NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

Abstract

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O 2•-source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O 2•-molecule and half a H 2 O 2 molecule per NADH molecule, at rates 3 times those observed for XO (29.2 ± 1.6 and 9.38 ± 0.31 min -1 , respectively). EPR spectra of NADHreduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 ± 1.36 lM -1 min -1 ) was found to be higher than that of the XO specificity constant (1.07 ± 0.09 lM -1 min -1 ). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O 2•-source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O 2•-than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.Keywords Xanthine oxidoreductase Á Xanthine oxidase Á Xanthine dehydrogenase Á Rat liver Á Reactive oxygen species Á NADH Abbreviations AFR Activity-to-flavin ratio AO Aldehyde oxidase EPR Electron paramagnetic resonance FAD Flavin adenine dinucleotide ROS Reactive oxygen species Sim Simulated XD Xanthine dehydrogenase XO Xanthine oxidase XOR Xanthine oxidoreductase

Aldehyde Oxidase Functions as a Superoxide Generating NADH Oxidase: An Important Redox Regulated Pathway of Cellular Oxygen Radical Formation

“…The K m values of NADH oxidation and ${\text{O}}_2^{•-}$ production are not necessarily the same and a two- to three-fold variation for human milk XDH has been observed (39). While the K m value for NADH oxidation reported here is very close to the literature value (28 M) (32), the K m value for ${\text{O}}_2^{•-}$ generation is found to be twice the value (5.1 μM) reported recently (40); however, our V max value is higher. This discrepancy may be attributed to the different buffer solutions, enzyme conditions, and assays used.…”

Nitrite Reductase Activity of Rat and Human Xanthine Oxidase, Xanthine Dehydrogenase, and Aldehyde Oxidase: Evaluation of Their Contribution to NO Formation in Vivo

“…In addition, some assays were conducted with deflavo-RLXO and deflavo-RLAO, two enzyme forms whose FAD center was chemically removed but that retain the molybdenum and Fe/S centers intact. 86,111 The presence of allopurinol completely inhibited the NO formation catalyzed by both native and deflavo forms of XO and XD of rat and human liver (not shown). Moreover, the NO formation rate was directly proportional to the XO and AO AFR, a ratio that reflects the concentration of XO/AO molecules with an active molybdenum center, per FAD center.…”

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

“…XO hydroxylates allopurinol to oxypurinol (1H-pyrazolo [3,4-d]pyrimidine-4,6-diol), which binds tightly to the reduced molybdenum, blocking it and inhibiting the XO hydroxylase activity [40,49,50]. The formation of the oxypurinol-XO complex, however, does not interfere with any reaction occurring at the two Fe/S or at the FAD site, as shown by the NADH oxidation by molecular oxygen (which occurs at the flavin site) in the presence of the inhibitor [51]. Therefore, the Fe/S and FAD of allopurinol-inhibited XO can be reduced with NADH, in spite of the molybdenum being blocked.…”

“…I. Dithionite reduces both enzymes, whereas NADH reduces only the XO, through the FAD site, leading to molybdenum reduction through intramolecular electron transfer [51]. Once the molybdenum is reduced, Mo 4?…”