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Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase

Significance Crystal structures of intermediates in the reaction of oxygen (O2) with cytochrome c oxidase (CcO) have never been determined, limiting our understanding of the coupling between O2 reduction and proton translocation. With time-resolved serial femtosecond crystallography, the structure of a catalytic intermediate was obtained in the reaction of reduced microcrystals of bovine CcO with O2. This intermediate, identified as the PR species formed in the R → A → PR reaction, is composed of a heme a3 ferryl (Fe4+ = O2−) structure and an electronic configuration of the metal centers of [a34+ = O2−, CuB2+-OH−, a3+, CuA1+]. There is a 120° rotation of the heme a farnesyl side chain in PR, suggesting its role in proton gating in mammalian CcOs. Cytochrome c oxidase (CcO) 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 CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2−) configuration, and heme a and CuB are oxidized while CuA 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.