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 site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ 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 Ca2+ ligand.

Calcium ion binds reversibly with cytochrome c oxidase from beef heart mitochondria (K d∼2 μM) shifting α‐ and γ‐absorption bands of heme a to the red. Two sodium ions compete with one Ca2+ for the binding site with an average dissociation constant ∼3.6 mM. The Ca2+‐induced spectral shift of heme a is specific for mammalian cytochrome c oxidase and is not observed in bacterial or yeast aa 3 oxidases although the Ca2+‐binding site has been revealed in the bacterial enzyme [Ostermeier, C., Harrenga, A., Ermler, U. and Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547–10553]. As His‐59 and Gln‐63 involved in Ca2+ binding with Subunit I of P. denitrificans oxidase are not conserved in bovine oxidase, these residues have to be substituted by alternative ligands in mammalian enzyme, which is indeed the case as shown by refined structure of bovine heart cytochrome oxidase (S. Yoshikawa, personal communication). We propose that it is interaction of Ca2+ with the species‐specific ligand(s) in bovine oxidase that accounts for perturbation of heme a. The Ca2+/Na+‐binding site may be functionally associated with the exit part of `pore B' proton channel in subunit I of mammalian cytochrome c oxidase.