Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium.
A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with alpha2beta2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 microM and with a turnover number around 2900 (min(-1)). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.
Ishii
M
M
Department of Biotechnology, the University of Tokyo, Japan.
Takishita
S
S
Iwasaki
T
T
Peerapornpisal
Y
Y
Yoshino
J
J
Kodama
T
T
Igarashi
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001420:Bacteria, Aerobic / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D006361:Heating; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008670:Metals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D017421:Sequence Analysis
D000596:Amino Acids; D008565:Membrane Proteins; D008670:Metals; D006859:Hydrogen; D006864:Hydrogenase