Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium

被引:28
作者
Ishii, M
Takishita, S
Iwasaki, T
Peerapornpisal, Y
Yoshino, J
Kodama, T
Igarashi, Y [1 ]
机构
[1] Univ Tokyo, Dept Biotechnol, Bunkyo Ku, Tokyo 1138657, Japan
[2] Nippon Med Sch, Dept Biochem & Mol Biol, Bunkyo Ku, Tokyo 1138602, Japan
[3] Chiang Mai Univ, Dept Biol, Chiangmai 50200, Thailand
[4] Shinshu Univ, Fac Text Sci & Technol, Ueda, Nagano 3860018, Japan
关键词
membrane-bound hydrogenase; quinone reduction; EPR; hydrogen bacterium; Hydrogenobacter;
D O I
10.1271/bbb.64.492
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 alpha(2)beta(2) 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 fi, of around 300 mu M 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.
引用
收藏
页码:492 / 502
页数:11
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