TY - JOUR
T1 - Aldehyde oxidase functions as a superoxide generating NADH oxidase
T2 - An important redox regulated pathway of cellular oxygen radical formation
AU - Kundu, Tapan K.
AU - Velayutham, Murugesan
AU - Zweier, Jay L.
PY - 2012/4/3
Y1 - 2012/4/3
N2 - 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.
AB - 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.
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U2 - 10.1021/bi3000879
DO - 10.1021/bi3000879
M3 - Article
C2 - 22404107
AN - SCOPUS:84859385695
SN - 0006-2960
VL - 51
SP - 2930
EP - 2939
JO - Biochemistry
JF - Biochemistry
IS - 13
ER -