Abstract
Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.
Highlights
Xanthine oxidoreductase is a complex enzyme comprising two identical subunits of Mr 145,000 each containing one molybdenum, one FAD, and two nonidentical iron sulfur centers
In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase
DHR is oxidized by the hydroxyl radical but not by superoxide radical anion or hydrogen peroxide [32] that are produced during XO turnover (Reactions 1–3) (4 – 8)
Summary
Xanthine oxidoreductase is a complex enzyme comprising two identical subunits of Mr 145,000 each containing one molybdenum, one FAD, and two nonidentical iron sulfur centers (reviewed in Refs. 1–3). Both xanthine dehydrogenase and XO can reduce molecular oxygen with production of superoxide radical anion and hydrogen peroxide at proportions that depend on the substrate and oxidation conditions (Reactions 1–3) (4 – 8). XO has been shown to be able to use both NADH and xanthine to reduce nitrite to nitric oxide [11,12,13,14,15], and the concomitant production of the superoxide radical anion, which reacts with nitric oxide in a diffusion-controlled process, results in peroxynitrite formation [16]. Turnover [17] to be able to generate yet another strong one-electron oxidant, the carbonate radical anion Formation of this radical through Fenton chemistry was proposed to explain the low level chemiluminescence detected in incubations of acetaldehyde, XO, and bicarbonate at pH 10.2 (Reactions 1–9) [17, 18]. In addition to being considered an important mediator of peroxynitrite effects in vivo (reviewed in Refs. 20 and 21), the carbonate radical anion has been proposed as the diffusible oxidant produced during the peroxidase activity of the enzyme Cu,Zn-superoxide dismutase [22,23,24,25,26,27], whose relationship to the familial amyothophic lateral sclerosis disease remains under scrutiny
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