Hydrogenases comprise a variety of enzymes that catalyze the reversible oxidation of molecular hydrogen. Out of this group, [FeFe]-hydrogenase shows the highest activity for hydrogen production which is, therefore, of great interest in the field of renewable energies. Unfortunately, this comes with the flaw of a generally very high sensitivity against molecular oxygen that irreversibly inhibits this enzyme. While many studies have already addressed the mechanism of hydrogen formation by [FeFe]-hydrogenase, little is kown about the molecular and mechanistic details leading to enzyme inactivation by O-2. In order to elucidate this process, we performed density functional theory calculations on several possible O-2 adducts of the catalytic center - the so-called H-cluster - and show that the direct interaction of the [2Fe](H) subsite with dioxygen is an exothermic and specific reaction in which O-2 most favorably binds in an end-on manner to the distal Fed. Based on the results, we propose a protonation mechanism that can explain the irreversibility of dioxygen-induced enzyme inactivation by water release and degradation of the ligand environment of the H-cluster.