$^{63}$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. ... mehrThe 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.