Distinguishing between Cl^- and O₂^2- as the bridging element between Fe³⁺ and Cu²⁺ in resting-oxidized cytochrome c oxidase

Fully oxidized cytochrome c oxidase (CcO) under enzymatic turnover is capable of pumping protons, while fully oxidized CcO as isolated is not able to do so upon one-electron reduction. The functional difference is expected to be a consequence of structural differences: [Fe³⁺-OH^-] under enzymatic turnover versus [Fe³⁺-O₂^2--Cu²⁺] for the as-isolated CcO. However, the electron density for O₂^2- is equally assignable to Cl^-. An anomalous dispersion analysis was performed in order to conclusively demonstrate the absence of Cl^- between the Fe³⁺ and Cu²⁺. Thus, the peroxide moiety receives electron equivalents from cytochrome c without affecting the oxidation states of the metal sites. The metal-site reduction is coupled to the proton pump.