Studies on the metabolism of a sulfur-oxidizing bacterium VIII. Purification and characterization of soluble components indispensables for sulfur oxidation by Thiobacillus thiooxidans

The soluble fraction required for the sulfur-oxidizing systemof Thiobacillus thiooxidans was resolved into two componentsthrough ammonium sulfate fractionation, Amberlite CG-50, DEAEcellulose column chromatography and Sephadex gel filtration.Both components (A and B) were indispensable for the sulfur-oxidizingsystem. Component A with a molecular weight of 120,000 is a non-hemeiron protein. The absorption maximum of the oxidized form wasat 410 nm but was shifted to 420 nm by reduction. ComponentB is a new flavoprotein containing non-heme iron. Although thesame absorption change was seen at 410 nm (as with componentA) the shoulder at 485 nm in the oxidized form disappeared uponreduction. The molecular weight of component B was calculatedas 23,000 using the gel nitration method. The sulfur-oxidizing activity of the reconstituted system wasmarkedly inhibited by such metal-chelating agents as EDTA, DDCand o-phenanthroline. Several nucleotides, which are known flavoproteininhibitors, inhibited the sulfur-oxidizing activity. Removalof the Fe in the soluble fraction components by KCN or DDC treatmentdecreased the sulfur-oxidizing activity of the reconstitutedsystem. Based on these evidences we concluded that iron and flavin inthe soluble components may have an important role in the elementalsulfur-oxidizing system of Thiobacillus thiooxidans. (Received May 2, 1975; )