Probing structural changes in the alpha and beta domains of copper- and silver-substituted metallothionein by emission spectroscopy and electrospray ionization mass spectrometry.

Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted alpha and beta domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag(I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag(I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted alpha and beta domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag(I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted alpha and beta fragments than when the Ag(I)-MT species were formed from the apo-alpha and apo-beta fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag(I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.