Distinguishing between Cl ^- and O ^2- ₂ as the bridging element between Fe ³⁺ and Cu ²⁺ 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: [Fe ³⁺-OH ^-] under enzymatic turnover versus [Fe ³⁺-O ₂ ^2--Cu ²⁺] 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 Fe ³⁺ and Cu ²⁺. 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.