(63)Cu(I) binding to human kidney (68)Zn(7)-βα MT1A: determination of Cu(I)-thiolate cluster domain specificity from ESI-MS and room temperature phosphorescence spectroscopy

Mammalian metallothioneins (MTs) are important proteins in Zn(II) and Cu(I) homeostasis with the Zn(II) and Cu(I) binding to the 20 cysteines in metal-thiolate clusters. Previous electrospray ionization (ESI) mass spectrometric (MS) analyses of Cu(I) binding to Zn(7)-MT were complicated by significant overlap of the natural abundance isotopic patterns for Zn(II) and Cu(I) leading to impossibly ambiguous stoichiometries. In this paper, isotopically pure (63)Cu(I) and (68)Zn(II) allowed determination of the specific stoichiometries in the (68) Zn,(63)Cu-βα MT1A species formed following the stepwise addition of (63)Cu(I) to (68)Zn(7)-βα MT1A. These species were characterized by ESI-MS and room temperature emission spectroscopy. The key species that form and their emission band centres are Zn(5)Cu(5)-βα MT1A (λ = 684 nm), Zn(4)Cu(6)-βα MT1A (λ = 750 nm), Zn(3)Cu(9)-βα MT1A (λ = 750 nm), Zn(2)Cu(10)-βα MT1A (λ = 750 nm), and Zn(1)Cu(14)-βα MT1A (λ = 634 nm). The specific domain stoichiometry of each species was determined by assessing the species forming following (63)Cu(I) addition to the (68)Zn(3)-β MT1A and (68)Zn(4)-α MT1A domain fragments. The domain fragment emission suggests that Zn(5)Cu(5)-βα MT1A contains a Zn(1)Cu(5)-β cluster and the Zn(4)Cu(6)-βα MT1A, Zn(3)Cu(9)-βα MT1A, and Zn(2)Cu(10)-βα MT1A each contain a Cu(6)-β cluster. The species forming with >10 mol. eq. of (63)Cu(I) in βα-MT1A exhibit emission from the Cu(6)-β cluster and an α domain cluster. This high emission intensity is seen at the end of the titrations of (68)Zn(7)-βα MT1A and the (68)Zn(4)-α MT1A domain fragment suggesting that the initial presence of the Zn(II) results in clustered Cu(I) binding in the α domain.