Oxygen Sensitivity of Carbon Monoxide-Dependent Hydrogen Production Activity in Citrobacter sp.

A newly isolated Citrobacter sp. Y19 catalyzes the CO-dependent H-2 production (biological water-gas shift reaction) by the actions of CO dehydrogenase (CODH) and hydrogenase. Y19 requires O-2 for fast growth, but its H-2 production activity is significantly inhibited by O-2. In the present study, the effect of O-2 on the activities of CODH and hydrogenase was investigated quantitatively in both whole cells and broken cells, based on CO-dependent or methyl viologen (MV)-dependent H-2 production in addition to CO-dependent MV reduction. In crude cell extracts, CODH activity was mostly found in the soluble fraction. Inactivation of CODH and hydrogenase activities by O-2 followed the first-order decay kinetics, and the dependence of the rate constants on O-2 partial pressure could be expressed by the Michaelis-Menten equation. In whole cells, the maximum deactivation rate constants (k(d.max)) of hydrogenase and CODH were quite similar: 0.07 +/- 0.03 min' and 0.10 +/- 0.04 min(-1), respectively. However, the first-order rate constant (k(d.max)/K-s) of CODH (0.25 min(-1) atm(-1)) at low O-2 partial pressures was about 3-fold higher than that of the hydrogenase, since the half-saturation constant (K-s) of CODH was about half of that of hydrogenase. In broken cells, both enzymes became significantly more sensitive to O-2 compared to the unbroken cells, while k(d,max)/K-s increased 37-fold for hydrogenase and 6.7-fold for CODH. When whole cells were incubated under anaerobic conditions after being exposed to air for 1 h, hydrogenase activity was recovered more than 90% in 2 h suggesting that the deactivation of hydrogenase by O-2 was reversible. On the contrary, CODH activity was not recovered once deactivated by O-2, and the only way to recover the activity was to synthesize new CODH. This study indicates that O-2 sensitivity of H-2 production activity of Citrobacter Y19 is an important drawback as in other H-2-producing bacteria.