Ferryl and hydroxy intermediates in the reaction of oxygen with reduced cytochrome c oxidase

Abstract

CYTOCHROME c oxidase catalyses the 4-electron reduction of dioxygen to water and translocates protons vectorially across the inner mitochondrial membrane. Proposed reaction pathways for the catalytic cycle of the O₂ reduction^1–3 are difficult to verify without knowing the structures of the intermediates, but we now have such information for the catalytic intermediates in the first steps of the reaction of O₂ with cytochrome c oxidase from resonance Raman spectroscopy^4–6, a technique that enables iron-ligand stretching modes to be identified^4–7. Here we report on two more key intermediates: a ferryl–oxo (Fe⁴⁺=O²⁻) and a ferric-hydroxy (Fe³⁺–OH⁻) intermediate at the level of 3- and 4-electron reduction, respectively. We identified these intermediates by their characteristic iron-oxygen stretching frequencies (786cm⁻¹ for Fe⁴⁺=O²⁻, and 450cm⁻¹ for Fe³⁺–OH⁻) and oxygen and deuterium isotope shifts. The oxo atom in the ferryl intermediate is hydrogen-bonded and the iron–oxygen bond in the hydroxy intermediate is anomalously weak. With the identification of the primary, ferryl and hydroxy intermediates, the predominant structures at almost all stages of O2 reduction are now known and the catalytic pathway can be described with more certainty.