Metallothionein is a ubiquitous protein with a wide range of proposed physiological roles, including
the transport, storage and detoxification of essential and nonessential trace metals. The amino
acid sequence of isoform 2a of rabbit liver metallothionein, the isoform used in our spectroscopic
studies, includes 20 cysteinyl groups out of 62 amino acids. Metallothioneins in general represent
an impressive chelating agent for a wide range of metals. Structural studies carried out by a
number of research groups (using H1 and Cd113 NMR, X-ray crystallography, more recently EXAFS,
as well as optical spectroscopy) have established that there are three structural motifs for metal
binding to mammalian metallothioneins. These three structures are defined by metal to protein
stoichiometric ratios, which we believe specifically determine the coordination geometry adopted
by the metal in the metal binding site at that metal to protein molar ratio. Tetrahedral geometry is
associated with the thiolate coordination of the metals in the M7-MT species, for M = Zn(II), Cd(II),
and possibly also Hg(II), trigonal coordination is proposed in the M11-12-MT species, for M = Ag(I),
Cu(I), and possibly also Hg(II), and digonal coordination is proposed for the metal in the M17-18-MT
species for M = Hg(II), and Ag(I). The M7-MT species has been completely characterized for M =
Cd(II) and Zn(II). Cd113 NMR spectroscopic and x-ray crystallographic data show that mammalian
Cd7-MT and Zn7-MT have a two domain structure, with metal-thiolate clusters of the form M4(Scys)11
(the α domain) and M3(Scys)9 (the β domain). A similar two domain structure involving Cu6(Scys)11
(α) and Cu6(Scys)9 (β) copper-thiolate clusters has been proposed for the Cu12-MT species.
Copper-, silver- and gold-containing metallothioneins luminesce in the 500-600 nm region from
excited triplet, metal-based states that are populated by absorption into the 260-300 nm region of
the metal-thiolate charge transfer states. The luminescence spectrum provides a very sensitive
probe of the metal-thiolate cluster structures that form when Ag(I), Au(I), and Cu(I) are added to
metallothionein. CD spectroscopy has been used in our laboratory to probe the formation of
species that exhibit well-defined three-dimensional structures. Saturation of the optical signals
during titrations of MT with Cu(I) or Ag(I) clearly show formation of unique metal-thiolate structures
at specific metal:protein ratios. However, we have proposed that these M=7, 12 and 18 structures
form within a continuum of stoichiometries. Compounds prepared at these specific molar ratios
have been examined by X-ray Absorption Spectroscopy (XAS) and bond lengths have been
determined for the metal-thiolate clusters through the EXAFS technique. The stoichiometric ratio
data from the optical experiments and the bond lengths from the XAS experiments are used to
propose structures for the metal-thiolate binding site with reference to known inorganic
metal-thiolate compounds.
