Nitric oxide regulates cytochrome P450 2D6 and 3A4 activity via concentration-dependent modulation of heme loading

Cytochrome P450 enzymes (CYPs) play diverse roles in human health and disease, and although their activities depend on their heme contents, the cellular mechanisms governing CYP heme levels are unclear. Because CYP activities are influenced by biological nitric oxide (NO), we investigated how a range of NO exposures would impact the heme levels and activities of CYP2D6 and 3A4 expressed in Chinese hamster ovary cells and in the human liver cell line HepG2. Following expression, both CYPs were present as a 60:40 mix of heme-free and heme-bound forms. A low range of NO concentrations (approximately 1-10 nM) generated in cultures by a chemical NO donor or by added activated macrophages caused cells to allocate heme into their heme-free CYP3A4 and 2D6 populations such that the levels of heme-replete and active CYPs increased by twofold to threefold. NO concentrations above this range (approximately 25-100 nM) gradually lost the positive effect and at the higher level caused heme loss from the CYPs and corresponding losses in activity. The positive or negative effects of NO began within the first 2 h of exposure and were completed within 6 h. The NO-driven increase in CYP heme content relied on a GAPDH-heme complex forming and chaperone heat shock protein 90 activity in the cells. Thus, NO can upregulate or downregulate cellular CYP3A4 and 2D6 activities by exerting a concentration-dependent change in their heme contents. These findings may help explain how NO generation in disease or inflammation can change CYP activities and impact drug pharmacokinetics and the generation of immune-active metabolites.