Interplay between CN^– Ligands and the Secondary Coordination Sphere of the H‑Cluster in [FeFe]-Hydrogenases

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4Fe_H) linked to a binuclear iron–sulfur cluster (2Fe_H) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2Fe_H) subsite features a diatomic coordination sphere composed of three CO and two CN^– ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN^– signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN^– ligands tightly anchoring the (2Fe_H) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN^– ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN^– ligands of the (2Fe_H) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN^– ligand (i.e., closest to the (4Fe_H) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.