Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn( II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circu lar dichroism (CD) and luminescence spectra in particular, provide rich det ail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguous ly identify key metal to protein stoichiometric ratios that result in well- defined structures. Electrospray ionization-mass spectrometry data are repo rted for reactions in which Hg(II) binds to ape-MT 2A as previously describ ed from CD data. Emission spectra in the 450-750 nm region have been report ed for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescenc e of Cu-MT can also be detected directly-from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) at oms to Zn-MT indicates a first-order mechanism over a concentration range o f 5-50 mu M. Three-dimensional modeling was carried out using the results o f the CD and EXAFS studies, model calculations for Zn-7-MT, Hg-7-MT, and Cu -12-MT are described. (C) 2000 Elsevier Science Inc. All rights reserved.