The mechanisms of nitric oxyde synthesis by nitric oxide synthases

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Nitric oxide synthases (NOSs) synthesize the signaling and oxidative stress molecule nitric oxide (NO). The long-term objectives of this research program are to determine the structural, functional and dynamic properties of the active site of NOSs to understand the catalytic cycle and mechanisms of regulation of the enzymatic activity at the molecular level. The NO synthesized by NOSs is used as a signaling molecule in the vascular and neural systems. It is also a toxic molecule used by the immune system in host defenses against pathogens and cancer cells. The involvement of NO in many aspects of human physiology and diseases, including atherosclerosis, Alzheimer's and Parkinson's, means that isoform-specific inhibitors are needed to regulate neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). We expect that gaining a thorough understanding of the catalytic mechanisms of NOSs, including providing a detailed picture of heme-ligand substrate interactions and identifying key kinetic intermediates, will improve our comprehension of the chemistry of NOSs and, in the long term, will help in the development of potent, isoform-specific inhibitors of human NOSs. The specific objectives of this proposal thus are: A-Characterize by resonance Raman spectroscopy the first oxygenated intermediate formed during the catalytic cycle of NOSs using non-natural substrates and characterize the oxygenated complex of active site mutants. These studies will help determine the importance in the catalytic cycle of polar and steric interactions between substrates and the heme-bound dioxygen at the active site and will also help decipher the role played by the proximal cysteine ligated to the heme. B- Characterize the oxygenated form of NOS mutants to determine the involvement of protein dynamics in the catalytic cycle. C-Characterize catalytic intermediates formed by the reaction with peroxide. The latter two studies aim to get a better understanding of the mechanisms of NOS synthesis by NOSs. D-Characterize the interactions between heme-bound ligands (O2, NO and CO) and NOS inhibitors with all three mammalian NOSs to decipher the structural basis for inhibitor binding and isoform specificity. Together, these studies will provide an understanding at the molecular level of the mechanism of NO synthesis, identify important factors modulating the enzymatic activity and identify structural determinants for isoform-specificity of NOSs.