Zn– and Cd–Metallothionein Recombinant Species from the Most Diverse Phyla May Contain Sulfide (S²⁻) Ligands

Abstract

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 ...

Evidence of Native Metal–S2−–Metallothionein Complexes Confirmed by the Analysis of Cup1 Divalent‐Metal‐Ion Binding Properties

“…This preparation exhibited a spectropolarimetric fingerprint (Figure 4 A), also typical of S 2À -devoid Cd-MT complexes due to the absence of absorption bands at approximately d = 280 nm. [8] Taken together, these results evidenced that this preparation consisted of a major canonical Cd 5 -Cup1 species and completely lacked S 2À -containing complexes.…”

“…Therefore, we considered n2Cd-Cup1 (F1B peak) to be representative of this preparation strategy. Significantly, the CD fingerprint of this sample exhibited a positive absorption around d = 280 nm, to which Cd-S 2À -Cup1 chromophores contributed, [8] with the expected signals at approximately d = 250 nm (Figure 4 A).…”

“…[8] Our results clearly showed that the highest S 2À content was incorporated by Cu-thioneins when coordinating divalent metal ions, significantly Cd II ions. However, the importance of this finding remained obscured by the possibility that the presence of S 2À ligands in metal-MT complexes could be a mere consequence of their synthesis in a heterologous bacterial host.…”

“…Subsequently, we undertook characterization of the Zn IIand Cd II -binding abilities of recombinant Cup1 to fully interpret these results and to validate those results obtained from the recombinant synthesis of a considerable number of MTs. [8] Cloning, synthesis, and purification of recombinant metalCup1 complexes: DNA sequencing confirmed that amplification by polymerase chain reaction (PCR) on VC-sp6 DNA yielded as the product a complementary DNA (cDNA) strand that encoded a protein with the sequence of the native mature (N-term processed) form of Cup1, that is, starting at Gln9 (Gln = glutamine) of the translated polypeptide . [12] Small-scale expression assays with the pGEX-CUP1 construct enabled the analysis of the recombinant apo-Cup1 peptide by ESI-MS measurements after acidification at pH 1.5 of the complexes obtained from Zn II -supplemented cultures.…”

“…The presence of labile S 2À ligands was confirmed not only by the discrepancy between normal and acidic ICP-AES measurements but also by GC-FPD analyses (Table 1). Unlike rZn-Cup1, the recombinant Cd-Cup1 (rCd-Cup1) preparation was chiroptically active, thus exhibiting a CD spectrum mainly composed of two bands: the exciton coupling centered at approximately d = 250 nm, attributable to the Cd-Scys (cys = cystein) chromophores, and the Gaussian band at approximately d = 280 nm, which corresponds to the presence of Cd-S 2À entities [8] (Figure 4). Strikingly, we observed that the CD and UV/Vis spectra of both types of fresh Cd-Cup1 preparation (native n2Cd-Cup1 and rCdCup1, denoted by [g] in Table 1) varied in shape over time so that two main different CD fingerprints were identified.…”