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 enz yme. A P450 reductase domain is tethered to an oxygenase domain containing the heme, the substrate (L-arginine) binding site, and a cofactor, tetrahyd robiopterin (BH4). This "triad", located at the distal heme pocket, is the center of oxygen activation and enzyme catalysis. To probe the relationship s among these three components, we examined the binding kinetics of three d ifferent small heme ligands in the presence and absence of either L-arginin e, BH4, or both. Imidazole binding was strictly competitive with L-arginine , indicating a domain overlap. BH4 had no obvious effect on imidazole bindi ng but slightly increased the k(on) for L-arginine. L-Arginine decreased th e k(on) and k(off) for cyanide by two orders, indicating a "kinetic obstruc tion" mechanism. BH4 slightly enhanced cyanide binding. Nitric oxide (NO) b inding kinetics were more complex. Increasing the L-arginine concentration decreased the NO binding affinity at equilibrium. In both BH4-abundant and BH4-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.