Molecular Basis of the Biological Function of Molybdenum

Abstract Livers of tungsten-treated rats have been shown to contain inactive protein which cross-reacts with antibody prepared against native rat liver sulfite oxidase. The cross-reacting material (CRM) was shown to be immunologically identical to the native enzyme by Ouchterlony double immunodiffusion. Difference spectrophotometry, acrylamide gel electrophoresis, and immunological analysis of the proteins of the mitochondrial intermembranous space from livers of control and tungsten-treated rats showed that, like native sulfite oxidase, the CRM was localized in this compartment. CRM was quantitated in terms of the amount of the b5-type heme of sulfite oxidase which was precipitable by the antibody and was shown to be present in amounts corresponding to almost 70% of control. Sulfite oxidase CRM was purified by the procedure established for native enzyme and shown to contain the b5 heme in stoichiometric amounts and no molybdenum. Tungsten was quantitated by a sensitive colorimetric technique and was found to be incorporated into 35% of the molybdenum-free molecules. Tungsten-containing sulfite oxidase was shown to have no detectable cytochrome c reductase activity. Tungsten in the enzyme could be visualized by EPR spectroscopy after reduction by dithionite. The signal at g = 1.87 was very similar in line shape to the g = 1.97 signal of molybdenum (V) in native sulfite oxidase. The native and inactive proteins were indistinguishable on acrylamide gels in the presence or absence of sodium dodecyl sulfate. The absorption spectra of the two proteins were quite similar in both the oxidized and reduced states, although the conditions required for reduction of the heme chromophore differed. The native enzyme was reduced by sulfite within 1 min; the inactive protein showed 30 to 40% reduction in the course of 30 min in the presence of sulfite, with full reduction achieved only upon the addition of dithionite. The difference spectrum of oxidized native sulfite oxidase minus inactive protein revealed subtle differences in the environments of the heme prosthetic group, hydrophobic aromatic residues, and possibly sulfhydrylmolybdenum linkages. Activity assays of the native enzyme complexed with antibody showed loss of ability to reduce O2 and cytochrome c but normal ferricyanide activity. EPR studies revealed that the molybdenum center of the enzyme in the complex was rapidly reducible by sulfite and maintained its sensitivity to anionic and pH effects. Sulfite oxidase in other rat tissues was shown to be antigenically similar to the liver enzyme.