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.