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 ( O2•−) generation. NADH was found to be a good substrate for AO with apparent Km and Vmax values of 29μM and 12 nmol min−1 mg−1, respectively. From O2•− generation measurements by cytochrome c reduction the apparent Km and Vmax 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 O2•− generation. Diphenyleneiodonium completely inhibited AO-mediated O2•− production confirming that this occurs at the FAD site. Inhibitors of this NADH-derived O2•− 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, the levels of AO and NADH, O2•− 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.

The Na + -Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae

“…While complex I harbors a noncovalently FMN as a mediator for electron transport between NADH and the [2Fe-2S] cluster, Na ϩ -NQR contains an FAD for NADH oxidation, with a [2Fe-2S] center in close proximity (3). Thus, as reported for complex I and aldehyde oxidase (55), one-and two-electron-reduced FAD may act as an electron donor for superoxide formation in the Na ϩ -NQR. In this case, cytoplasmic superoxide formation would mainly depend on NADH oxidation activity catalyzed by FAD in the cytoplasmic domain of subunit NqrF (Fig.…”

Potent Inhibition of Aldehyde Dehydrogenase-2 by Diphenyleneiodonium: Focus on Nitroglycerin Bioactivation

“…A similar mechanism was described for inhibition of cytochrome P450 reductase (Tew, 1993), and numerous papers published in the past two decades indicate that DPI is a nonselective inhibitor of NAD(P)H-dependent enzymes using flavins as redox cofactors (Aldieri et al, 2008). These include NO synthases, mitochondrial complex I, cytochrome P450 reductase, xanthine oxidase, and, described recently, the related molybdo-flavoprotein aldehyde oxidase (Kundu et al, 2012). The affinity constants found in the literature vary from 50 nM to 2.8 mM, with IC 50 values in the range of 0.3-5.6 mM for inhibition of NADPH oxidase (Aldieri et al, 2008).…”

“…In addition to flavin prosthetic groups, metal-bound porphyrins were identified as targets of aryliodonium compounds, suggesting that modification of flavocytochrome b may contribute to NADPH oxidase inhibition (Doussiere et al, 1999). Mechanism-based modification of flavin or heme prosthetic groups explains inhibition by DPI of a wide variety of flavoproteins, including NADH:ubiquinone oxidoreductase, NO synthases, xanthine oxidase, cytochrome P450 reductase (see Aldieri et al, 2008, for review), and aldehyde oxidase (Kundu et al, 2012).…”

Aldehyde Oxidase 4 Plays a Critical Role in Delaying Silique Senescence by Catalyzing Aldehyde Detoxification

“…Additionally, Ibdah et al (2009), in their AAO4 activity assay, employed NAD + as a cosubstrate together with the aldehyde substrate. However, the use of NAD + by plants, or for that matter by any eukaryotic AO, is not known (Nishino and Nishino, 1989;Terao et al, 1998;Garattini et al, 2009;Kundu et al, 2012;Zarepour et al, 2012;Li et al, 2014). Moreover, Coelho et al (2012) recently demonstrated that the lack of residues leading to a flexible loop around the FAD domain in mammalian AO crystal structure can account for the fact that AOs are pure oxidases and cannot use NAD + as the final acceptor of reducing equivalents.…”