Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide: NITRIC OXIDE CONVERTS REDUCED XANTHINE-OXIDIZING ENZYMES INTO THE DESULFO-TYPE INACTIVE FORM

Kohji Ichimori,Masami Fukahori,Hiroe Nakazawa,Ken Okamoto,Takeshi Nishino

doi:10.1074/jbc.274.12.7763

Kohji Ichimori, Masami Fukahori + Show 3 more

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https://doi.org/10.1074/jbc.274.12.7763

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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 degrees 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 iron-sulfur 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.

Highlights

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol
Much attention has been paid to XO in connection with the pathogenesis of ischemia/ reperfusion injury since it has been proposed that superoxide/ H2O2 production by XO is enhanced by the accelerated conversion of XDH to XO [6], the accumulation of ATP degradation products that are substrates for XO [7], and the up-regulation of XDH/XO mRNA [8]
Aerobic Treatment of Enzymes with Nitric Oxide—XO (AFR Ͼ 200) or XDH (AFR ϭ 109.00) in 0.2 M Hepes buffer containing 1 mM EDTA and 1 mM NADH was mixed with saturated Nitric oxide (NO) solution, which was prepared by bubbling NO gas into the anaerobic buffer, under air at 25 °C

Summary

Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide

NITRIC OXIDE CONVERTS REDUCED XANTHINE-OXIDIZING ENZYMES INTO THE DESULFO-TYPE INACTIVE FORM*. The oxidative hydroxylation of xanthine to uric acid takes place at the molybdenum center, and reducing equivalents 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 NADϩ, but low reactivity toward O2, whereas XO has high reactivity toward O2, but negligible reactivity toward NADϩ [2]. The inhibitory kinetics of XDH were examined to obtain basic data on the interaction of NO and the superoxide-generating system in the event of reduced oxygen supply, such as ischemia

EXPERIMENTAL PROCEDURES

RESULTS

TABLE I Inactivation of XO by NO under anaerobic conditions

ND ϩXanthineb ϩ NO ϩAllopurinolb ϩ NO

DISCUSSION