Removal of H2O2 and generation of superoxide radical: Role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe(3+)cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H2O2) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe(3+)cyt c in the presence of H2O2. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe(3+)cyt c, and H2O2 in the presence of methyl-beta-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-beta-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe(3+)cyt c increased with NADH and H2O2 concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe(3+)cyt c oxidizes NADH to NAD(center dot), which in turn donates an electron to O-2, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe(3+)cyt c is reduced in the presence of both NADH and H2O2. Our results suggest that Fe(3+)cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe(3+)cyt c may play a key role in the mitochondrial ROS-induced ROS-release signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage. (C) 2011 Elsevier Inc. All rights reserved.