The nitric oxide synthases (NOS) are hemoproteins with a cytochrome P450-like active site that catalyze the oxidation of arginine to nitric oxide and citrulline. The explosion of research in this area has demonstrated that nitric oxide fulfills a large range of biological functions as both a messenger and a cytotoxic factor. There are three basic types of nitric oxide synthases: (a) a soluble constitutive enzyme found in high concentrations in the brain, (b) a constitutive endothelial enzyme that is membrane bound, and (c) an inducible enzyme associated with the cytotoxic function of macrophages. The nitric oxide synthases incorporate several domains in a single polypeptide: (a) the P450-like heme domain that binds a tetrahydrobiopterin cofactor and is the site of oxidation of arginine to nitric oxide and citrulline, (b) a P450-reductase domain that has binding sites for FMN, FAD, and NADPH and is responsible for providing electrons to the heme domain, and (c) a connecting peptide between the heme and flavin domains that binds calmodulin in a Ca2+-dependent manner for the endothelial (eNOS) and neuronal (nNOS) forms and essentially Ca2+-independent manner in the inducible enzyme (iNOS).

The focus of our work over the given period has been on the factors that control the electron transfer chain in the nitric oxide synthases. The electron transfer takes electrons provided by NADPH sequentially through the FAD and FMN cofactors to the heme and the tetrahydrobiopterin cofactors. We have established that the maximum rate of turnover of the isoforms is linked to the maximum ability of the flavin domain to deliver electrons. Thus, the reductase domain of eNOS delivers electrons more slowly than that of nNOS or iNOS, and the maximum rate of synthesis of NO by nNOS and iNOS is much slower. We have specifically shown that the rate of electron transfer is controlled, at least in part, by the presence of an insert in the eNOS flavin domain. This insert is also present in nNOS and is partially responsible for the Ca2+ dependence of eNOS and nNOS, but is not present in iNOS, which binds calmodulin independently of the Ca2+ concentration. Interestingly, the electrons from the reductase domains can be diverted to the reduction of exogenous substrates and have potential in the reductive activation of quinone anticancer agents.

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