IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Electronic structure of CuA sites explored by NMR spectroscopy
Autor/es:
LUCIANO A. ABRIATA; GABRIELA N. LEDESMA; ALEJANDRO J. VILA
Lugar:
Hotel do Frade, Angra dos Reis, Brasil
Reunión:
Congreso; 12th Nuclear Magnetic Resonance Users Meeting; 2009
Institución organizadora:
AUREMN
Resumen:
<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;} @page Section1 {size:595.3pt 841.9pt; margin:3.0cm 3.0cm 70.9pt 3.0cm; mso-header-margin:35.45pt; mso-footer-margin:35.45pt; mso-paper-source:0;} div.Section1 {page:Section1;} /* List Definitions */ @list l0 {mso-list-id:997224563; mso-list-type:hybrid; mso-list-template-ids:375046112 2143711044 201981977 201981979 201981967 201981977 201981979 201981967 201981977 201981979;} @list l0:level1 {mso-level-text:%1-; mso-level-tab-stop:36.0pt; mso-level-number-position:left; text-indent:-18.0pt;} ol {margin-bottom:0cm;} ul {margin-bottom:0cm;} -->      Cytochrome c oxidases (COX) couple oxygen reduction with proton translocation across the inner membrane of mitochondria and bacteria, contributing to the accumulation of energy in the form of an electrochemical gradient.1,2 Subunit II of cytochrome c oxidase contains a soluble subunit exposed to the outer part, which bears a dinuclear copper termed the CuA site, which functions as the electron entry port in this oxidase as well as in most terminal oxidases. Electrons arriving from docked cytochrome c molecules are shuttled through the CuA center to the catalytic O2-reducing center located inside subunit I, nearly 20Å away. This process takes place with high efficiency (k = 105 s-1) even if the driving force is minimum.      The CuA site features two copper ions strongly bound to two bridging cysteins, two terminal histidine ligands and two weakly bound axial ligands (a methionine and a backbone carbonyl).3,4 The Cu2S2 moiety forms a highly covalent core that delocalizes the unpaired electron in the oxidized protein giving raise to a mixed-valence cluster, as evidenced by 1H-NMR, 1H-ENDOR, XAS and EPR experiments together with DFT calculations.5-8   Figure 1. The 3D structure of Thermus thermophilus ba3 oxidase, its three subunits and cofactors. The CuA center is zoomed in the inset.        According to Marcus' semiclassical theory, high efficiency in long-range electron transfer processes with low driving force requires a low reorganization energy and a high electronic coupling between the donor and acceptor sites. We have employed NMR to study both backbone dynamics in the soluble fragment of COX that bears the CuA site, as well as the electronic structure of the center in the wild type protein and in mutants where the axial Met160 ligand has been replaced by His or Gln resulting in fine tuning of the redox potential.9      Standard T1, T2, NOE and T1r relaxation experiments were measured in the wild type protein in order to probe backbone dynamics in the ps-ms time scales. These studies revealed a highly rigid structure in both oxidation states, in agreement with the low reorganization energy of this metal center.      The electronic structure of the CuA center was studied by analyzing the paramagnetically shifted 1H, 15N and 13C resonances from nuclei close to it. A combined strategy of direct and indirect detection experiments allowed us to assign all resonances from 1H, 13C and 15N nuclei of the Cys and His ligands in the wild type protein and in the two mutants. Our results confirm the large degree of delocalization in the wild type protein.10 The similar amount of unpaired spin density found at the Cb nuclei of the Cys residues indicates that the extent of delocalization is similar in the three proteins, even though the shifts of the Cys’ Ca suggest that the geometry of the site is somewhat perturbed in the mutants. Having all ligand resonances assigned, we can quantify the unpaired spin densities on all the nuclei of the ligands. Completion of the 1H assignment allowed us to find that delocalization also occurs through a hydrogen-bond from one of the cysteine S atoms to a backbone proton conserved in all known CuA sites, a situation similar to that observed for blue copper proteins but previously unnoticed.      Temperature dependence studies allowed us to determine the value of the energy gap between the ground and first excited states and to map the electron density in each state for the three isoforms. In the wild type protein, ground and excited state spin densities for atoms at dihedral positions of the sulfur p orbitals are in agreement with the symmetry proposed by theoretical calculations. The energy gap separating the ground and excited states is 600 cm-1 for the wild type protein10 and is influenced by the identity of the axial ligand.      These results show NMR is a valuable tool which can yield information that is unretrievable by other spectroscopies, and that can be linked to the functioning of electron transfer centers.     REFERENCES   1-   Ferguson-Miller, S.; Babcock, G. T.; Chem. Rev. 1996, 96, 2889-2907. 2-   Wikstrom, M.;  Biochim. Biophys. Acta 2004, 1655, 241-247. 3-   Williams, P. A.; Blackburn, N. J.; Sanders, D.; Bellamy, H.; Stura, E. A.; Fee, J. A.; McRee, D. E.;  Nat. Struct. Biol. 1999, 6, 509-516. 4-   Beinert, H.;  Eur. J. Biochem. 1997, 245, 521-532. 5-   Kroneck, P. M.; Antholine, W. E.; Kastrau, D. H.; Buse, G.; Steffens, G. C.; Zumft, W. G.  FEBS Lett. 1990, 268, 274-276. 6-   de Beer, S.; Markus, M.; Wang, H.; Wang, H.; Cramer, S. P.; Lu, Y.; Tolman, W. B.; Hedman, B.; Hodgson, K. O.; Solomon, E. I.;  J. Am. Chem. Soc. 2001, 123(24), 5757-5767. 7-   Epel, B.; Slutter, C. S.; Neese, F.; Kroneck, P. M.; Zumft, W. G.; Pecht, I.; Farver, O.; Lu, Y.; Goldfarb, D.  J. Am. Chem Soc. 2002, 124(27), 8152-8162. 8-   a) Bertini, I.; Bren, K. L.; Clemente, A.; Fee, J. A.; Gray, H. B.; Luchinat, C.; Malmström, B. G.; Richards, J. H.; Sanders, D.; Slutter, C. E.;  J. Am. Chem. Soc. 1996, 118(46), 11658-11659. b) Fernandez, C. O.; Cricco, J. A.; Slutter, C. E.; Richards, J. H.; Gray, H. B.; Vila, A. J. ;  J. Am. Chem. Soc. 2001, 123(47), 11678-11685. 9-   Olsson, M.; Ryde, U.  J. Am. Chem. Soc. 2001, 123, 7866-7876. 10-Abriata, L.A.; Ledesma, G.N.; Pierattelli, R.; Vila, A.J.; J. Am. Chem. Soc. 2009, 131(5), 1939-1946.     Funding: CONICET, ANPCYT, NIH