Investigating the role of iron sulfur cluster binding residues of HYDF
[FeFe]-hydrogenases are metalloenzymes found in many bacteria and lower eukaryotes. The catalytic active site of [FeFe]-hydrogenases termed as H-cluster consists of a [4Fe-4S] cubane bridged to a 2Fe subcluster. The two iron atoms of the 2Fe subcluster are decorated by carbon monoxide, cyanide ligands as well as a bridging dithiolate ligand. The assembly of this complex H cluster involves the role of three accessory enzymes namely HydE, HydG and HydF. The maturase, HydF is a GTPase and contains two types of clusters, a [4Fe-4S] and a [2Fe-2S] cluster. The [2Fe-2S] cluster is transformed into an H-cluster precursor by action of HydE and HydG. It is suggested from EPR spectroscopic data of both reduced HydF DeltaEG and Oxidized HydF EG that the [2Fe-2S] cluster and the [4Fe-4S] cluster are not bound to each other. Since an H-cluster like signal was observed in oxidized HydF EG suggested that the two clusters are arranged in same manner as the H-cluster itself. This aforementioned hypothesis drove us to investigate the ligand arrangement of both a [4Fe-4S] and most importantly the [2Fe-2S] clusters in HydF. The apo HydF structure does not provide us with significant insights into Fe-S cluster coordination details, therefore we have attempted to experimentally identify the residues that act as ligands to both the clusters. To that end, we substituted each of the conserved Fe-S cluster binding residues and observed the effects of these mutations on both clusters by spectroscopic methods like UV-Vis spectroscopy and EPR. Our observations indicated that among the three conserved cysteines, C304 and C356 are absolutely quintessential for iron sulfur cluster assembly in HydF DeltaEG while C353 and H306 have some capacity to bind iron sulfur clusters. Further in vitro hydrogenase assays suggested importance of C353 residue as it affected the assembly of the 2Fe subcluster. Thus we propose a dimeric/ tetrameric model of HydF where both the [2Fe-2S] and the [4Fe-4S] clusters are ligated by eight conserved, four putative Fe-S cluster binding residues from each monomer. In our proposed model we discuss the possible occurrence of non cysteinyl ligation for iron sulfur clusters.