Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c.

Site-directed mutagenesis in subunit II of the cytochrome c oxidase (haem aa3) from Paracoccus denitrificans reveals that two carboxylic residues, Glu-246 and Asp-206 (corresponding to 198 and 158 in the bovine subunit II), are involved in the binding of cytochrome c. Spectrophotometric and polarographic measurements with the isolated enzymes of both mutant strains show a strongly reduced activity compared to wild-type oxidase, with the overall catalytic capacity (kcat/KM) of both mutants decreased about 8-fold. EPR spectra reveal no significant differences between the wild-type and the mutant enzymes, indicating that neither residue contributes significantly to the structure of the CuA centre. We conclude that Glu-246 and Asp-206 constitute an essential part of the binding site for cytochrome c.