Inhibition of xanthine oxidase and xanthine dehydrogenase by nitric oxide - Nitric oxide converts reduced xanthine-oxidizing enzymes into the desulfo-type inactive form

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 ina ctivated by nitric oxide. The second-order rate constant of the reaction be tween 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-dichlorophenoli ndophenol activity, and the oxypurinol-bound form of XO was partly protecte d from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and i ron-sulfur centers of inactivated XO were reduced by dithionite and reoxidi zed readily with oxygen, and inactivated XDH retained electron transfer act ivities from NADH to electron accepters, consistent with the conclusion tha t the flavin and iron-sulfur centers of the inactivated enzyme both remaine d 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, inactivate d XO reduced by dithionite exhibited the same signals for iron-sulfur cente rs as the normal enzyme. Inactivated XO recovered its activity in the prese nce of a sulfide-generating system. It is concluded that nitric oxide react s with an essential sulfur of the reduced molybdenum center of XO and XDH t o produce desulfo-type inactive enzymes.