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 replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the Cu-A center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native Cu-A center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate, The azurin 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 detectable hyperfines, similar to those of the Cu-A sites in COX of bovine heart and of Paracoccus denitrificans.