Binding of excess cadmium(II) to Cd-7-metallothionein from recombinant mouse Zn-7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis

A mouse metallothionein (MT) 1 expression system has been constructed that renders recombinant MT as a high parity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn-7-MT showed that it behaves like the native protein. Exposure of Cd-7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn-7-MT. Spectral data are compatible with a model in which the first four additional Cd ions are bound to Cd-7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd-7-MT appear to have lower binding constants as exposure of Cd-n-MT (n > 7) species to Chelex-100 retrieved Cd-7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd-5, Zn-2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd-7-MT proceeds by means of S-Met1-0-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible. (C) 1997 Elsevier Science Inc.