Spectroscopic Evidence of Reversible Disassembly of the [FeFe] Hydrogenase Active Site.

[FeFe] hydrogenases are extremely active and efficient H2 -converting biocatalysts. Their active site comprises a unique [2Fe] subcluster bonded to a canonical [4Fe-4S] cluster. The [2Fe] subsite can be introduced into hydrogenases lacking an assembled H-cluster through incubation with a synthesized [2Fe]H precursor, which initially produces the CO-inhibited state of the enzyme. We present FTIR spectroelectrochemical studies on the CO-inhibited state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, DdHydAB. At very negative potentials, disassembly of the H-cluster and dissociation of the [2Fe] subcluster is observed. Subsequently raising the potential allows cofactor rebinding and H-cluster reassembly. This demonstrates how the stability of the [2Fe]-[4Fe-4S] intercluster bond depends on the applied potential and the presence of an inhibiting CO ligand on the [2Fe] subcluster. These results provide insight into the mechanisms of CO inhibition and H-cluster assembly in [FeFe] hydrogenases. A fundamental understanding of these properties will provide clues for designing better H2 -converting catalysts.