Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin

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 (e = 3730), 530 (e = 3370), 360 (e = 550), and 770 nm (e = 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 (Δe = −118 deg M-1 cm-1 T-1) and 530 nm (Δe = 155 deg M-1 cm-1 T-1) and a negative feature occurring at 810...