Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

Abstract

bRecently, 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 mol 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 M 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 .

Atmospheric Hydrogen Scavenging: from Enzymes to Ecosystems

“…Though the number of clusters is the same as for the well-described group 1 [NiFe]-hydrogenases, the ligands of the proximal cluster and the configuration of the medial cluster differ (31). The metal content and cofactor structures observed in the structure are consistent with those derived from spectroscopic studies (29,30).…”

“…Genes encoding factors involved in genetic mobility have consistently been observed in the vicinity of hhyLS genes in 10 species (25,27). Genes encoding this enzyme have also been acquired in the pHG1 megaplasmid of the model aerobic hydrogenotroph Ralstonia eutropha (also known as Cupriavidus necator) (28), likely through horizontal gene transfer, but the hydrogenase does not yet appear to have acquired full functionality in this betaproteobacterial host (29).…”

“…A preliminary 2.8-Å resolution crystal structure of the low-affinity group 5 [NiFe]-hydrogenase from Ralstonia eutropha provides a revealing insight into structure-function relationships of this group of enzymes (29,30 (30). Though the number of clusters is the same as for the well-described group 1 [NiFe]-hydrogenases, the ligands of the proximal cluster and the configuration of the medial cluster differ (31).…”

A soil actinobacterium scavenges atmospheric H 2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases

“…In such conditions, H 2 may substitute as the primary electron donor and be used to maintain redox balance and a membrane potential. The finding that group 5 [NiFe] hydrogenases can sustain catalytic activity at a wide range of pH values, temperatures, and O 2 concentrations further suggests that these enzymes confer robustness and flexibility in response to environmental shifts (20). Hyd1 and Hyd2 share many characteristics, including similar expression profiles (23), high-affinity oxidation activities, and membrane association.…”

“…only have low to medium affinities for H 2 (e.g., Streptomyces scabiei, Streptomyces griseoflavus) (17). Furthermore, the recently purified group 5 [NiFe] hydrogenase from the β-proteobacterium R. eutropha is a low-affinity, low-activity enzyme (20).…”