Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase

Recent structure determinations suggested a new binding site for a non-redo x active metal ion in subunit I of cytochrome c oxidase both of mitochondri al and of bacterial origin, We analyzed the relevant metal composition of t he bovine and the Paracoccus denitrificans enzyme and of bacterial site-dir ected mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was fo und, 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 additio n, which was reversed by Na ions. This spectral behavior, largely comparabl e to that of the mitochondrial enzyme, was not observed for the bacterial W T oxidase, Further structure refinement revealed a tightly bound water mole cule as an additional Ca2+ ligand. (C) 1999 Federation of European Biochemi cal Societies.