The application of [FeFe] hydrogenases for renewable energy production is limited by the generally high O2-sensitivity of this class of enzymes. In this work, we report energy differences in oxygen coordination at the active sites of [FeFe] and monoiron (Hmd) hydrogenase as obtained from DFT calculations. Whereas O2 addition is clearly exothermic in the case of [FeFe] hydrogenase, it is endothermic for monoiron hydrogenase. By applying a recently proposed concept [Inorg. Chem.2010, 49, 5818], which allows us to structurally and electronically relate the two active sites, we show that an inversion in oxygen affinity is observed as a result of mutually exchanging the first ligand sphere of the central iron atoms of both catalytic centers. Modified Hmd active sites can bind O2 exothermically, whereas O2 addition to the altered H-cluster variants is less exothermic than in the wild-type configuration. Our results show that oxygen affinity is not very strong when measured in terms of the coordination energy. Hence, changes in the first ligand shell of the reactive iron atoms with retention of the similarity of the ligand spheres, that is, having the same type of ligands in the same positions of the coordination octahedron, can render oxygen coordination endo- or exothermic. This observation could explain the difference in oxygen sensitivity of Hmd and [FeFe] hydrogenases.