer and Interdomain Interactions. Coord Chem Rev 256:393-411
Li, Wenbing; Chen, Li; Fan, Weihong et al. (2012) Comparing the temperature dependence of FMN to heme electron transfer in full length and truncated inducible nitric oxide synthase proteins. FEBS Lett 586:159-62
Li, Wenbing; Fan, Weihong; Elmore, Bradley O et al. (2011) Effect of solution viscosity on intraprotein electron transfer between the FMN and heme domains in inducible nitric oxide synthase. FEBS Lett 585:2622-6
Feng, Changjian; Fan, Weihong; Ghosh, Dipak K et al. (2011) Role of an isoform-specific substrate access channel residue in CO ligand accessibilities of neuronal and inducible nitric oxide synthase isoforms. Biochim Biophys Acta 1814:405-8
Astashkin, Andrei V; Fan, Weihong; Elmore, Bradley O et al. (2011) Pulsed ENDOR determination of relative orientation of g-frame and molecular frame of imidazole-coordinated heme center of iNOS. J Phys Chem A 115:10345-52
Sempombe, Joseph; Galinato, Mary Grace I; Elmore, Bradley O et al. (2011) Mutation in the flavin mononucleotide domain modulates magnetic circular dichroism spectra of the iNOS ferric cyano complex in a substrate-specific manner. Inorg Chem 50:6859-61

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