Electron transfer and catalytic activity of nitric oxide synthases. Chimeric constructs of the neuronal, inducible, and endothelial isoforms.
The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of .NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of .NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and L-arginine on electron transfer rates are mediated exclusively by heme domain interactions.
Nishida
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CR
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Ortiz de Montellano
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PR
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D019281:Dimerization; D004533:Egtazic Acid / Q000494:pharmacology; D004579:Electron Transport / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D003574:Cytochrome c Group; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001708:Biopterins; D009569:Nitric Oxide; D004533:Egtazic Acid; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat; C003402:sapropterin