Structural characterization of the P_CO/O2 compound of cytochrome c oxidase

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 806 cm^-1 was observed in PCO/O2 produced by adding CO and O₂ to the resting enzyme. The vibrational band shifted to 771 cm ^-1 upon isotopic substitution of ¹⁶O₂ with ¹⁸O₂. The same modes at 806 and 771 cm^-1 were present simultaneously when the mixed isotope, ¹⁸O¹⁶O, was employed, indicating that in PCO/O2 the O-O bond is cleaved, resulting in a Fe⁴⁺O^2- structure. This result unifies the nature of the three equilibrium analogues of the P_R intermediate.