${}^{63}$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.