Cytochrome c oxidase (C c O) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine C c O. It is assigned to the P _R -intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a ₃ iron atom is in a ferryl (Fe ⁴⁺ = O ²⁻ ) configuration, and heme a and Cu _B are oxidized while Cu _A is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.