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

Abstract

Human metallothioneins (MTs) are involved in binding the essential elements, Cu(I) and Zn(II), and the toxic element, Cd(II), in metal-thiolate clusters using 20 reduced cysteines. The brain-specific MT3 binds a mixture of Cu(I) and Zn(II) in vivo. Its metallation properties are critically important because of potential connections between Cu, Zn and neurodegenerative diseases. We report that the use of isotopically pure ⁶³ Cu(I) and ⁶⁸ Zn(II) greatly enhances the element resolution in the ESI-mass spectral data revealing species with differing Cu:Zn ratios but the same total number of metals. Room temperature phosphorescence and circular dichroism spectral data measured in parallel with ESI-mass spectral data identified the presence of specific Cu(I)-thiolate clusters in the presence of Zn(II). A series of Cu(I)-thiolate clusters form following Cu(I) addition to apo MT3: the two main clusters that form are a Cu₆ cluster in the β domain followed by a Cu₄ cluster in the α domain. ⁶³ Cu(I) addition to ⁶⁸ Zn₇ -MT3 results in multiple species, including clustered Cu₅ Zn₅ -MT3 and Cu₉ Zn₃ -MT3. We assign the domain location of the metals for Cu₅ Zn₅ -MT3 as a Cu₅ Zn₁ -β cluster and a Zn₄ -α cluster and for Cu₉ Zn₃ -MT3 as a Cu₆ -β cluster and a Cu₃ Zn₃ -α cluster. While many reports of the average MT3 metal content exist, determining the exact Cu,Zn stoichiometry has proven very difficult even with native ESI-MS. The work in this paper solves the ambiguity introduced by the overlap of the naturally abundant Cu(I) and Zn(II) isotopes. Contrary to other reports, there is no indication of a major fraction of Cu₄ -β-Zn_n -α-MT3 forming.

Keywords: Cu homeostasis; Cu-thiolate clusters; ESI-MS; Zn homeostasis; metallothionein-3; phosphorescence spectroscopy.