Abstract
The alpha- and beta-polypeptides of human metallothionein (isoform 2), obtained by chemical synthesis, were converted into their respective zinc/thiolate clusters, and each domain was investigated separately. Proton titration data for the N-terminal beta-domain fit a simple model with three ionizations of the same apparent pK(a) value of 4.9 and a collective binding constant for zinc of 5 x 10(-12) M at pH 7.0. The zinc cluster in the C-terminal alpha-domain is more stable than that in the beta-domain. Its pH titration is also more complex, indicating at least two classes of zinc sites with different affinities. The whole molecule is stabilized with regard to the individual domains. Chemical modification implicates lysine side chains in both the stabilization of the beta-domain cluster and the mutual stabilization of the domains in the whole molecule. The two zinc clusters also differ in the reactivity of their cysteine sulfurs and their potential to donate zinc to an acceptor molecule dependent on its type and characteristics. The isolated beta-domain cluster reacts faster with Ellman's reagent and is a better zinc donor toward zinc-depleted sorbitol dehydrogenase than is the isolated alpha-domain cluster, whereas the reverse is observed when a chelating agent is the zinc acceptor. Thus, although each cluster assembles independently of the other, the cumulative properties of the individual domains do not suffice to describe metallothionein either structurally or functionally. The two-domain structure of the whole molecule is important for its interaction with ligands and for control of its reactivity and overall conformation.