Preferential binding of copper to the beta domain of metallothionein.

Proteolytic studies of rat liver metallothionein reconstituted in vitro with Cu salts revealed that the 2 metal centers fill in an ordered fashion. The B cluster in the NH2-terminal beta domain fills prior to Cu binding in cluster A. This is in contradistinction to cluster formation induced by the binding of Cd or Zn ions in which cluster A is the center of initial binding. The formation of metal cluster B by Cu occurs in a cooperative fashion yielding a saturated cluster with approximately 6 Cu+ ions bound. The B cluster is saturated with Cd or Zn after binding of only 3 metal ions. The preferential binding of Cd and Cu to the alpha and beta domains, respectively, and the tolerance toward proteolysis of these 2 different half saturated molecules permit the isolation of each domain. The metal cluster in each isolated domain can be reversibly formed with predicted stoichiometries of Cd and Cu. The folding of the polypeptide therefore appears to create each cluster independently. The metal binding data suggest that Cu-metallothionein contains 11-12 Cu ions, 6 bound in the beta domain and 5-6 in the alpha domain. In contrast, Cd-metallothionein contains 7 Cd ions, 3 bound to beta and 4 to alpha.