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
相关论文
共 52 条
  • [21] Biological activation of hydrogen
    Happe, RP
    Roseboom, W
    Pierik, AJ
    Albracht, SPJ
    Bagley, KA
    [J]. NATURE, 1997, 385 (6612) : 126 - 126
  • [22] Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis
    Higuchi, Y
    Yagi, T
    Yasuoka, N
    [J]. STRUCTURE, 1997, 5 (12) : 1671 - 1680
  • [23] 2-METHYLTHIO-1,4-NAPHTHOQUINONE, A UNIQUE SULFUR-CONTAINING QUINONE FROM A THERMOPHILIC HYDROGEN-OXIDIZING BACTERIUM, HYDROGENOBACTER-THERMOPHILUS
    ISHII, M
    KAWASUMI, T
    IGARASHI, Y
    KODAMA, T
    MINODA, Y
    [J]. JOURNAL OF BACTERIOLOGY, 1987, 169 (06) : 2380 - 2384
  • [24] PURIFICATION AND CHARACTERIZATION OF ATP - CITRATE LYASE FROM HYDROGENOBACTER-THERMOPHILUS TK-6
    ISHII, M
    IGARASHI, Y
    KODAMA, T
    [J]. JOURNAL OF BACTERIOLOGY, 1989, 171 (04) : 1788 - 1792
  • [25] Purification and characterization of ferredoxin from Hydrogenobacter thermophilus strain TK-6
    Ishii, M
    Ueda, Y
    Yoon, KS
    Igarashi, Y
    Kodama, T
    [J]. BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 1996, 60 (09) : 1513 - 1515
  • [26] METHIONAQUINONE IS A DIRECT NATURAL ELECTRON-ACCEPTOR FOR THE MEMBRANE-BOUND HYDROGENASE IN HYDROGENOBACTER-THERMOPHILUS STRAIN-TK-6
    ISHII, M
    OMORI, T
    IGARASHI, Y
    ADACHI, O
    AMEYAMA, M
    KODAMA, T
    [J]. AGRICULTURAL AND BIOLOGICAL CHEMISTRY, 1991, 55 (12): : 3011 - 3016
  • [27] Ishizaka K, 1987, Int Rev Immunol, V2, P1, DOI 10.3109/08830188709044744
  • [28] IWASAKI T, 1994, J BIOL CHEM, V269, P29444
  • [29] JACOBSON FS, 1982, J BIOL CHEM, V257, P3385
  • [30] JUSZCZAK A, 1991, J BIOL CHEM, V266, P13834