[FeFe]-hydrogenases are powerful hydrogen evolution biocatalysts because of the activity of their unique metallocofactor, the H-cluster, derived from the association of a [4Fe-4S] cluster and a [2Fe]-subcluster, containing 2 CO and 2 CN- ligands as well as an aza-propanedithiolate (adt(2-)) ligand bridging the two Fe atoms. Various analogues of the H-cluster have been assembled in the iron-sulfur protein HydF, containing a [4Fe-4S] cluster, by reaction with a series of synthetic di-iron complexes mimicking the [2Fe]-subcluster of hydrogenases. The mimics contain a mixture of CO and CN- ligands and various dichalcogenide (S or Se) bridging ligands. The resulting hybrid proteins have been characterized and shown to behave as active artificial hydrogenases. On the basis of the present structure-activity relationship study, we report that the most active and stable artificial hydrogenase was obtained with the mimic containing a propane diselenol bridging ligand. This opens the possibility to developing a class of hydrogen-evolving enzymes with a synthetic active site mimicking that of the natural hydrogenase.