tailieunhanh - Báo cáo khóa học: Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae

We studied the interaction of several metal ions with the copper chaperone fromEnterococcus hirae(EhCopZ). We show that the stoichiometry of the protein–metal complex varies with the experimental conditions used. At high con-centration of the protein in a noncoordinatingbuffer, a dimer, (EhCopZ)2–metal, was formed. The presence of a potentially coordinatingmolecule L in the solution leads to the formation of a monomeric ternary complex, EhCopZ– Cu–L, where L can be a buffer or a coordinatingmole-cule (glutathione, tris(2-carboxyethyl)phosphine). . | Eur. J. Biochem. 271 993-1003 2004 FEBS 2004 doi Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae Agathe Urvoas1 Mireille Moutiez1 Clement Estienne1 Joel Couprie1 Elisabeth Mintz2 and Lo ic Le Clainche1 1 Departement d lngeniierie et d Etudes des Proteines Direction des Sciences du Vivant CEA Saclay Gif sur Yvette France 2Laboratoire de Biophysique Moleculaire et Cellulaire UMR 5090 CFA-CNRS Universite Joseph Fourier Direction des Sciences du Vivant CEA Grenoble France We studied the interaction of several metal ions with the copper chaperone from Enterococcus hirae EhCopZ . We show that the stoichiometry of the protein-metal complex varies with the experimental conditions used. At high concentration of the protein in a noncoordinating Uuffer a dimer EhCopZ 2-metal was formed. The presence of a potentially coordinating molecule L in the solution leads to the formation of a monomeric ternary complex EhCopZ-Cu-L where L can be a buffer or a coordinating moee-cule glutathione tris 2-carboxyethyl phosphine . This was demonstrated in the presence of glutathione by electrospray ionization MS. The presence of a tyrosine close to the metalbindingsite allowed us to follow the bindingof cadmium to EhCopZ by fluorescence spectroscopy and to determine the corresponding distociation fontlanl Kd 30 dm . Competition experiments were performed with mercury copper and cobalt and the correspondingdissociation constants were calculated. A high preference for copper was found with an upper limit for the dissociation constant of 10 12 M. These results confirm the capacity of EhCopZ to bind copper at very low concentrations in livingcells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ. Keywords copper transport CopZ metal binding metal-lochaperone selectivity. Copper is a first-row transition metal which plays a fundamental role