PURIFICATION AND CHARACTERIZATION OF A PERIPLASMIC THIOSULFATE DEHYDROGENASE FROM THE OBLIGATELY AUTOTROPHIC THIOBACILLUS SP W5

A periplasmic thiosulfate dehydrogenase (EC 1.8.2.2) was purified to h omogeneity from the neutrophilic, obligately chemolithoautotrophic Thi obacillus sp. W5. A five-step procedure resulted in an approximately 2 ,300-fold purification. The purified protein had a molecular mass of 1 20 +/- 3 kDa, as determined by gel filtration. It is probably a tetram er containing two different subunits with molecular masses of 33 +/- 1 kDa and 27 +/- 0.5 kDa, as determined by SDS-PAGE. UV/visible spectro scopy revealed that the enzyme contained haem c; haem staining showed that both subunits contained haem c. A haem c content of 4 mol per mol of enzyme was calculated using the pyridine haemochrome test. The pH optimum of the enzyme was 5.5. At pH 7.5, the K-m and V-max were 120 /- 10 mu M and 1, 160 +/- 30 U mg(-1), respectively. The absence of 2- heptyl-4-hydroquinoline-N-oxide (HQNO) inhibition for the oxidation of thiosulfate by whole cells suggested that the electrons enter the res piratory chain at the level of cytochrome c. Comparison with thiosulfa te dehydrogenases from other Thiobacillus species showed that the enzy me was structurally similar to the thiosulfate dehydrogenase of the ac idophilic, facultatively chemolithoautotrophic Thiobacillus acidophilu s, but not to the thiosulfate dehydrogenases published for the obligat ely chemolithoautotrophic Thiobacillus tepidarius and Thiobacillus thi oparus.