title: Oxygen-tolerant H2 oxidation by membrane-bound [NiFe] hydrogenases of ralstonia species. Coping with low level H2 in air.
abstract: 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.
authors.affiliation: Institut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom.
authors.forename: Marcus
authors.initials: M
authors.lastname: Ludwig
authors.affiliation: Institut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Cracknell
authors.affiliation: Institut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom.
authors.forename: Kylie A
authors.initials: KA
authors.lastname: Vincent
authors.affiliation: Institut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom.
authors.forename: Fraser A
authors.initials: FA
authors.lastname: Armstrong
authors.affiliation: Institut fu¨r Biologie/Mikrobiologie, Humboldt-Universita¨t zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany and the Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom. Electronic address: oliver.lenz@rz.hu-berlin.de.
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D006859:Hydrogen / Q000737:chemistry* / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D043367:Ralstonia / Q000201:enzymology* / Q000254:growth & development
chemical_list: D001426:Bacterial Proteins; D006859:Hydrogen; D006864:Hydrogenase; D010100:Oxygen