Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide

Abstract

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 ؎ 1.4 M ؊1 s ؊1 at 25°C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and ironsulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no "very rapid" spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes.Mammalian xanthine oxidase (XO 1 ; EC 1.1.3.22) and xanthine dehydrogenase (XDH; EC 1.1.1.204), which are alternative forms of the same gene product (for a recent review, see Ref. 1), are complex flavoproteins composed of two identical subunits of M r 145,000; each subunit contains one molybdenum center, two non-identical Fe 2 S 2 -type iron-sulfur centers, and one FAD center. The enzymes catalyze oxidation of xanthine to uric acid with concomitant reduction of NAD ϩ or molecular oxygen. The oxidative hydroxylation of xanthine to uric acid takes place at the molybdenum center, and reducing equivalents thus introduced into enzymes are transferred rapidly via intramolecular electron transfer to FAD, where physiological oxidation occurs (2). Both enzymes can reduce molecular oxygen to superoxide and hydrogen peroxide (1), but XDH is characterized by high reactivity toward NAD ϩ , but low reactivity toward O 2 , whereas XO has high reactivity toward O 2 , but negligible reactivity toward NAD ϩ (2). Mammalian XDH can be converted to XO either by the oxidation of sulfhydryl groups or by limited proteolysis (3, 4). XO is thought to be one of the key enzymes for cellular injury by superoxide and related active oxygen species (5). In particular, much attention has been paid to XO in connection with the pathogenesis of ischemia/ reperfusion injury since it has been proposed t...

Neuronal nitric oxide synthase negatively regulates xanthine oxidoreductase inhibition of cardiac excitation-contraction coupling

“…XOR contains multiple redox centers essential for enzyme activity that are plausible targets of NO attack (10). In this regard, NO directly binds with an essential sulfur of the reduced molybdenum center of XOR to produce desulfo-type inactive enzymes (9,10).…”

“…Mechanisms for NO inhibition of XOR have been described (10). XOR contains multiple redox centers essential for enzyme activity that are plausible targets of NO attack (10).…”

“…In intact animals, nonspecific inhibition of NO synthase (NOS) stimulated XOR activity causing cardiac dysfunction. Indeed, earlier studies had suggested that NO inhibits XOR activity (10,11). Accordingly, the aims of the present study were to test the hypothesis that endogenous cardiac NOSs exert tonic inhibition over cardiac XOR.…”

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

“…One should bear in mind, however, that nitric oxide at low concentrations (formed by constitutive forms of NOS) might act as a pseudo antioxidant (see the review [35]). Nitric oxide inhibits aconitase, ribonucleotidyl reductase, glutathione peroxidase, cytochrome c oxidoreductase, NAD(P)H oxidase, xanthine oxidase or caspases [20,27,28,32]. On the contrary shear stress which accompanies injured muscle also activates endothelial NAD(P)H oxidase with the superoxide anion radical being formed …”

“…Apparently, in control conditions, L6 muscle cells started to form myotubes after 10 days of incubation (multinucleated postmitotic cells) but the process was significantly accelerated (8. day) after addition of Dex (2 nM). Higher doses (20,200 nM) eventually caused cell death before any symptoms of muscle cell fusion occurred (6th or 4th day of treatment, respectively). Nevertheless, some antioxidants could prevent cell elimination.…”