Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Abstract

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. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable O_H state, and a reductive phase, in which O_H is reduced back to the R state. During each phase, two protons are translocated across the membrane. 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. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a₃ iron and Cu_B in the active site of the O state, like those in the O_H state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, 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 insights into the proton translocation mechanism of CcO.