Distinguishing between Cl- and O22- 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+-O-2(2-)-Cu2+] for the as-isolated CcO. However, the electron density for O-2(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.