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

The copper(I) and silver(I) binding properties of the beta fragment of reco mbinant mouse metallothionein 1 have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of th e species formed was confirmed by electrospray mass spectrometry. The behav iour 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-bet a MT, Cu-7-beta MT and Cu-10-beta MT. In the first stage of the titration o f Zn-3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-be ta MT was detected. In contrast, the titration of Zn-3-beta MT at pH 7.5 an d of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pat hways, 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 correspon ding to species with the same stoichiometric ratio, Ag-6-beta MT and Ag-9-b eta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between c opper and silver binding to metallothionein may be questioned. (C) 1999 Els evier Science Inc. All rights reserved.