Oxygen sensitivity of carbon monoxide-dependent hydrogen production activity in Citrobacter sp

A newly isolated Citrobacter sp. Y19 catalyzes the CO-dependent H₂ production (biological water-gas shift reaction) by the actions of CO dehydrogenase (CODH) and hydrogenase. Y19 requires O₂ for fast growth, but its H₂ production activity is significantly inhibited by O₂. In the present study, the effect of O₂ 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 H₂ 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 O₂ followed the first-order decay kinetics, and the dependence of the rate constants on O₂ 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^-1 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 O₂ 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 O₂ 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 O₂ was reversible. On the contrary, CODH activity was not recovered once deactivated by O₂, and the only way to recover the activity was to synthesize new CODH. This study indicates that O₂ sensitivity of H₂ production activity of Citrobacter sp. Y19 is an important drawback as in other H₂-producing bacteria.