Transformation of the Cu-A redox site in cytochrome c oxidase into a mononuclear copper center

Subunit II Of the aa(3) type cytochrome c oxidase contains a binuclear copper center (CUA) which functions as the entry point for electrons donated by cytochrome c. We have introduced site-specific mutations in residues liganding the Cu-A center in the oxidase of the bacterium Paracoccus denitrificans; the purified, fully assembled enzyme complexes were analyzed by various techniques, including EPR, optical spectroscopy, and total-reflection X-ray fluorescence spectrometry, to determine metal to protein ratios. In the C216S mutant, the binuclear Cu-A Site is transformed into a mononuclear copper center. In contrast to wild type, the C216S mutant does no longer exhibit the characteristic absorption band in the near-infrared region of the optical spectrum that has been assigned to Cu-A. These major changes in the CUA Site Of this mutant correlate with an almost complete loss in catalytic activity.