In vitro reconstitution of demolybdosulfite oxidase by a molybdenum cofactor from rat liver and other sources.

A molybdenum cofactor which is capable of reconstituting demolybdosulfite oxidase purified from livers of tungstentreated rats has been identified in rat liver and various organisms. Reconstitution with the rat liver cofactor is directly proportional to cofactor and demolybdoenzyme concentrations and proceeds most efficiently in 0.01 M potassium phosphate buffer, pH 7.4. The activation process is inhibited by high concentrations of anions and by reduction of the demolybdoenzyme and requires incubation temperatures higher than 30”. The population of demolybdo molecules reconstituted by the molybdenum cofactor has been shown to be distinct from those which contain tungsten substituted at the molybdenum site and are reconstituted by inorganic molybdate (Jones, H. P., Johnson, J. L., and Rajagopalan, K. V. (1977) J. Biol. Chem. 252, 4988-49!K3); only aposulfite oxidase molecules with vacant molybdenum centers are reconstituted by the molybdenum cofactor. Cofactor-dependent reconstitution of sulfite oxidase, in contrast to the molybdate-dependent process, is not affected by preincubation of the enzyme at 37”. Reconstitution achieved in the presence of both cofactor and molybdate is equal to the sum of activation by the two processes individually. The molybdenum cofactor present in rat liver has been localized on the mitochondrial outer membrane. Quantitative transfer of molybdenum from isolated mitochondrial outer membranes to sulfite oxidase has been demonstrated. The rat liver molybdenum cofactor has been shown to correspond to a labile pool of the metal in this tissue with a halflife of about 1 day. The molybdenum cofactor and the two molybdoenzymes, sulfite oxidase and xanthine oxidase. account for essentially all molybdenum present in rat liver. A molybdenum cofactor capable of reconstituting aposulfite oxidase has been identified in many other rat tissues and in such diverse sources as Escherichia coli, Neurospora crassa, and human tissue. In all cases the cofactor is present in a particulate cell fraction or associated with a macromolecule. The labile molybdenum cofactor released from acidified milk xanthine oxidase will also reconstitute aposulfite oxidase.