Spectroscopic Characterization of an Engineered Purple Cu_A Center in Azurin

Spectroscopic characterization of a purple Cu_A center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple Cu_A azurin hereafter) is presented. Both electrospray mass spectrometry and copper analysis indicated the protein binds two copper ions per protein. The electronic absorption (UV-vis) magnetic circular dichroism (MCD). multifrequency electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectra of the purple Cu_A azurin are strikingly similar to other native or engineered Cu_A centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a Cu_A center with absorption bands at 485 (ε = 3730), 530 (ε = 3370), 360 (ε = 550), and 770 nm (ε = 1640 M^-1 cm^-1) The MCD spectrum of purple Cu_A azurin is dominated by a pair of intense, oppositely-signed features occurring at 480 nm (Δε = -118 deg M^-1 cm^-1 T^-1) and 530 nm (Δε = 155 deg M^-1 cm^-1 T^-1) and a negative feature occurring at 810 nm (Δε = -52 deg M^-1 cm^-1 T^-1). Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g_∥ region, typical of a delocalized mixed-valence [Cu(1.5)⋯ Cu(1.5)] binuclear center. Compared with other delocalized mixed-valence Cu_A centers, this purple Cu_A azurin has a relatively high energy near-IR Cu-Cu σ → σ* absorption at 770 nm. the largest A→ at 55 G, and the shortest Cu-Cu distance at 2.39 Å. These results may reflect a more sterically compressed Cu_A center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear Cu_A center. and are consistent with the general trend between the near-IR Cu-Cu σ → σ* absorption and the degree of Cu₂(SR)₂ core contraction.