H2 In Air Research Articles

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 H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 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 O2 inhibition of H2 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 O2 sensitivity. Michaelis constants for H2 (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 O2 concentrations.

Optical recognition of CO and H2 by use of gas-sensitive Au–Co3O4 composite films

M. Ando, T. Kobayashi, S. Iijima and M. Haruta, J. Mater. Chem., 1997, 7, 1779 DOI: 10.1039/A700125H

Expression of hydrogenase activity in free-living Rhizobium japonicum

A medium is described on which selected Rhizobium japonicum strains express hydrogenase (H(2) uptake) activity under free-living conditions. Low concentrations of carbon substrates, decreased oxygen tension, and the quantity of combined nitrogen in the medium were major factors influencing hydrogenase expression. Hydrogenase activity was dependent upon a preincubation period in the presence of H(2) under conditions such that the cells did not exhibit nitrogenase activity. H(2) uptake rates were easily measured amperometrically in aerobically or anaerobically prepared suspensions from free-living cultures. Six R. japonicum strains that formed nodules with the ability to utilize H(2) oxidized this gas when grown in free-living cultures. In comparison six randomly chosen strains forming nodules that lost H(2) in air either showed no or low capacity to take up H(2) under free-living conditions. The reduction of triphenyltetrazolium chloride in an agar medium was used to detect strains capable of oxidizing H(2). This method has enabled us to isolate a spontaneous R. japonicum mutant strain that has lost the ability to utilize H(2). This mutant strain forms nodules that evolve H(2) but other symbiotic characteristics appear normal. This strain will be useful in evaluating the importance of the hydrogenase system in the nitrogen-fixing process of legumes.