α-Ketoglutarate (αKG)-dependent nonheme iron enzymes utilize a high-spin (HS) ferrous center to couple the activation of oxygen to the decarboxylation of the cosubstrate αKG to yield succinate and CO2, and to generate a high-valent ferryl species that then acts as an oxidant to functionalize the target CH bond. Herein a detailed analysis of the electronic-structure changes that occur in the oxygen activation by this enzyme was performed. The rate-limiting step, which is identical on the septet and quintet surfaces, is the nucleophilic attack of the distal O atom of the O2 adduct on the carbonyl group in αKG through a bicyclic transition state (5, 7TS1). Due to the different electronic structures in 5, 7TS1, the decay of 7TS1 leads to a ferric oxyl species, which undergoes a rapid intersystem crossing to form the ferryl intermediate. By contrast, a HS ferrous center ligated by a peroxosuccinate is obtained on the quintet surface following 5TS1. Thus, additional two single-electron transfer steps are required to afford the same FeIV–oxo species. However, the triplet reaction channel is catalytically irrelevant. The biological role of αKG played in the oxygen-activation reaction is dual. The αKG LUMO (CO π*) serves as an electron acceptor for the nucleophilic attack of the superoxide monoanion. On the other hand, the αKG HOMO (C1C2 σ) provides the second and third electrons for the further reduction of the superoxide. In addition to density functional theory, high-level ab initio calculations have been used to calculate the accurate energies of the critical points on the alternative potential-energy surfaces. Overall, the results delivered by the ab initio calculations are largely parallel to those obtained with the B3LYP density functional, thus lending credence to our conclusions.