Journal
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
Volume 114, Issue 5, Pages 479-484Publisher
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2012.05.018
Keywords
O-2-stability; H-2-oxidation; Membrane-bound [NiFe]hydrogenase; Cytochrome b(560); Citrobacter sp S-77; Kinetic parameters
Funding
- World Premier International Research Center Initiative (WPI) [18065017, 19205009, 23655053]
- Global COE Program, Science for Future Molecular Systems from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan
- Basic Research Programs CREST Type, Development of the Foundation for Nano-Interface Technology from Japan Science and Technology Agency (JST), Japan
- Grants-in-Aid for Scientific Research [23655053] Funding Source: KAKEN
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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 degrees 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. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.
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