Metallothioneins (MT), a class of protein characterized by a high cysteine content, low molar mass, and lack of aromatic amino acid residues, have been isolated from mammals, yeasts, fungus, and crustaceans [1-3]. The 20 cysteines out of a total of 61 or 62 amino acids in rabbit liver Mf bind a remarkably wide range of metals, including, significantly, cadmium, copper, and mercury. The binding constants for metal binding follow the order found for inorganic thiolates, Hg(II) > Ag(I) approximate to Cu(I) > Cd(II) > Zn(II). In mammalian Cd-7-MT and Zn-7-MT the metals are tetrahedrally co-ordinated in two isolated domains with stoichiometries of M(4)S(11) and M(3)S(9). Optical spectra, in particular circular dichroism and luminescence spectra, have provided rich details of a complicated metal binding chemistry when metals are added directly to the metal free- or zinc- containing protein. The absence of aromatic amino acids is important because spectral data can be measured for the thiolate to metal charge transfer transitions that occur between 220 and 350 nm, a region that would be completely masked by the presence of aromatic groups. The CD technique is sensitive to changes in the orientation of the peptide chain induced by changes in the metal binding site as a result of metal binding or metal exchange. In particular, the CD spectral changes are extensive when the metal coordination geometry changes, for example from the tetrahedral of Zn-7-MT to accommodate metals like Cu(I) and Ag(I), metals that generally exhibit trigonal or digonal co-ordination geometries. Absorption, emission, MCD, and CD spectra provide considerable detail about the stoichiometries of complexes that form as metal are added to either apo-MT or the Zn(II) in Zn-7-MT. Cu(I) and Hg(II) bind strongly to the cysteinyl thiolates in metallothionein both in vivo and in vitro. Structural information about mercury-containing metallothioneins is currently limited to optical and x-ray absorption (XAS, XANES, XAFS) studies. Emission spectra in the 450-750 run region have been reported for metallothioneins containing Ag(I), Au(I), Cu(I), and Pt(II), at both room temperature and cryogenic temperatures. Excitation in the 250-300 nm results in emission intensity in the 500-700 nm region for Cu(I), Ag(I), and Au(I) metallothioneins. The most well known emission of the metallothioneins is the orange luminescence observed at room temperature for copper-containing metallothioneins. The emission is generally characterized by lifetimes of the order a few microseconds. Recently the complex function of the emission intensity at 600 nm on the Cu(I):MT ratio has been interpreted. When Cu(I) binds to rabbit liver Zn-7-MT, Zn(II) is first displaced from both domains on a statistical basis at all temperatures. Over time, the Cu(I) redistributes into the beta domain forming the domain specific product As a result of an imbalance in quantum yields between the two domains, the redistribution of Cu(I) from the alpha domain to the beta domain can be monitored in real time. The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. XAFS structural data on a number of metallothioneins have been reported. The availability of XAFS data from both the coordinating thiolate sulfur and the bound metal provides information unavailable from other techniques. Three structural motifs have been identified for rabbit liver metallothionein following analysis of spectroscopic data for protein containing Zn(II)), Cu(I), Ag(I), Co(II)), and Hg(II). In these species the peptide chain forms metal thiolate clusters with stoichiometries of M(7)-S-20, M(12)-S-20, and M(18)-S-20. The precise determination of the stoichiometric ratio between the bound metals and the number of accessible cysteinyl sulfurs is important in understanding the chemistry of these proteins. Because the formation of metal-thiolate clusters involving terminal and/or bridging cysteinyl thiolate groups characterizes all metallothioneins, the protein's tertiary structure is dominated by the cross-linking imposed by these clusters. Key metal binding properties for metallothioneins isolated from all sources are (i) the metal to sulfur stoichiometry, (ii) the domain preference in the two-domain class 1 proteins, and (iii) the coordination geometry of the sulfur around the metal. In addition, for a protein that binds multiple metals, answers to a number of questions are needed. First, what is the form of the metal binding site with very few metals bound? Second, how does the clustering proceed? Third, how does metal exchange occur between different sites and different domains?
