63Cu(I) binding to human kidney 68Zn7-βα MT1A: determination of Cu(I)-thiolate cluster domain specificity from ESI-MS and room temperature phosphorescence spectroscopy

Abstract

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 analyses of Cu(I) binding to Zn7-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 63Cu(I) and 68Zn(II) allowed determination of the specific stoichiometries in the 68Zn,63Cu-βα MT1A species formed following the stepwise addition of 63Cu(I) to 68Zn7-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 Zn5Cu5-βα MT1A (λ = 684 nm), Zn4Cu6-βα MT1A (λ = 750 nm), Zn3Cu9-βα MT1A (λ = 750 nm), Zn2Cu10-βα MT1A (λ = 750 nm), and Zn1Cu14-βα MT1A (λ = 634 nm). The specific domain stoichiometry of each species was determined by assessing the species forming following 63Cu(I) addition to the 68Zn3-β MT1A and 68Zn4-α MT1A domain fragments. The domain fragment emission suggests that Zn5Cu5-βα MT1A contains a Zn1Cu5-β cluster and the Zn4Cu6-βα MT1A, Zn3Cu9-βα MT1A, and Zn2Cu10-βα MT1A each contain a Cu6-β cluster. The species forming with >10 mol. eq. of 63Cu(I) in βα-MT1A exhibit emission from the Cu6-β cluster and an α domain cluster. This high emission intensity is seen at the end of the titrations of 68Zn7-βα MT1A and the 68Zn4-α MT1A domain fragment suggesting that the initial presence of the Zn(II) results in clustered Cu(I) binding in the α domain.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry

“…These results are in line with those from the Stillman group, who studied Cu­(I) binding to apoMT1a and showed that the α-domain binds four Cu­(I) ions, while the β-domain binds six Cu­(I), forming copper-thiolate clusters. , In their recent research, they showed that Cu­(I) (not Cu­(II)) binds to Zn 7 MT3 forming multiple species, with no preference for Cu 4 Zn 4 MT3 . In another study, Melenbacher et al studied Cu­(I) binding to Zn 7 MT1a instead of apoMT1a . Their results suggested the formation of two main complexes, βZn 1 Cu 5 αZn 4 MT1a and βCu 6 αZn 4 MT1a.…”

“…38 In another study, Melenbacher et al studied Cu(I) binding to Zn 7 MT1a instead of apoMT1a. 39 Their results suggested the formation of two main complexes, βZn 1 Cu 5 αZn 4 MT1a and βCu 6 αZn 4 MT1a. Furthermore, in the presence of excess glutathione (GSH), Austin and co-workers reported, based on ITC analysis, that Zn 7 MT2 and Zn 7 MT3 can form Cu(I) 4 -thiolate clusters, resulting in βCu 4 αCu 4 MT2 ox and βCu 4 αCu 4 MT3 ox .…”

Metallothionein‐3: 63Cu(I) binds to human 68Zn7‐βα MT3 with no preference for Cu4‐β cluster formation

“…Phosphorescence has long been used to identify the presence of Cu(I)-thiolate clusters in MTs [63]. Both room temperature and 77 K emission data have been reported [35,[52][53][54]59,60,[62][63][64][65][66]. Emission spectra are often measured at 77 K as the metal-dependent charge transfer band blue shifts from ~700 nm (room temperature) to ~500-600 nm and intensifies at low temperatures (77 K).…”

“…Some of the above-mentioned reports do include ESI-MS data [52,53]. However, due to the overlapping naturally abundant isotopes of Cu(I) and Zn(II) (Table 1) and the broad nature of the metallothionein protein peak, it is essentially impossible to determine accurate Cu, Zn-MT stoichiometries using natural abundance Cu(I) and Zn(II) ions [62]. As such, many studies on Cu, Zn-MTs that do include ESI-mass spectral results tend to only report the total number of metals bound to the protein because the data cannot discriminate between the relative fractions of Cu(I) and Zn(II) [70][71][72].…”

“…In our recent paper, we have shown through both simulations and experiments that the use of isotopically pure 63 Cu(I) and 68 Zn(II) greatly increases our ability to resolve species with the same total number of metals but differing Cu:Zn ratios [62]. This allows for the accurate determination of the ratios of both Cu(I) and Zn(II) bound to the MT by ESI-MS at neutral pH.…”