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 homogeneity from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5. A five-step procedure resulted in an approximately 2,300-fold purification. The purified protein had a molecular mass of 120 +/- 3 kDa, as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 33 +/- 1 kDa and 27 +/- 0.5 kDa, as determined by SDS-PAGE. UV/visible spectroscopy 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 Km and Vmax were 120 +/- 10 microM 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 respiratory chain at the level of cytochrome c. Comparison with thiosulfate dehydrogenases from other Thiobacillus species showed that the enzyme was structurally similar to the thiosulfate dehydrogenase of the acidophilic, facultatively chemolithoautotrophic Thiobacillus acidophilus, but not to the thiosulfate dehydrogenases published for the obligately chemolithoautotrophic Thiobacillus tepidarius and Thiobacillus thioparus.
Visser
J M
JM
Department of Microbiology and Enzymology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
de Jong
G A
GA
Robertson
L A
LA
Kuenen
J G
JG
D001426:Bacterial Proteins / Q000302:isolation & purification; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors; D011487:Protein Conformation; D011725:Pyridines / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013466:Sulfurtransferases; D013855:Thiobacillus / Q000201:enzymology*; D013885:Thiosulfates / Q000378:metabolism
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D011725:Pyridines; D013885:Thiosulfates; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; D013466:Sulfurtransferases; C022747:thiosulfate-dithiol sulfurtransferase