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 A₄₈₀/A₅₃₀ 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.