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 (MCS), 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) center, indicating that they all share similar geometric and electronic structures. Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g(parallel) 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 CuBCu absorption at 770nm, the largest A(paralle l) a t 55 G, and the shortest CuBCu distance at 2.39A. 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 CuBCu absorption and the degree of Cu2[SR]2 core contraction.
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