Structural Basis of Electron Transfer Modulation in the Purple Cu_A Center

Abstract

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.

pH-dependent transition between delocalized and trapped valence states of a Cu A center and its possible role in proton-coupled electron transfer

“…It is well known that the reduction potential of azurin is pH dependent due to the protonation͞deprotonation equilibrium of the uncoordinated His-35 and His-83 residues (45,46). Because no significant perturbation was observed in the position of His-35 and His-83 in Cu A azurin upon introduction of the new metal-binding site by using loop-directed mutagenesis (12), protonation͞ deprotonation of His-35 and His-83 in Cu A azurin should affect the reduction potential to a similar extent as in WT azurin. However, the difference in reduction potential (70 mV) of Cu A azurin and His120Ala mutant at high pH (Ͼ 5.5) should purely represent the difference in reduction potential of the Cu A center in the totally delocalized state (Cu A with His-120 ligation) and in the valence-trapped state (His120Ala) in the same overall protein environment.…”

“…1) (5)(6)(7)(8)(9). The distance between the two copper ions is short (Ϸ2.4 Å) (5,(10)(11)(12)(13)(14), suggesting a weak CuOCu bond (6). Each copper is also coordinated by a N␦ (His) , with weak axial ligand interactions approximately perpendicular to the plane defined by the Cu 2 (S Cys ) 2 core.…”

Axial interactions in the mixed-valent Cu A active site and role of the axial methionine in electron transfer

“…Fig. S5) (11). This model consists of the protein backbone loop connecting the two bridged S(Cys) residues as well as the equatorial His residues and both axial S(Met) and carbonyl backbone axial ligands to the Cu centers (93 total atoms).…”

“…The first coordination sphere of the classic BC sites [e.g., plastocyanin (Pc) and azurin (Az)] consists of a trigonally distorted tetrahedral environment where Cu resides in an equatorial plane formed by one S(Cys) and two N(His) ligands and has an axial S (Met) ligand ( (Fig. 1B) (8)(9)(10)(11). Both sites carry out rapid, efficient longrange ET with rates on the order of 10 3 -10 5 s −1 (12,13).…”

Recent Applications of Hybrid Ab Initio Quantum Mechanics – Molecular Mechanics Simulations to Biological Macromolecules

“…However, several distinct experimental results were reported. For example, in the engineered azurin, in which the Cu A site was incorporated to mimic that of CcO, the roles of the Met residue were analyzed by measuring the redox potentials of several mutants (Hwang et al, 2005;Robinson et al, 1999). This analysis revealed only slight changes in the redox potential of the mutants.…”