Iron(II)-substituted metallothionein: evidence for the existence of iron-thiolate clusters.

Metallothioneins (MT's) are unique low molecular weight (Mr 6000-7000) metal- and cysteine-rich proteins characterized by two tetrahedral tetrathiolate clusters containing three and four metal ions. Naturally occurring proteins usually contain the diamagnetic metal ions Zn(II) and/or Cd(II). We have now succeeded in substituting these ions by paramagnetic Fe(II). Rabbit liver MT-1 in which all seven metal binding sites were occupied by Fe(II) ions displays absorption features typical of tetrahedral tetrathiolate Fe(II) coordination. This is documented by the presence of a ligand field 5E----5T2 transition in the near-infrared region centered at about 1850 nm (epsilon Fe approximately 100 M-1 cm-1) and a broad charge-transfer absorption in the UV region with a shoulder at 314 nm. A metal-thiolate cluster structure is inferred from the 7 to 20 ratio of metal ions to cysteine residues and from spectral studies in which successive increments of Fe(II) were incorporated into the metal-free protein. Thus, to about 4 equiv, the charge-transfer absorption and magnetic circular dichroism (MCD) features of the complexes formed resemble closely those of reduced rubredoxin from Desulfovibro gigas in which tetrahedral tetrathiolate Fe(II) coordination is documented. However, upon further addition of Fe(II) ions, the charge-transfer absorption bands undergo a progressive red-shift until the full metal occupancy of seven Fe(II) ions per molecule is reached. The bathochromic shift which is also manifested in the MCD spectra can be ascribed to the transformation of some of the terminal thiolate ligands to bridging when the full complement of Fe(II) is bound.(ABSTRACT TRUNCATED AT 250 WORDS)