title: Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH.
abstract: 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 Fe³⁺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 Fe³⁺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, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-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 Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD(•), which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺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, Fe³⁺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.
authors.affiliation: Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, and Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, OH 43210, USA. Murugesan.Velayutham@osumc.edu
authors.forename: Murugesan
authors.initials: M
authors.lastname: Velayutham
authors.forename: Craig
authors.initials: C
authors.lastname: Hemann
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D000818:Animals; D003497:Cyclic N-Oxides / Q000737:chemistry; D045304:Cytochromes c / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006736:Horses; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000378:metabolism; D019097:Spin Trapping; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D013481:Superoxides / Q000737:chemistry* / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000737:chemistry
chemical_list: D003497:Cyclic N-Oxides; D017382:Reactive Oxygen Species; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D009243:NAD; D013481:Superoxides; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D007501:Iron; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D010100:Oxygen