Thiosulfate oxidase from an Alcaligenes grown on mercaptosuccinate

Abstract

The soil isolate Alcaligenes sp. (M1) was able to use mercaptosuccinate (MS) as a sole source of carbon, sulfur, and energy. Resting cells oxidized the sulfur moiety of MS completely to sulfate. Thiosulfate oxidase, rhodanese, sulfite oxidase, and adenosine-5-phosphosulfate reductase were all detected in crude extracts. However, only thiosulfate oxidase appeared to be induced by growth of the organism on mercaptosuccinate. The intracellular thiosulfate oxidase was purified 139 to 170-fold by a procedure which included MnCl2 precipitation, treatment with calcium phosphate gel, and successive column chromatography on diethylaminoethyl (DEAE) Sephadex and Sephadex G-150, respectively. The resultant enzyme had a pH optimum which appeared to be below 5.0 and an optimum temperature of 40 °C using ferricyanide as electron acceptor. However, with cytochrome c as electron acceptor the pH optimum was 7.2 to 7.6. No activity was observed with other electron acceptors such as methylene blue, nicotinamide-adenine dinucleotide, 2,6-dichlorophenol-indophenol, and flavine-adenine dinucleotide. Enzymatic activity was inhibited 100% by 10−3 M mercuric chloride and 90% by 10−3 M p-chloromercuribenzoate (p-CMB). The molecular weight of the enzyme was found to be about 90 000. Resting cells and thiosulfate oxidase converted thiosulfate labeled in the inner sulfur atom to tetrathionate as the only detectable product, thereby suggesting that thiosulfate oxidase does not play a direct role in sulfate formation from mercaptosuccinate.