RESUMEN
CuA centers perform efficient long-range electron transfer. The electronic structure of native CuA sites can be described by a double-potential well with a dominant σu* ground state in fast equilibrium with a less populated πu ground state. Here, we report a CuA mutant in which a lysine was introduced in the axial position. This results in a highly unstable protein with a pH-dependent population of the two ground states. Deep analysis of the high-pH form of this variant shows the stabilization of the πu ground state due to direct binding of the Lys residue to the copper center that we attribute to deprotonation of this residue.
RESUMEN
Electron transfer is the simplest chemical reaction and constitutes the basis of a large variety of biological processes, such as photosynthesis and cellular respiration. Nature has evolved specific proteins and cofactors for these functions. The mechanisms optimizing biological electron transfer have been matter of intense debate, such as the role of the protein milieu between donor and acceptor sites. Here we propose a mechanism regulating long-range electron transfer in proteins. Specifically, we report a spectroscopic, electrochemical, and theoretical study on WT and single-mutant Cu(A) redox centers from Thermus thermophilus, which shows that thermal fluctuations may populate two alternative ground-state electronic wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways. These findings suggest a unique role for alternative or "invisible" electronic ground states in directional electron transfer. Moreover, it is shown that this energy gap and, therefore, the equilibrium between ground states can be fine-tuned by minor perturbations, suggesting alternative ways through which protein-protein interactions and membrane potential may optimize and regulate electron-proton energy transduction.