Spectroscopic study of the CuA fragment of T. thermophilus ba3 oxidase: wild type protein and mutants of the Met160 residue

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

Rosario, Santa Fe, Argentina

Congreso; XXXV Reunion Anual de la Sociedad Argentina de Biofísica; 2006

Sociedad Argentina de Biofísica

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 (l) arising from a rather rigid electron transfer site and/or the electronic coupling (HAB) 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. The electronic structure of the CuA site in its oxidized state was studied by paramagnetic NMR, and the redox potentials of different mutants replacing the weak axial ligand Met160 were measured. 15N 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. Variations in the axial Met exert a pronnunced effect on the corresponding redox potentials when compared to the wild type protein, in contrast with the small modifications observed in variants of an engineered CuA center in azurin[1]. However, the Met160 mutants 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 [2,3].   References: [1]  Hwang, H. J., Berry, S. M., Nilges, M.J. and Lu, Y., J. Am. Chem. Soc. 2005, 127, 7274-7275. [2] Bertini, I.; Bren, K. L.; Clemente, A.; Fee, J. A.; Gray, H. B.; Luchinat, C.; Malmstrom, B. G.; Richards, J. H.; Sanders, D. and Slutter, C. E. J. Am. Chem. Soc. 1996, 118, 11658-11659. [3]  Fernández, C.O.; Cricco, J. A.; Slutter, C. E.; Richards, J. H.; Gray, H. B.; Vila, A. J., J. Am. Chem. Soc. 2001, 123, 11678-11685.   Acknowledgments: CONICET, ANPCyT and NIH are acknowledged for financial support.