[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 Hox-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 Hox state, i.e. the catalytically active form, characterized by a vacant external coordination site. The other state (HLI) is probably characterized by dissociation of the bridging CO ligand. The photodissociation of the Hox-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, HLI. 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 HLI is intermediate between that of Hox and Hox-CO.