Structural rearrangements upon assembly of CuA centers

LUCIANO A. ABRIATA; MARÍA-EUGENIA ZABALLA; ANTONIO DONAIRE; ALEJANDRO J. VILA

Firenze, Italia

Congreso; EUROMAR 2010 and 17th ISMAR Conference; 2010

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:IT; mso-fareast-language:IT;} p.MsoBodyTextIndent, li.MsoBodyTextIndent, div.MsoBodyTextIndent {mso-style-noshow:yes; margin:0cm; margin-bottom:.0001pt; text-align:justify; text-indent:14.2pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:IT;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Cytochrome c oxidase (COX) is a terminal oxidase present in all aerobic organisms, which shuttles electrons from cytochrome c to oxygen using the released energy to pump protons across the membrane and thus contribute to its potential. It is embedded in the mitochondrial inner membrane of eukaryotic cells and in the cytoplasmic membrane of prokaryotes. The function of COX depends critically on the correct disposition of two copper centers (CuA and CuB) and two heme moieties (b/a and a3), hence the assembly of these cofactors is assisted by (co)chaperone proteins. Mutations on genes coding for these proteins lead to several fatal disorders in humans, related to inefficient energy production by mitochondria. We have previously shown that a thiol:disulfide oxidoreductase and a Cu(I) metallochaperone are required for in vitro maturation of the CuA site in Thermus thermophilus ba3 oxidase (TtCuA). During that work we gathered evidence that the structure and dynamics of the apoprotein are perturbed compared to the copper-bound form, in line with the fact that the copper center is embedded inside the holoprotein but the ligand loops must be somewhat exposed for metallation to occur in the apoprotein. Our current goal is to study the solution structure and dynamics of apoTtCuA relative to the holoprotein in the reduced state. All the backbone and most of the side chain resonances have been completely assigned for 118 out of 126 residues. Broadening is observed for most resonances assigned to nuclei in two ligand loops, while residues lacking resonance assignments are located in these loops. These observations indicate enhanced dynamics in these regions compared to the holoprotein, which is highly rigid along the entire sequence. These studies will be complemented aiming to describe the rearrangements that occur in the apoprotein upon copper insertion at atomic level and to provide a qualitative assessment of the contribution of metal binding to protein folding and stability.