IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
TUNING THE ELECTRONIC STRUCTURE OF ELECTRON TRANSFER COPPER PROTEINS THROUGH LOOP ENGINEERING
Autor/es:
ANDRÉS ESPINOZA CARA, LUCIANO ABRIATA, ALEJANDRO J. VILA
Lugar:
Rosario, Santa Fe
Reunión:
Congreso; XVIII Congreso Argentino de Fisicoquímica y Química Inorgánica; 2013
Resumen:
Nature employs metal ions for a wide variety of biological functions. Copper proteins play an important role in electron transfer processes. There are two types of copper electron transfer sites in nature: (a) mononuclear type I centers (T1), also known as blue sites, and (b) the dinuclear CuA center. Proteins with T1 and CuA centers share a conserved fold (cupredoxin fold) despite being from different organisms, revealing a common evolutionary origin. The functional properties of these redox sites cannot be compared directly because of being in different protein matrixes. The T1 and CuA centers presents and ideal situation for loop changing as they share the same protein fold and most of the metal ligands are present in loops. There are few examples using this strategy, but all of them suggest that the ligand-containing loops gather most of the structural information that regulates the function of the redox site. In this project we employ loop directed mutagenesis in order to introduce different T1 loops in the soluble fragment of the subunit II of the cytochrome ba3 oxidase from Thermus thermophilus, naturally harboring a CuA site (TtCuA). Two T1 variants were constructed, employing the loops from amicyanin (Ami-TtCuA) and azurin (Az-TtCuA). These proteins have distinct characteristics from the native sites. Using distinct biophysical approaches such as optical spectroscopy, EPR and NMR we determined that both proteins have properties that vary along those of rhombic T1 sites. Both proteins can also bind exogenous imidazole in a reversible way giving rise to a copper site similar spectral features as those found in nitrosocyanin. Taken together, these observations discard the ?loop defines everything? hypothesis and show how reversible binding of small molecules can be elicited in an otherwise occluded metal site.