A New Insight into Metallothionein (MT) Classification and Evolution: The in vivo and in vitro metal binding features of Homarus americanus recombinant MT

We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc- and copper-supplemented medium was achieved for MTH, the two domains beta beta MTH and beta alpha MTH and three site-directed mutants, beta beta C9H, beta alpha C37H, and beta alpha E31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M-6(II)-MTH for the entire MTH and M-3(II)-beta beta MTH and M-3(II)-beta alpha MTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the in vivo synthesized MTH was located in the beta alpha domain. Additionally, minor M-4(II) species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their in vivo zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. In vivo synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-AIT species and molecular phylogenetic analysis.