PERTURBATION OF THE CU-A SITE IN CYTOCHROME-C-OXIDASE OF PARACOCCUS-DENITRIFICANS BY REPLACEMENT OF MET227 WITH ISOLEUCINE

Subunit II of cytochrome-e oxidase contains a redox centre, Cu-A, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site-directed mutant of cytochrome-e oxidase from Paracoccus denitrificans in which the Cu-A site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady-state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the Cu-A site can be observed in the EPR and near-infrared optical spectra. It is concluded that in the mutant Cu-A is still binuclear bur that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5)....Cu(1.5)] centre of the wild type into a localized [Cu(I)....Cu(II)] system. Changes in the overall kinetics of the mutant an correlated with a diminished electron transfer rate between Cu-A and heme a.