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 reduction1-3 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 spectroscopy4-6, a technique that enables iron-ligand stretching modes to be identified4-7. 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.
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