Oxygen-tolerant H2 Oxidation by Membrane-bound [NiFe] Hydrogenases of Ralstonia Species COPING WITH LOW LEVEL H2 IN AIR

Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H-2 using O-2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H-2 in the presence of O-2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O-2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O-2 inhibition of H-2 oxidation (K-I(app)(O2)) for each enzyme. These values were at least 2 orders of magnitude higher than those of "standard" [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O-2 sensitivity. Michaelis constants for H-2 (K-M(H2)) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased K-M(H2) or decreased K-I(app)(O2) values were associated with impaired lithoautotrophic growth in the presence of high O-2 concentrations.