Structural basis for functional properties of cytochrome c oxidase

Cytochrome c oxidase (CcO) 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 synthesis¹. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized O state, and a reductive phase, in which O is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes². However, if O is allowed to relax to the resting oxidized state (O), a redox equivalent to O, its subsequent reduction to R is incapable of driving proton translocation^2,3. How the O state structurally differs from O remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)⁴, we show that the heme a₃ iron and Cu_B in the active site of the O state, like those in the O state^5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three Cu_B ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.