Nitric oxide synthase (NOS) is a complex enzyme responsible for the synthesis of nitric oxide (NO), a key signaling molecule. The enzyme contains several tightly bound redox cofactors, including a cytochrome P-450-type heme, and is structurally and functionally related to NADPH cytochrome P-450 reductase. NOS has three isoforms: constitutive (Ca²+/CaM-dependent) and inducible (iNOS), which do not require Ca²+/CaM. The constitutive isoforms require Ca²+ and calmodulin (CaM) for activity, while iNOS has CaM as a tightly bound subunit. NOS also contains a reduced pterin, which is essential for its function. The reaction catalyzed by NOS involves the oxidation of L-arginine to produce NO and citrulline, with molecular oxygen and NADPH as substrates. The enzyme's reaction mechanism involves a heme ferric peroxide intermediate and the reduction of ferrous oxy-heme to form a radical species that leads to NO production. NOS is structurally distinct from the P-450 superfamily, despite sharing a heme chromophore, and has evolved to use this chromophore for its unique chemistry. The NOS isoforms are closely related proteins, with constitutive isoforms being Ca²+/CaM-dependent and inducible isoforms being expressed in response to stimuli. The constitutive isoforms are cytosolic, while the inducible isoform is cytosolic and dimeric. The interaction of CaM with NOS is crucial for electron transfer and catalysis, with constitutive isoforms showing a strong dependence on Ca²+/CaM. The potential for end product inhibition by NO is still under investigation, as NO can form stable complexes with heme, but the stability of these complexes in the presence of oxygen is a concern. Overall, NOS has evolved to efficiently synthesize NO, a key signaling molecule, using parts of previously characterized proteins. The enzyme's structure and function are closely related, and further studies are needed to fully understand its mechanisms.Nitric oxide synthase (NOS) is a complex enzyme responsible for the synthesis of nitric oxide (NO), a key signaling molecule. The enzyme contains several tightly bound redox cofactors, including a cytochrome P-450-type heme, and is structurally and functionally related to NADPH cytochrome P-450 reductase. NOS has three isoforms: constitutive (Ca²+/CaM-dependent) and inducible (iNOS), which do not require Ca²+/CaM. The constitutive isoforms require Ca²+ and calmodulin (CaM) for activity, while iNOS has CaM as a tightly bound subunit. NOS also contains a reduced pterin, which is essential for its function. The reaction catalyzed by NOS involves the oxidation of L-arginine to produce NO and citrulline, with molecular oxygen and NADPH as substrates. The enzyme's reaction mechanism involves a heme ferric peroxide intermediate and the reduction of ferrous oxy-heme to form a radical species that leads to NO production. NOS is structurally distinct from the P-450 superfamily, despite sharing a heme chromophore, and has evolved to use this chromophore for its unique chemistry. The NOS isoforms are closely related proteins, with constitutive isoforms being Ca²+/CaM-dependent and inducible isoforms being expressed in response to stimuli. The constitutive isoforms are cytosolic, while the inducible isoform is cytosolic and dimeric. The interaction of CaM with NOS is crucial for electron transfer and catalysis, with constitutive isoforms showing a strong dependence on Ca²+/CaM. The potential for end product inhibition by NO is still under investigation, as NO can form stable complexes with heme, but the stability of these complexes in the presence of oxygen is a concern. Overall, NOS has evolved to efficiently synthesize NO, a key signaling molecule, using parts of previously characterized proteins. The enzyme's structure and function are closely related, and further studies are needed to fully understand its mechanisms.