Mössbauer studies on the metal‐thiolate cluster formation in Fe(II)‐metallothionein

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Abstract
The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using Mossbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting .DELTA.EQ and isolmer shift .delta. as found for reduced rubredoxin (Rdrcd) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in prvious electronic absorption studies [M. Good and M. Vasak (1986) Biochemistry 25,8353-8356]. The Mossbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in Mossbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the Mossbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existance of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187,, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cystein residues in metal binding, the appearance of Mossbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(*I) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.