Outer-Sphere Effects on Reduction Potentials of Copper Sites in Proteins: The Curious Case of High Potential Type 2 C112D/M121E Pseudomonas aeruginosa Azurin

Redox and spectroscopic (electronic absorption, multifrequency electron paramagnetic resonance (EPR), and X-ray absorption) properties together with X-ray crystal structures are reported for the type 2 CuII C112D/M121E variant of Pseudomonas aeruginosa azurin. The results suggest that CuII is constrained from interaction with the proximal glutamate; this structural frustration implies a “rack” mechanism for the 290 mV (vs NHE) reduction potential measured at neutral pH. At high pH (∼9), hydrogen bonding in the outer coordination sphere is perturbed to allow axial glutamate ligation to CuII, with a decrease in potential to 119 mV. These results highlight the role played by outer-sphere interactions, and the structural constraints they impose, in determining the redox behavior of transition metal protein cofactors.