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 ββMTH and βαMTH and three site-directed mutants, ββC9H, βαC37H, and βαE31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of MII6-MTH for the entire MTH and MII3-ββMTH and M II3-βαMTH 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 βα domain. Additionally, minor MII4 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-MT species and molecular phylogenetic analysis.