The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5).Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.

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 (UVvis), 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 ( ) 3730), 530 ( ) 3370), 360 ( ) 550), and 770 nm ( ) 1640 M -1 cm -1 ). The MCD spectrum of purple Cu A azurin is dominated by a pair of intense, oppositelysigned features occurring at 480 nm (∆ ) -118 deg M -1 cm -1 T -1 ) and 530 nm (∆ ) 155 deg M -1 cm -1 T -1 ) and a negative feature occurring at 810 nm (∆ ) -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 f 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 f σ* absorption and the degree of Cu 2 (SR) 2 core contraction.

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