Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.

Influence of the fusion of two subunits of the F420-non-reducing hydrogenase of Methanococcus voltae on its biochemical properties.

In Methanococcus voltae, one of the two [NiFeSe] hydrogenases is unusual in that the large subunit is split into two subunits, each contributing two ligands to the [NiFe] center that catalyzes the heterolytic cleavage of the dihydrogen molecule. We have engineered a fusion of these two subunits. The resulting new enzyme showed no significant difference in hydrogen uptake activity or in the Ni-C or Ni-L EPR spectra compared to the the wild-type enzyme, but exhibited a tenfold increase in both the Km for hydrogen and the Ki for the competitive inhibitor carbon monoxide.

Fusion of two subunits does not impair the function of a [NiFeSe]-hydrogenase in the archaeon Methanococcus voltae.

[NiFe]-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit. The [NiFeSe]-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild-type enzyme. M. voltae carries the genetic information for four different [NiFe]-hydrogenases. In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified. Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium-containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc andfrc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.