title: Purified NADH-cytochrome b5 reductase is a novel superoxide anion source inhibited by apocynin: sensitivity to nitric oxide and peroxynitrite.
abstract: Cytochrome b5 reductase (Cb5R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb5R acting in concert with other cellular components. In this work, we have purified Cb5R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb5R at 37°C are Vmax = 3.0 ± 0.5 μmol/min/mg of purified Cb5R and KM(NADH) = 2.8 ± 0.3 μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb5R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the KM(NADH) value of Cb5R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb5R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb5R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb5R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb5R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb5R for NADH.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06006 Badajoz, Spain. Electronic address: aksamhan@unex.es.
authors.forename: Alejandro K
authors.initials: AK
authors.lastname: Samhan-Arias
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Sciences, University of Extremadura, 06006 Badajoz, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Gutierrez-Merino
mesh_terms: D000098:Acetophenones / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D042966:Cytochrome-B(5) Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry; D016166:Free Radical Scavengers / Q000494:pharmacology; D008099:Liver; D062105:Molecular Docking Simulation; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors; D009569:Nitric Oxide / Q000494:pharmacology*; D009580:Nitroblue Tetrazolium / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013552:Swine
chemical_list: D000098:Acetophenones; D000975:Antioxidants; D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D009243:NAD; D013481:Superoxides; D030421:Peroxynitrous Acid; D009580:Nitroblue Tetrazolium; D009569:Nitric Oxide; D045304:Cytochromes c; C056165:acetovanillone; D009247:NADH, NADPH Oxidoreductases; D042966:Cytochrome-B(5) Reductase; C014227:ferricyanide reductase
keywords: Apocynin; Cytochrome b(5) reductase; Free radicals; NADH-dependent superoxide anion production; Nitric oxide; Nonmitochondrial ROS production; Peroxynitrite