Nitric oxide synthases (NOS) are haem-containing oxidoreductases and exist as homodimers in three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). They produce NO through a 5-step oxidation of its substrate L-arginine to L-citrulline, using molecular oxygen. The C-terminal reductase domain of NOS contains binding sites for FAD, FMN and the electron donor, NADPH (Fig 3). The N-terminal oxygenase domain contains the haem group and binding sites for the substrate, arginine and the cofactor, BH4. The reductase domain generates electron flow from NADPH through the flavins FAD and FMN and then transfers the electrons to the oxygenase domain of the other monomer, where L-arginine oxidation occurs at the haem group in the active site .
In the vasculature, eNOS is the main NOS isoform, constitutively expressed in normal endothelium, while iNOS expression is normally low but may be increased in certain disease states, such as atherosclerosis . eNOS is localized to invaginations of the plasma membrane called caveolae. Its activity is regulated through multiple integrated pathways, including activation by calcium-camodulin, membrane localization in caveolae through lipid modifications, protein-protein interactions with caveolin and hsp90, phosphorylation at key serine and threonine residues, and subcellular trafficking between caveolae and cytosol .
BH4 is an essential cofactor for all 3 NOS isoforms. BH4 binds close to the haem active site at the interface between the two monomers (Fig 3). In vitro experiments have shown an important role of BH4 in maintaining and stabilizing the active dimeric form of NOS [9,68]. BH4 also participates in the transfer of electrons to L-arginine, serving as an electron donor to the haem group [67,75]. Biochemical and cellular studies have demonstrated that when BH4 levels are reduced or absent, eNOS dimerization is destabilized, leading to a reduction in the relative proportion of eNOS dimers versus monomers in the cell. eNOS catalytic activity also becomes 'uncoupled'. The stoichiometric coupling between the reductase domain and the L-arginine oxidation site is lost. The electron transfer from NADPH through flavins to molecular oxygen results in the formation of superoxide and/or hydrogen peroxide [12,80]. Besides BH4, oxidation status of the Zn 2+-thiolate centre  and substrate L-arginine availability are also involved in the NOS uncoupling.
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