Structural basis for functional properties of cytochromecoxidase

Abstract

Cytochromecoxidase (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. 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 oxidizedOHstate, and a reductive phase, in whichOHis reduced back to theRstate. During each of the two phases, two protons are translocated across the membranes. However, ifOHis allowed to relax to the resting oxidized state (O), the redox equivalent toOH, its subsequent reduction toRis incapable of driving proton translocation. How theOstate structurally differs fromOHremains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the hemea3iron and CuBin the active site of theOstate, like those in theOHstate, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuBligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishesOfromOH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics ofOprovide new insights into the proton translocation mechanism of CcO.