A QM/MM investigation of the active-ready (Hox) form of FeFe-hydrogenase from D. desulfuricans, in which the electronic properties of all Fe-S clusters (H, F and F') have been simultaneously described using DFT, was carried out with the aim of disclosing a possible interplay between the H-cluster and the accessory iron-sulfur clusters in the initial steps of the catalytic process leading to H2 formation. It turned out that one-electron addition to the active-ready form leads to reduction of the F'-cluster and not of the H-cluster. Protonation of the H-cluster in Hox is unlikely, and in any case it would not trigger electron transfer from the accessory Fe4S4 clusters to the active site. Instead, one-electron reduction and protonation of the active-ready form trigger electron transfer within the protein, a key event in the catalytic cycle. In particular, protonation of the H-cluster after one-electron reduction of the enzyme lowers the energy of the lowest unoccupied molecular orbitals localized on the H-cluster to such an extent that a long-range electron transfer from the F'-cluster towards the H-cluster itself is allowed.