The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases

Abstract

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN− ligands. The biogenesis of this diiron site, termed [2FeH], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2FeH] precursor, termed [2FeP], to the [FeFe]-hydrogenase. The binding site of [2FeP] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2FeP]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2FeP]. Surprisingly, our results suggest that binding or transfer of [2FeP] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.