Purification and biochemical characterization of a membrane-bound [NiFe]-hydrogenase from a hydrogen-oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH-24.
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 H(2) oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H(2) oxidation/H(2) evolution) was 1.61 x 10(2) at pH 7.0. The optimal temperature was 75 degrees C. The enzyme was quite stable under air atmosphere (the half-life of activity was c. 48 h at 4 degrees 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 degrees C. The activity increased up to 2.5-fold during incubation at 50 degrees C under H(2). Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V(max)=336 U mg(-1), k(cat)=560 s(-1), and k(cat)/K(m)=2.24 x 10(7) 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 H(2)-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga.
Yoon
Ki-Seok
KS
Department of Bioresource Science, School of Agriculture, Ibaraki University, Ibaraki, Japan.
Sakai
Yukiko
Y
Tsukada
Natsuki
N
Fujisawa
Kiyoshi
K
Nishihara
Hirofumi
H
D000595:Amino Acid Sequence; D052818:Autotrophic Processes; D002462:Cell Membrane / Q000201:enzymology*; D042621:Comamonadaceae / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D016415:Sequence Alignment
D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase