Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

Abstract

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-αMT 1 and Zn₃-βMT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–αMT 1 and Zn₃-βMT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag _x –MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag _x –MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the Μ _x Zn _y –MT (0≤y<7) species found, only M^I ₄Zn₅–MT [(Zn₄)^α(Μ₄Zn₁)^β] and M^I ₇Zn₃–MT [(Μ₃Zn₂)^α(Μ₄Zn₁)^β], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).