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.
Ludwig
Marcus
M
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.
Cracknell
James A
JA
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.
Vincent
Kylie A
KA
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.
Armstrong
Fraser A
FA
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.
Lenz
Oliver
O
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.
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
D001426:Bacterial Proteins; D006859:Hydrogen; D006864:Hydrogenase; D010100:Oxygen