title: Novel, oxygen-insensitive group 5 [NiFe]-hydrogenase in Ralstonia eutropha.
abstract: Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.
authors.affiliation: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Berlin, Germany.
authors.forename: Caspar
authors.initials: C
authors.lastname: Schäfer
authors.forename: Bärbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
mesh_terms: D020118:Cupriavidus necator / Q000201:enzymology* / Q000254:growth & development; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D021122:Protein Subunits; D009580:Nitroblue Tetrazolium; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen