The structural properties of a key transient oxygen intermediate of cytochrome c oxidase, P R , remain an enigma, although inferences have been drawn from its equilibrium analogues, PCO/O2, P H and P M . With resonance Raman spectroscopy, an oxygen isotope-sensitive band at 806cm −1 was observed in PCO/O2 produced by adding CO and O 2 to the resting enzyme. The vibrational band shifted to 771cm −1 upon isotopic substitution of 16 O 2 with 18 O 2 . The same modes at 806 and 771cm −1 were present simultaneously when the mixed isotope, 18 O 16 O, was employed, indicating that in PCO/O2 the O–O bond is cleaved, resulting in a Fe 4+ O 2− structure. This result unifies the nature of the three equilibrium analogues of the P R intermediate.