Purification of Thiobacillus novellus sulfite oxidase. Evidence for the presence of heme and molybdenum.
Sulfite oxidase from Thiobacillus novellus has been purified 206-fold. The enzyme reduced both ferricyanide and cytochrome c. The ferricyanide activity was 3-5% of the cytochrome c activity. During purification, the absorbance ratio of A413 nm/A280 nm showed a continual increase, suggesting the presence of heme in the T. novellus sulfite oxidase molecule. The absorption spectrum of the enzyme is very similar to that of rat liver sulfite oxidase which contains cytochrome b5 type heme. Gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate revealed a protein staining band of approximately 41,000 Da. Gel filtration chromatography of the enzyme in aqueous media indicated a molecular weight of 38,000. These results suggest that T. novellus sulfite oxidase is a monomer of approximately 40,000 Da. Moreover, analysis of the visible absorption spectrum of the purified enzyme and the co-elution of 413 and 280 nm absorbing material during high pressure liquid chromatography gel chromatography provided clear evidence for the presence of heme in T. novellus sulfite oxidase. EPR spectroscopy of the enzyme revealed a characteristic molybdenum spectrum, which was observed only in the presence of sulfite. Analysis of the T. novellus sulfite oxidase molybdenum cofactor showed a fluorescence spectrum indistinguishable from that displayed by the molybdenum cofactor of chicken liver sulfite oxidase. Therefore, it is concluded that T. novellus sulfite oxidase is a monomeric (Mr approximately 40,000) molybdohemoprotein.
D006418:Heme / Q000032:analysis*; D007700:Kinetics; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013855:Thiobacillus / Q000201:enzymology*
D006418:Heme; D008982:Molybdenum; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors