62 - Generation of superoxide radical by human cytochrome b5 reductase is modulated by cytochrome b5

Abstract

Cytochrome b5 reductase (CYB5R) plays a central role in many diverse metabolic reactions by coupling NAD(P)H oxidation to reduction of proteins, such as cytochrome b5 (CYB5). Together, CYB5R and CYB5 transfer electrons to terminal oxidases, such as the mitochondrial amidoxime reducing component (mARC) or hemoglobin. Previous studies have demonstrated that nitric oxide (NO) production by the mARC/CYB5/CYB5R system is quenched by molecular oxygen (O2) and suggested that superoxide radical production was involved, as the addition of Cu,Zn-superoxide(SOD1) dismutase partially recovered NO production. Flavin proteins are known to generate superoxide, suggesting that the CYB5R was the likely site of superoxide radical production. However, the direct formation of superoxide by human CYB5R had not been studied. Here, studies were performed to understand the related mechanisms of radical generation by CYB5R in the absence and presence of CYB5. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with CYB5R and NADH. An EPR spectrum corresponding to the superoxide radical adduct of DMPO (DMPO-OOH) was obtained. This EPR signal was quenched by the addition of the SOD1. The amount of DMPO-OOH formed from the oxidation of NADH by the CYB5R increased with NADH concentration. Importantly, the formation of DMPO-OOH was abrogated in the presence of CYB5. Spectral kinetics demonstrate that NADH consumption is O2-dependent, as no NADH consumption was observed in the absence of O2. From these results, we propose a mechanism in which NADH-reduced CYB5R catalyzes superoxide radical formation. Our results demonstrate that human CYB5R catalyzes the O2-dependent oxidation of NADH to generate superoxide radical. Additionally, CYB5 may protect biological targets from oxidative damage, by inhibiting CYB5R superoxide radical production. The ability of CYB5R to catalyze superoxide radical production in response to O2 has potential pathophysiological implications for the ischemia/reperfusion induced damages, endothelial dysfunction, and cardiovascular and neurological diseases.