Spectroscopic characterization of a purple CuA center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple CuA 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 CuA azurin are strikingly similar to other native or engineered CuA centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a CuA 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 CuA 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 CuA centers, this purple CuA 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 CuA center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear CuA 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.
All Science Journal Classification (ASJC) codes
- Physical and Theoretical Chemistry
- Inorganic Chemistry