Nitric oxide synthases (NOSs) are heme-and flavin-containing enzymes that catalyze the synthesis of NO through two serial monooxygenase reactions analogous to those of the NADPH-dependent cytochrome P450 oxidoreductase (CYPOR) systems. Electrons are transferred from NADPH, through the flavins FAD and FMN, to the heme iron, where molecular oxygen is bound and activated. All NOSs share 50-60% overall amino acid sequence homology1 and have similar cofactor requirements. The NOSs are functional dimers, with each monomer containing an N-terminal oxygenase domain with binding sites for arginine, tetrahydrobiopterin (H4B), and a tetracoordinated zinc atom, and a reductase domain with an autoinhibitory region and binding sites for FMN, FAD, and NADPH connected by a linker containing a Ca2+/calmodulin (CaM) binding site. nNOS and eNOS are directly activated by agonist-induced elevation of intracellular Ca2+, binding of Ca2+ to CaM, and subsequent binding of CaM to NOS. The constitutive NOS isoforms are also indirectly regulated by H4B synthesis and by other proteins through direct binding to NOS, subcellular localization, and phosphorylation in neurons, skeletal muscle (nNOS), or endothelial cells (eNOS), whereas iNOS is transcriptionally activated by endotoxins and cytokines in macrophages, hepatocytes, and vascular smooth muscle cells.

This review, which is not intended to be comprehensive, is focused on the modes of regulation of the isoforms of NOS. As the structures of these enzymes have been revealed, albeit piecemeal in the form of domains of the intact proteins, and as the biochemistry and physiology of the NOS isoforms have revealed various interesting differences among them, it has become obvious that several regulatory mechanisms must be operating. The elucidation of the three-dimensional structures of all three NOS isoform heme domain dimers by several laboratories2-6 has presented meager insight into the differences in electron-transport capacities among them. This review will focus attention on the many differences in sequence and thus, potentially, structure and function demonstrated by our laboratory and others among the flavoprotein domains of the NOSs. While it would seem logical that an obvious control mechanism for an enzyme involved in oxygenation reactions would involve electrontransfer processes, only recently have experimental approaches been directed to the flavin-mediated enzymatic reactions.