Structural basis of electron transfer modulation in the purple Cu-A center

The X-ray structure of an engineered purple Cu-A center in azurin from Pseu domonas aeruginosa has been determined and refined at 1.65 Angstrom resolut ion. Two independent purple Cu-A azurin molecules are in the asymmetric uni t of a new P2(1) crystal, and they have nearly identical conformations (rms d of 0.27 Angstrom for backbone atoms). The purple Cu-A azurin was produced by the loop-engineering strategy, and the resulting overall structure is u nperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. T he high-resolution structure reveals the detailed environment of the deloca lized mixed-valence [Cu(1.5)... Cu(1.5)] binuclear purple Cu-A center, whic h serves as a useful reference model for other native proteins, and provide s a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-C u distances of 2.42 and 2.35 Angstrom (with respective concomitant adjustme nts of ligand-Cu distances) are consistent with that (2.39 Angstrom) obtain ed from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complex es. A comparison of the purple Cu-A azurin structure with those of other Cu -A centers reveals an important relationship between the angular position o f 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 strong ly suggests that the fine structural variation of different Cu-A centers ca n be correlated with the angular positions of the two histidine rings becau se, from these positions, one can predict the relative axial ligand interac tions, which are responsible for modulating the Cu-Cu distance and the elec tron transfer properties of the Cu-A centers.