Many of the active sites involved in electron transfer (ET) in biology have more than one metal and are mixed valent in at least one redox state. These include Cu(A), and the polynuclear Fe-S clusters which vary in their extent of delocalization. In this tutorial review the relative contributions to delocalization are evaluated using S K-edge X-ray absorption, magnetic circular dichroism and other spectroscopic methods. The role of intra-site delocalization in ET is considered.

Using a combination of electronic spectroscopies and DFT calculations, the effect of pH perturbation on the geometric and electronic structure of the CuA site has been defined. Descriptions are developed for high pH (pH = 7) and low pH (pH = 4) forms of CuA azurin and its H120A mutant which address the discrepancies concerning the extent of delocalization 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 CuA site. However, in going from high pH to low pH, a seven-line hyperfine coupling pattern associated with complete delocalization 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 approximately 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 delocalization 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.

For the CuA site in the protein, sigmau* and piu are the ground and lowest energy excited-states, respectively. EPR data on CuA proteins show a low g parallel value of 2.19 which derives from spin-orbital coupling between sigmau* and piu which requires an energy gap between sigmau* and piu of 3000-4500 cm-1. On the other hand, from paramagnetic NMR studies, it has been observed that the first excited-state is thermally accessible and the energy gap between the ground state and the thermally accessible state is approximately 350 cm-1. This study addressed this apparent discrepancy and evaluated the roles of the two electronic states, sigmau* and piu, in electron transfer (ET) of CuA. The potential energy surface calculations show that both NMR and EPR results are consistent with the electronic/geometric structure of CuA. The anti-Curie behavior observed in paramagnetic NMR studies of CuA results from the thermal equilibrium between the sigmau* and piu states which are at very close energies in their respective equilibrium geometries. Alternatively, the EPR g-value analysis involves the sigmau* ground state in the geometry with a short dCu-Cu where the piu state is a Frank-Condon excited-state with the energy of 3200 cm-1. The protein environment plays a role in maintaining CuA in the sigmau* state as a lowest-energy state with the lowest reorganization energy and high-covalent coupling to the Cys and His ligands for efficient intra- and intermolecular ET with a low-driving force.