Structural basis for functional properties of cytochrome c oxidase

Cytochrome c oxidase (C c O) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis[1][1]. The full turnover of the C c O reaction involves an oxidative phase, in which the reduced enzyme ( R ) is oxidized by molecular oxygen to the metastable oxidized O H state, and a reductive phase, in which O H is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes[2][2]. However, if O H is allowed to relax to the resting oxidized state ( O ), a redox equivalent to O H , its subsequent reduction to R is incapable of driving proton translocation[2][2],[3][3]. How the O state structurally differs from O H remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)[4][4], we show that the heme a 3 iron and CuB in the active site of the O state, like those in the O H state[5][5],[6][6], are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O H , where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of C c O. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6