After much multidisciplinary research into metallothioneins (MTs), the ubiquitous metal-binding proteins first described by Vallee and Margoshes [1] in 1957, there is still little information on the structures and functions [2,3] of the biological metal-MT complexes. There are two main obstacles to studying the physiological features of MTs. First, although MTs are present in all living organisms except Eubacteria, most of the existing data refers to mammalian MTs, which precludes any homology-driven structural, functional, or evolutionary inference because of the extreme sequence heterogeneity of this family of metalloproteins (see http:// www.biochem.unizh.ch/mtpage/MT.html). Second, the difficulties encountered when trying to obtain homogeneous native preparations have led to the common utilization of in vitro reconstituted metal-MT complexes, based on the assumption that they represent genuine structural and functional native MT species. Thus, most of the data available to date, especially referring to MT structure, comes from nonbiological characterization of metal-MT complexes. [4] Following the MT discovery, another class of eukaryote metal-coordinating molecules was reported in plants and fungi: the enzymatically synthesized g-glutamyl (g-EC) peptides, also called phytochelatins and cadystins, which have always been considered as providing a very different mechanism of metal detoxification compared to the geneencoded MTs, sharing few, if any, structural and functional features with them. From the chemical point of view, MTs bind a variety of metal ions giving rise to individual polynuclear clusters that are linked exclusively to cysteine residues through thiolate bonds. In contrast, g-EC peptides create oligomeric clusters with a variable number of units that, most significantly, include acid-labile sulfide (S 2À ) ions as additional ligands which induce the clusters to evolve to peptide-coated particles, so-called crystallites. [5] Herein, we report the first definite evidence that sulfide ions are also present in nearly all the recovered Zn II -MT and Cd II -MT complexes, but never in the Cu I -MT species of a wide range of recombinant metal-MT aggregates, thus sulfide ions are found in species formed in vivo, that is, in a physiological, although heterologous, environment. We have determined the presence of the acid-labile S 2À ligands both qualitatively and quantitatively by analytical, spectroscopic, and spectrometric techniques, and it is clear that the features of the recovered Zn II -MT and Cd II -MT complexes correlate well with those reported for plant and yeast Zn-or Cd-gglutamyl peptides, [5] therefore bridging the behavior gap between both types of metal-binding molecules.Recombinant expression in E. coli has permitted the routine synthesis of a large number of proteins that are difficult or even impossible to obtain in their native forms. MTs stand out among them because of their extreme complexity and heterogeneity. Nearly ten years ago we developed an E. coli expression system that allows ...

Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA , MtnB , MtnC , MtnD , all of which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. Here we show, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the individual metallothionein mutants, the most prominent distinction between them was that MtnA-defective flies were the most sensitive to copper load, while MtnB-defective flies were the most sensitive to cadmium. Using various reporter gene constructs and mRNA quantification, we show that the MtnA promoter is preferentially induced by copper, while the MtnB promoter is preferentially induced by cadmium. Such a metal preference is also observed at the protein level as the stoichiometric, spectrometric and spectroscopic features of the copper and cadmium complexes with MtnA and MtnB correlate well with a greater stability of copper-MtnA and cadmium-MtnB. Finally, MtnC and MtnD, both of which are very similar to MtnB, display lower copper and cadmium binding capabilities compared to either MtnA or MtnB. In accordance with these binding studies, Drosophila mutants of MtnC or MtnD have a near wild type level of resistance against copper or cadmium load. Furthermore, eye-specific over-expression of MtnA and MtnB , but not of MtnC or MtnD , can rescue a "rough eye" phenotype caused by copper load in the eye. Taken together, while the exact roles of MtnC and MtnD remain to be determined, the preferential protection against copper and cadmium toxicity by MtnA and MtnB, respectively, are the result of a combination of promoter preference and metal binding.

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In this work, we have analyzed both at stoichiometric and at conformational level the Cd(II)-binding features of a type 2 plant metallothionein (MT) (the cork oak, Quercus suber, QsMT). To this end four peptides, the wild-type QsMT and three constructs previously engineered to characterize its Zn(II)- and Cu(I)-binding behaviour, were heterologously produced in Escherichia coli cultures supplemented with Cd(II), and the corresponding complexes were purified up to homogeneity. The Cd(II)-binding ability of these recombinant peptides was determined through the chemical, spectroscopic and spectrometric characterization of the recovered clusters. Recombinant synthesis of the four QsMT peptides in cadmium-rich media rendered complexes with a higher metal content than those obtained from zinc-supplemented cultures and, consequently, the recovered Cd(II) species are nonisostructural to those of Zn(II). Also of interest is the fact that three out of the four peptides yielded recombinant preparations that included S(2-)-containing Cd(II) complexes as major species. Subsequently, the in vitro Zn(II)/Cd(II) replacement reactions were studied, as well as the in vitro acid denaturation and S(2-) renaturation reactions. Finally, the capacity of the four peptides for preventing cadmium deleterious effects in yeast cells was tested through complementation assays. Consideration of all the results enables us to suggest a hairpin folding model for this typical type 2 plant Cd(II)-MT complex, as well as a nonnegligible role of the spacer in the detoxification function of QsMT towards cadmium.

Zn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT.