The presence of a reducible disulfide bond in milk xanthine oxidase.
The stoichiometry of reducing equivalents per protomer for the complex molybdoflavoprotein xanthine oxidase has been re-examined by reductive titrations with sodium dithionite and anaerobic reoxidation with cytochrome c and phenazine methosulfate of dithionite- or photo-reduced enzyme. It is found that 8.0 +/- 0.1 reducing equivalents are taken up (or given up) by the enzyme, a value of 2 eq greater than expected on the basis of the known oxidation-reduction centers in the enzyme. The reaction of reduced xanthine oxidase with [14C]iodoacetate indicates that, in the reduced form of the enzyme, additional cysteine residues are available for reaction. These results, in conjunction with the observation that reaction of oxidized enzyme with sulfite results in the appearance of an additional equivalent of thiol capable of reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) or iodoacetate, indicate the presence of a disulfide linkage in the enzyme that can be reduced by dithionite or photochemically employing EDTA and 5-deazaflavin. Neither xanthine nor lumazine, however, is capable of reducing this oxidation-reduction center, suggesting that the disulfide does not play a role in the catalytic reactions of the enzyme. These results resolve discrepancies in the literature which indicated that greater than 6 reducing equivalents were consistently needed to bring about the complete reduction of xanthine oxidase.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004227:Dithionite; D004228:Dithionitrobenzoic Acid; D005260:Female; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008892:Milk / Q000201:enzymology*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000378:metabolism*
D003574:Cytochrome c Group; D004220:Disulfides; D007461:Iodoacetates; D004227:Dithionite; D004228:Dithionitrobenzoic Acid; D014969:Xanthine Oxidase; D019807:Iodoacetic Acid