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 circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence 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) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.
Stillman
M J
MJ
Department of Chemistry, The University of Westertn Ontario, London, Canada. stillman@uwo.ca
Thomas
D
D
Trevithick
C
C
Guo
X
X
Siu
M
M
D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008628:Mercury / Q000032:analysis / Q000378:metabolism*; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009942:Organometallic Compounds / Q000737:chemistry; D011487:Protein Conformation; D011817:Rabbits; D013053:Spectrophotometry
D009942:Organometallic Compounds; C048402:copper(I)-thiolate; D003300:Copper; D008668:Metallothionein; D008628:Mercury