Characterization of interactions among the heme center, tetrahydrobiopterin, and L-arginine binding sites of ferric eNOS using imidazole, cyanide, and nitric oxide as probes.
Endothelial nitric oxide synthase (eNOS) is a self-sufficient P450-like enzyme. A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahydrobiopterin (BH(4)). This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis. To probe the relationships among these three components, we examined the binding kinetics of three different small heme ligands in the presence and absence of either L-arginine, BH(4), or both. Imidazole binding was strictly competitive with L-arginine, indicating a domain overlap. BH(4) had no obvious effect on imidazole binding but slightly increased the k(on) for L-arginine. L-Arginine decreased the k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruction" mechanism. BH(4) slightly enhanced cyanide binding. Nitric oxide (NO) binding kinetics were more complex. Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium. In both BH(4)-abundant and BH(4)-deficient eNOS, half of the NO binding sites showed a sizable decrease of the binding rate by L-arginine, with the rate of NO binding at the other half of the sites remaining essentially unaltered by L-arginine, implying that the two heme centers in the eNOS dimer are functionally distinct.
Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, 77030, USA.
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D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001667:Binding, Competitive; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry*; D002384:Catalysis; D002417:Cattle; D003198:Computer Simulation; D003486:Cyanides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry*; D005415:Flavins / Q000737:chemistry; D005822:Genetic Vectors / Q000138:chemical synthesis; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D008024:Ligands; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding
D003486:Cyanides; D005290:Ferric Compounds; D005415:Flavins; D007093:Imidazoles; D008024:Ligands; D001708:Biopterins; D009569:Nitric Oxide; D006418:Heme; C029899:imidazole; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin