Reversible Switching of Redox Active Molecular Orbitals and Electron Transfer Pathways in CuA Sites.

ULISES ZITARE; DAMIÁN ÁLVAREZ-PAGGI; MARCOS N. MORGADA; LUCIANO A. ABRIATA; ALEJANDRO J. VILA; DANIEL H. MURGIDA

Chascompús

Congreso; Latin American Meeting on Biological Inorganic Chemistry; 2014

INTRODUCTIONThe CuA site from cytochrome c oxidase is a redox hub that participates in electron transfer reactions in the mitochondrial electron transport chain. Previous studies in our lab show 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. To understand the fine tuning between them, we work on a CuA redox centers from the soluble subunit ba3 of Thermus thermophilus.A combination of spectroscopic and electrochemical characterizations performed on first and second sphere mutants have allowed us to experimentally detect the switching between those two alternative electronic states.EXPERIMENTAL METHODSProtein film voltammetry were performed in order to measure the electron transfer rate of adsorbed proteins.To follow changes in the alternative ground-states populations with pH, UV/Vis spectra were acquired from several samples solutions.Resonance Raman (RR) spectra were acquired using 514 nm excitation in order to analyze the behavior of several normal modes of frozen samples at varying pH.RESULTS AND DISCUSSIONThe M160H variant of a native CuA center unveils a pH transition responsible for fine tuning the energy gap between both electronic states, providing means for probing both states in the same protein species.Alternation between both states exerts a dramatic impact on the kinetic parameters of the electron transfer reaction.In particular, the electronic coupling may vary widely, and also overcompensates the changes on the reorganization energy.Moreover, the position of the ν2 band (260 cm-1) at RR spectra of WT and M160H is sensitive to pH and exhibits an apparent pKa of 3,1. We have previously reported a pH-dependent reversible transition with a similar pKa value in WT that exerted no impact on the UV/Vis spectra. This transition was ascribed to a faster exchange rate of the His 157 proton with the solvent3. Considering that the first-sphere ligand set is preserved, this change arises from small structural perturbations of the protein matrix. The ν2 band corresponds to an A1g vibrational mode that presents a significant contribution of Cu-N, Cu-S and Cu-Cu stretching, the latter having been proposed as the main reaction coordinate for the alternative ground-state transition4. Thus, ν2 is sensitive to perturbations in residue His157 through the contribution of the Cu-N stretching.The ν5 band (360 cm-1), which corresponds to a normal mode involving almost exclusively the Cu and S atoms, is not affected by pH in any protein variant.Alternation between both alternative ground-states is not observed in the WT variant presumably due to the larger energy gap that appears to render it insensitive to the perturbation taking place upon pH modification.CONCLUSIONThis hints at the alternation between electronic states as the underlying molecular mechanism behind the efficiency and directionality of the electron transfer reactions of CuA sites in vivo and may prove useful for the development of molecular electronics.