Redox properties of an engineered purple CuA azurin

Abstract Purple Cu A centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu A protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu A center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation–reduction midpoint potential ( E m ) values of the Cu A azurin of +399±10 and +380±2 mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E m value of native azurin is pH dependent, the E m value of Cu A azurin is not, as expected for the Cu A center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu A centers in cytochrome c oxidase and Cu A azurin.