A new insight into the Ag+ and Cu+ binding sites in the metallothionein β domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein 1 have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn-3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu-6-beta MT, Cu-7-beta MT and Cu-10-beta MT. In the first stage of the titration of Zn-3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn-3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag-6-beta MT and Ag-9-beta MT or Ag-3-beta MT, Ag-6-beta MT and Ag-9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag-6-beta MT and Ag-9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned. (C) 1999 Elsevier Science Inc. All rights reserved.