Neuronal nitric oxide synthase catalyzes the reduction of 7-ethoxyresorufin

Nitric oxide synthase (NOS: EC 1.14.13.39) catalyzes L-arginine oxidation to generate nitric oxide (NO) and L-citrulline. Recently, 7-ethoxyresorufin (7-ER), a specific substrate of cytochrome P-4501A1, was used as a cytochrome P-450 inhibitor to study the mechanism underlying the vasodilatation caused by some drugs, and was suggested to inhibit nitric oxide-mediated relaxation. Herein we demonstrate that 7-ER inhibits NO synthesis by uncoupling neuronal nitric oxide synthase (nNOS). 7-ER is a noncompetitive inhibitor of nNOS with respect to L-arginine with a Ki value of 0.76 +/- 0.06 microM. The decrease in NO formation is inversely correlated with an increase in NADPH oxidation. 7-ER binds to nNOS with a Km value of 0.68 +/- 0.07 microM, as calculated from the nNOS-dependent NADPH oxidation in the absence of L-arginine. nNOS catalyzes the reduction of 7-ER at the expense of NADPH. The flavoprotein inhibitor, diphenyleneiodonium chloride (100 microM), completely inhibited nNOS-dependent 7-ER reduction. While nitro-L-arginine (1 mM) and N(G)-nitro-L-arginine methyl ester (1 mM), specific inhibitors of nNOS, and phenylisocyanide (0.1 mM), a specific heme iron ligand, did not affect the reduction of 7-ER. These results indicate that the reductase domain, but not the oxygenase domain, of nNOS is involved in the reduction of 7-ER. 7-ER uncouples nNOS, shunting electrons from the reductase domain to the oxygenase domain of the enzyme. As a consequence, NO synthesis is inhibited.