Unraveling the electronic properties of the photoinduced states of the H-cluster in the [FeFe] hydrogenase from D. desulfuricans

[FeFe] hydrogenases belong to a class of enzymes that catalyze the reversible heterolytic splitting of molecular hydrogen. The structure of their active site is rather unusual consisting of a CN^- and CO-coordinated [2Fe] subcluster connected to a ferredoxin-like [4Fe4S] subcluster through one of the Cys-S ligands. The iron, distal to the "cubane" has an open coordination site that is believed to be the substrate binding site. In the active forms of the H-cluster, this site can be inhibited by carbon monoxide, which results in an EPR-active state, called H_ox-CO. At temperatures below 100 K, illumination in the visible range causes conversion of this state into two other EPR-active states. One of these is the H_ox state, i.e. the catalytically active form, characterized by a vacant external coordination site. The other state (H_LI) is probably characterized by dissociation of the bridging CO ligand. The photodissociation of the H_ox-CO state provides a convenient way to study changes in the electronic structure caused by dissociation of various CO ligands. In this article, we show results of an advanced EPR study of the light-induced species of the active site, in particular, H_LI. On the basis of the resolved hyperfine signals of the ¹H, ¹⁴N, ¹³C, and ⁵⁷Fe nuclei, we obtained a detailed picture of the electronic structure of the H-cluster. It turned out that dissociation of the bridging CO ligand causes a major rearrangement of the structure of the H-cluster and large changes in the distribution of the unpaired spin. Moreover, it appears that the extent of spin delocalization in the 2Fe subcluster in the case of H_LI is intermediate between that of H_ox and H_ox-CO.