Open AccessMetal‐binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae
Author(s) -
Urvoas Agathe,
Moutiez Mireille,
Estienne Clément,
Couprie Joël,
Mintz Elisabeth,
Le Clainche Loïc
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04001.x
Subject(s) - enterococcus hirae , copper , chemistry , dissociation constant , electrospray ionization , stoichiometry , dimer , inorganic chemistry , crystallography , mass spectrometry , chromatography , organic chemistry , biochemistry , receptor , enterococcus , antibiotics
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 buffer, 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 molecule (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 metal‐binding site allowed us to follow the binding of cadmium to EhCopZ by fluorescence spectroscopy and to determine the corresponding dissociation constant ( K d = 30 n m ). Competition experiments were performed with mercury, copper and cobalt, and the corresponding dissociation 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 living cells and may provide new clues in the determination of the mechanism of the uptake and transport of copper by the chaperone EhCopZ.