Characterization of the [FeFe]-Hydrogenase Maturation Protein HydF by EPR Techniques: Insights into the Catalytic Mechanism

A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF

“…n .) reported by a number of different laboratories. ,,,− ,,, Evidence for a [2Fe-2S] cluster on HydF Δ E G has come from EPR and XAS spectroscopic studies; ,, however, the EPR data have been disputed, with the suggestion that the g ≈ 2.0 EPR signal arose from a [3Fe-4S] + cluster or a protein radical. − The nature of the FeS cluster species bound to HydF prior to its interaction with HydE and HydG is directly relevant to the process of maturation, as it determines the identity of species that must be transferred from HydE and HydG, and the processes that must occur during assembly of the 2Fe precursor of the H-cluster on HydF. We therefore set out to provide a more definitive description of the clusters present on HydF Δ E G .…”

Overview of the Maturation Machinery of the H-Cluster of [FeFe]-Hydrogenases with a Focus on HydF

“…The crystal structure of HydF from T. melaniensis revealed that the fourth ligand of the cluster in the absence of the synthon comes from a highly conserved acidic residue (Glu in this organism) coordinating via the carboxylate [ 56 ]. HYSCORE spectroscopy studies had already shown that the fourth ligand is an oxygen in a variety of organisms and that this ligand is easily exchangeable, as expected since the fourth position is the one where the [2Fe] H cluster is anchored [ 52 , 54 , 55 ]; to date, the only known exception coming from the cluster of Clostridium acetobutylicum , where a nitrogen was found, however the possibility that the His-tag at the N-terminus of the recombinant protein, which is located spatially close to the cluster, interferes with the natural coordination must be taken into account. The putative binding pocket of the [2Fe] H cluster is located in a cleft at the interface between the second and third domains and is characterized by a positively charged surface.…”

“…The structure of a recombinant HydF from Thermotoga neapolitana (pdb.id 3QQ5) has been solved without the [4Fe-4S] cluster [ 48 ], which is known to be part of the holo-protein as derived by EPR of the protein in solution-state [ 37 , 49 , 50 , 51 , 52 , 53 , 54 , 55 ], and more recently complete with the cluster from Thermosipho melaniensis (pdb.id 5KH0, used for Figure 4 ) and from T. maritima (pdb.id 5LAD) [ 56 ]. HydF is a homodimeric protein with three domains per monomer.…”

From protein engineering to artificial enzymes – biological and biomimetic approaches towards sustainable hydrogen production

“…The preparation of semi-synthetic X -HydF, via the incorporation of [2Fe] subsite models 30–32 into the purified maturation protein HydF, did not only provide the first entry point into the generation of semi-synthetic hydrogenases, but also generated a model for the binding of the [2Fe] subsite precursor on this chaperone protein. Apart from the [2Fe] subsite precursor, HydF contains a [4Fe–4S] cluster with unknown function 195,199,201,224–226. Pulsed EPR spectroscopy of 13 CN – -labelled 31 -HydF in combination with DFT calculations supports a model in which the [2Fe] complex binds to the protein via a cyanide bridge to this [4Fe–4S] cluster (Fig.…”