An EPR/HYSCORE, M?ssbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe-S] clusters
文摘
The biosynthesis of the organometallic H cluster of [Fe–Fe]?hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe–S protein with GTPase activity. The [4Fe-S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe-S] cluster of HydF using M?ssbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe-S]2+/+ clusters. Our results show that M?ssbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe-S] cluster and the non-imidazole-bound [4Fe-S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6?MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe-S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.