In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 79, No. 17 ( 2013-09), p. 5137-5145
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 H 2 . 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 H 2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H 2 . 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 H 2 per min and mg of protein. However, an unexpectedly high Michaelis constant ( K m ) for H 2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low K m of group 5 hydrogenases and makes atmospheric H 2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H 2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O 2 .
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.01576-13
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2013
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
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