Perturbations to the geometric and electronic structure of the Cu _A site: Factors that influence derealization and their contributions to electron transfer

Using a combination of electronic spectroscopies and DFT calculations, the effect of pH perturbation on the geometric and electronic structure of the Cu_A site has been defined. Descriptions are developed for high pH (pH = 7) and low pH (pH = 4) forms of Cu_A azurin and its H120A mutant which address the discrepancies concerning the extent of derealization indicated by multifrequency EPR and ENDOR data (J. Am. Chem. Soc. 2005, 127, 7274; Biophys. J. 2002, 82, 2758). Our resonance Raman and MCD spectra demonstrate that the low pH and H120A mutant forms are essentially identical and are the perturbed forms of the completely delocalized high pH Cu_A site. However, in going from high pH to low pH, a seven-line hyperfine coupling pattern associated with complete derealization of the electron (S = 1/2) over two Cu coppers (I_Cu = 3/2) changes into a four-line pattern reflecting apparent localization. DFT calculations show that the unpaired electron is delocalized in the low pH form and reveal that its four-line hyperfine pattern results from the large EPR spectral effects of ∼1% 4s orbital contribution of one Cu to the ground-state spin wave function upon protonative loss of its His ligand. The contribution of the Cu-Cu interaction to electron derealization in this low symmetry protein site is evaluated, and the possible functional significance of the pH-dependent transition in regulating proton-coupled electron transfer in cytochrome c oxidase is discussed.