ALTERNATIVE GROUND STATES IN ELECTRON TRANSFER COPPER PROTEINS PROBED BY EPR AND NMR SPECTROSCOPIES

ESPINOZA CARA, AM; LEGUTO, AJ; LLASES, ME; MORGADA, MN; VILA, AJ

Santa Fe

Workshop; III Workshop on Magnetic Resonance; 2016

Universidad Nacional del Litoral

The dinuclear copper center CuA is the electron entry point of cytochrome c oxidase (COX). CuA funnels the electrons from reduced cytochrome c to the subunit I of COX where O2 is then reduced to water molecules. These electron transfer (ET) reactions are highly efficient and this efficiency is given by the characteristic coordination chemistry of this center. Paramagnetic spectroscopies of the CuA center in its oxidized state (Cu+1.5-Cu+1.5) revealed the presence of two electronic levels, with different orbital symmetries, σu* and πu. EPR spectroscopy studies of COX II from Thermus thermophilus (Tt-COX II) was compatible with a ΔEσ/π of ca. 5000 cm-1,1 thus the σu* has been considered as the only redox active. NMR spectroscopy later revealed the presence of signals with NonCurie temperature dependence behavior, indicating that these levels are very close in energy, ca. 600 cm-1, and both can be populated at room temperature.2Studies made with mutants of the first coordination sphere of the CuA site in Tt-COX II, showed that the energy gap between these two electronic levels can be fine tuned and mutations that cause an increase in the population of the πu state are not detrimental in the ET process, demonstrating that πu state is also redox active in the electron transfer process.3Recently we also found that mutations in the second coordination sphere also tune the energy gap, incrementing the population in the πu states, by replacing the loops surrounding the metal site in Tt-COX II by the ones present in an eukaryotic homologue, COX II*, maintaining the same ET capabilities.4Here we will show that a first coordination sphere mutations on COX II* tune theelectronic structure of the CuA site in the same direction as in Tt-COX II. In fact, combining both axial ligand mutations and second sphere perturbations we generated a soluble and ET functional mutant with completely degenerated molecular orbitals.This is the first time a COX II variant with equal populations in both alternative ground states is reported.