NMR Study of the CuA fragment of T. thermophilus ba3 Oxidase

ABRIATA, L. A.; LEDESMA, G. N.; FERNANDEZ, C. O.; VILA, A. J.

Göttingen, Alemania

Conferencia; XXII International Conference on Magnetic Resonance in Biological Systems; 2006

Comité Organizador de XXII International Conference on Magnetic Resonance in Biological Systems

Cytochrome c oxidases (COX) catalyze oxygen reduction by coupling electron transfer process with transmembrane proton pumping. The initial electron acceptor in COX is CuA, a delocalized mixed-valence dimetallic copper center. The metal ions are strongly coordinated to two bridging Cys and two terminal His ligands. Two weak axial ligands are also present: Met160 and a backbone carbonyl.Electron transfer through CuA occurs over long distances and is driven by small energy differences; nevertheless, it is an efficient process. According to semiclassical Marcus theory, this efficiency can be due to a small reorganization energy (lambda) arising from a rather rigid electron transfer site and/or the electronic coupling (H_AB) between donor and acceptor. We have tried to study the contributions of these factors by exploring by NMR the backbone dynamics and the spin electron delocalization, respectively. The system employed was the soluble fragment of T. thermophilus ba3 oxidase containing the CuA site.^15 N relaxation measurements revealed a low backbone mobility in both oxidation states, despite the fact that most of the metal ligands are located in loops. This is in agreement with the low reorganization energy measured by electrochemical methods. The electronic structure of the CuA site in its oxidized state was studied by paramagnetic NMR. Different mutants replacing the weak axial ligand Met160 were shown to display a delocalized, mixed valence Cu(1.5)–Cu(1.5) center, as the wild type protein, perturbed by the identity of the introduced residue. The energy of a low-lying excited state, that becomes populated at room temperatures, seems to be influenced by changes in this position, suggesting that weak axial ligands play a role in tuning the electronic structure of CuA sites in nature [1,2].References[1] Bertini et al., J. Am. Chem. Soc. 1996, 118, 11658-11659[2] Fernández et al., J. Am. Chem. Soc. 2001, 123, 11678-11685.AcknowledgementsCONICET, ANPCyT and NIH are acknowledged for financial support.