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

• Published in: The FEBS journal 2023-09, Vol.290 (17), p.4316-4341
• Main Authors: Melenbacher, Adyn, Stillman, Martin J
• Format: Article
• Language: English
• Subjects: Biocompatibility; Circular dichroism; Clusters; Copper; Dichroism; Isotopes; Metallothionein; Metallothioneins; Metals; Neurodegenerative diseases; Phosphorescence; Room temperature; Stoichiometry; Zinc
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.16812

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 63Cu(I) and 68Zn(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. 63Cu(I) addition to 68Zn7‐MT3 results in multiple species, including clustered Cu5Zn5‐MT3 and Cu9Zn3‐MT3. We assign the domain location of the metals for Cu5Zn5‐MT3 as a Cu5Zn1‐β cluster and a Zn4‐α cluster and for Cu9Zn3‐MT3 as a Cu6‐β cluster and a Cu3Zn3‐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‐Znn‐α‐MT3 forming.