CONSTRUCTION AND CHARACTERIZATION OF AN AZURIN ANALOG FOR THE PURPLE COPPER SITE IN CYTOCHROME-C-OXIDASE

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by re placing the loop containing the three ligands to the blue copper cente r with the corresponding loop of the Cu-A center in cytochrome c oxida se (COX) from Paracoccus denitrificans. The electronic absorption in t he UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the na tive Cu-A center in COX from Paracoccus denitrificans. The above spect ra can be obtained upon addition of a mixture of Cu2+ and Cu+. Additio n of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectr um can be converted to the spectrum of a pure purple copper by a prolo nged incubation in the air, or by addition of excess ascorbate, The az urin mutant reported here is an example of an engineered purple copper center with the A(480)/A(530) ratio greater than 1 and with no detect able hyperfines, similar to those of the Cu-A sites in COX of bovine h eart and of Paracoccus denitrificans.