Calmodulin-dependent nitric-oxide synthase. Mechanism of inhibition by imidazole and phenylimidazoles

Abstract

Calmodulin-dependent nitric-oxide synthase from bovine brain and GH3 pituitary cells is inhibited by imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole, with half-maximal inhibition occurring at 200, 25, 160, and 600 microM concentrations of inhibitor, respectively. Imidazole inhibits the maximal velocity of citrulline formation by the enzyme, but does not alter the concentration of arginine, calmodulin, or (6R)-5,6,7,8,-tetrahydro-L-biopterin required for expression of half-maximal activity. Imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole had no effect on calmodulin-dependent reduction of cytochrome c by the enzyme at concentrations up to 50-fold higher than those that inhibited citrulline formation. Imidazole inhibited calmodulin-dependent NADPH consumption by the enzyme with dissolved oxygen as the sole electron acceptor, with half-maximal inhibition occurring at a concentration of 225 microM. These observations are consistent with the proposal that imidazole and phenylimidazoles inhibit citrulline formation and oxygen reduction by acting as a sixth coordination ligand of the heme iron. This interaction prevents the formation of the activated reduced species of oxygen necessary for the formation of citrulline.