Nitric oxide synthase is a cytochrome P-450 type hemoprotein.
Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.
White
K A
KA
Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor 48109-1065.
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002248:Carbon Monoxide / Q000494:pharmacology; D002531:Cerebellum / Q000201:enzymology; D002956:Citrulline / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006420:Hemeproteins / Q000302:isolation & purification / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D019001:Nitric Oxide Synthase; D013053:Spectrophotometry
D006420:Hemeproteins; D002956:Citrulline; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases