Mossbauer and EPR studies of the hydrogen-cluster of the iron-hydrogenases from C. pasteurianum and of synthetic complexes designed to model the iron sites of the [(2)iron](H)-subcluster and Mossbauer studies of the transcription factor FNR of Escherichia

[Fe]-Hydrogenase II isolated from C. pasteurianum contains 14 Fe which are distributed among the so-called H-cluster (the catalytic center) and two [4Fe-4S] clusters. Insights gained from Mössbauer studies of M-[4Fe-4S]2+ cluster assemblies (M is a paramagnetic center) in sulfite reductase and carbon monoxide dehydrogenase have suggested that the H-cluster contains a [4Fe-4S]2+ cluster covalently linked to a smaller Fe-containing cluster. Recent X-ray studies of two [Fe]-hydrogenases, combined with the results of FTIR studies, have revealed that the H-cluster contains a novel binuclear Fe-cluster, [2Fe]_H, that is linked by a cysteinyl sulfur to a [4Fe-4S]-cluster; [2Fe]_H was found to have CO, CN−, and thiolate ligands. The analysis of the Mössbauer spectra of Hydrogenase II in the oxidized, reduced, and the CO-inhibited states has enabled us to assign the 57Fe magnetic hyperfine tensors observed by ENDOR and Mössbauer spectroscopy to the two subclusters. Given the environment of strong-field ligands in [2Fe]_H, the Fe III site must have low-spin configuration. While such an assignment is compatible with the EPR g-values, low-spin FeIII sites with g ≈ 2 commonly exhibit very anisotropic 57Fe A-tensors and thus the isotropic A-values of H_ox and H_ox-CO observed by ENDOR are difficult to explain. The study of complexes with coordination environment similar to that discovered for [2Fe]_H provided insight in the electronic structure of this unit. The coordination chemistry of Fe II and FeIII complexes with CN and CO ligands is very limited. We report the synthesis and characterization of the first iron(II)-thiolate complex with CN− and CO ligands, [Fe(II)PS ₃(CN)(CO)][PPh₄]₂ and the spectroscopic characterization of the corresponding FeIII complex, the first ferric complex with a bound CO ligand. EPR and Mössbauer studies of these complexes show that the states H_ox and H_ox-CO of the Fe-hydrogenases cannot contain a typical low-spin FeIII ion.; In addition, spectroscopic studies of the FNR transcription factor of E. coli and of the [4Fe-4S]2+ → [2Fe-2S] 2+ reversible conversion in vitro and in vivo are reported. These studies show that the cluster-conversion regulates the DNA-binding activity of FNR. Moreover, they demonstrate that 57Fe-Mössbauer spectroscopy can be employed to study the in vivo behavior of (over-expressed) proteins.