Experiments were done to define the stoichiometry of binding of Cu(I) to metallothionein (MT) and to determine its sites of binding in mixed-metal species. Spectrophotometric titrations of rabbit liver Cd,-MT 2, apoMT, and Cd-4-alpha-domain with Cu(I) revealed endpoints of 3-4, 4 and 8, and 4 and 6-7 added Cu(I)/mol of MT for the three species respectively. Observed endpoints depended on conditions of the titration and the wavelength chosen for absorbance measurement. Nevertheless, from metal and sulphydryl analyses of titrated proteins that were pretreated with Chelex-100 to remove metal ions from solution, almost all of the cadmium was displaced from Cd-7-MT by the addition of 7 Cu(I)/mol of MT. Similarly, 4 Cu(I)/mol of Cd-4-alpha-domain completely displaced bound cadmium. The Cu-4-alpha-domain was converted into a Cu-6-alpha species upon addition of two equivalents of Cu(I)/mol of alpha-domain. Reaction of Cd-7-MT with 7, 12 and 20 Cu/mol of MT, followed by reaction with Chelex resin, generated protein samples in each case with about 7 Cu/mol of MT. Cd-111-NMR analysis of the reaction of Cd-111(7)-MT with Cu(I) showed that nearly co-operative one-for-onk replacement of Cd-111 occurred and that the beta-domain cluster reacted before the alpha-domain cluster. Two mixed-metal MTs with Cu to Zn ratios approximating 3 to 4 and 6 to 4 were isolated from calf liver. After substitution of Zn with Cd-111, NMR spectra of each protein showed that Cd-111 was confined almost completely to the alpha-domain. By inference, about 3 or 6 Cu were bound in the beta-domain of these proteins. Supporting this segregation of metal ions into domains, reaction of Cu-6,Zn-4-MT with nitrilotriacetate removed zinc exclusively, whereas reaction of Cu-6,Cd-4-MT with 4,7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline extracted only Cu(I). Proteolytic digestion of both products followed by gel filtration demonstrated that Cu(I) and Cd were bound to fragments of the intact protein. Finally, reaction of rabbit liver Cd-111(7)-MT 2 With Cu-10-MT 2 resulted in interprotein metal exchange in which Cd-111 moved from the beta- to the a-domain according to NMR analysis. In contrast with the prevalent view that six Cu(I) bind to each domain of MT, the present results show that Cu(I) binds to MT with a minimum stoichiometry of about 7 Cu(I)/mol of MT and can bind to the alpha-domain with stoichiometries of 4 or 6 Cu(I)/mol of MT. Although MTs interacting with 12 or 20 Cu(I)/mol of MT are less stable than that binding about 7 Cu(I)/mol, it appears that MT can bind Cu(I) in multiple stoichiometries.
