O2-stable membrane-bound [NiFe]hydrogenase from a newly isolated Citrobacter sp. S-77.
Hydrogenases are of great interest due to their potential use in H(2)-based technology. However, most hydrogenases are highly sensitive to O(2), which have been the major bottleneck in hydrogenase studies. Here we report an O(2)-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5 kDa. The enzyme showed a specific activity for H(2)-oxidation of 661 U/mg, which is 35-fold greater than that for H(2)-production of 18.7 U/mg. Notably, the MBH showed a remarkable O(2)-stability, maintaining almost 95% of its original activity even after incubation for 30 h in air at 4°C. These results suggest that the O(2)-stable MBH may play an important role in the H(2)-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O(2)-stable MBH from the genus of Citrobacter.
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.
Shigenobu
S
Eguchi
Ki-Seok
KS
Yoon
Seiji
S
Ogo
D002462:Cell Membrane / Q000201:enzymology; D002954:Citrobacter / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D004795:Enzyme Stability; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010802:Phylogeny
C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen