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 synthesis(1). 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(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). 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(2,3). 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), we show that the heme a(3) iron and Cu(B) in the active site of the O state, like those in the O(H) 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(H), where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.