Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24

Ki Seok Yoon, Yukiko Sakai, Natsuki Tsukada, Kiyoshi Fujisawa, Hirofumi Nishihara

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11 Citations (Scopus)

Abstract

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100±10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H2 oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H2 oxidation/H2 evolution) was 1.61 × 102 at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c. 48 h at 4 °C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H2. Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were Vmax=336 U mg -1, kcat=560 s-1, and kcat/K m=2.24 × 107 M-1 s-1. The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe-4S]+ and [4Fe-4S]+ clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H2-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga.

Original languageEnglish
Pages (from-to)114-120
Number of pages7
JournalFEMS microbiology letters
Volume290
Issue number1
DOIs
Publication statusPublished - Jan 1 2009
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Genetics

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