Characterization of the Mechanism of Cytochrome P450 Reductase-Cytochrome P450-mediated Nitric Oxide and Nitrosothiol Generation from Organic Nitrates

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2-. The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2- generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2--dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed