title: Aldehyde oxidase functions as a superoxide generating NADH oxidase: an important redox regulated pathway of cellular oxygen radical formation.
abstract: The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress. Utilizing electron paramagnetic resonance spin trapping, we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH and the effects of inhibitors of AO on NADH-mediated superoxide (O(2)(•−)) generation. NADH was found to be a good substrate for AO with apparent K(m) and V(max) values of 29 μM and 12 nmol min(-1) mg(-1), respectively. From O(2)(•−) generation measurements by cytochrome c reduction the apparent K(m) and V(max) values of NADH for AO were 11 μM and 15 nmol min(-1) mg(-1), respectively. With NADH oxidation by AO, ≥65% of the total electron flux led to O(2)(•−) generation. Diphenyleneiodonium completely inhibited AO-mediated O(2)(•−) production, confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O(2)(•−) generation were studied with amidone the most potent exerting complete inhibition at 100 μM concentration, while 150 μM menadione, raloxifene, or β-estradiol led to 81%, 46%, or 26% inhibition, respectively. From the kinetic data, and the levels of AO and NADH, O(2)(•−) production was estimated to be ~89 and ~4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions. Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis.
authors.affiliation: Center for Biomedical EPR Spectroscopy and Imaging, the Davis Heart and Lung Research Institute, and Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210, United States.
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Kundu
authors.forename: Murugesan
authors.initials: M
authors.lastname: Velayutham
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D042931:Aldehyde Oxidase / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D013481:Superoxides; D042931:Aldehyde Oxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases