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Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial sitedirected mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca 2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca 2+ ligand. z 1999 Federation of European Biochemical Societies.

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Subunit II of the aa3 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 CuA 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 CuA 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 CuA. These major changes in the CuA site of this mutant correlate with an almost complete loss in catalytic activity.