Copper and zinc ion homeostasis in cells involves metallothioneins (MTs), which are low-weight proteins with a high cysteine residue content (~30%) and no aromatic residues. This protein family includes four main isoforms: MT1-MT4. Isoforms MT3 and MT4 were isolated from mouse cells in mixed copper-zinc complexes1,2. MT3 has recently been extensively studied for its interactions with Cu(I) and Cu(II) ions, but research on MT4 remains limited3. The research aims to examine the interactions and affinity of Cu(I)/Cu(II) with MT4 and the formation of reduced and oxidized mixed Cu(I)/Cu(II) complexes with Zn(II)- and metal-free MT4. The MT4 isoform was produced in a bacterial system (E. coli) with a chitin-binding tag (IMPACT). The obtained MT4 was reduced, purified, and reconstituted with Zn(II) ions to Zn7MT4. Interactions between MTs and Cu(I) ions were conducted under anaerobic conditions to prevent oxidation of Cu(I) ions and metallothionein-4. Spectroscopic methods were used to characterize the complexes formed with Cu(I), Cu(II), and Zn(II) ions. The affinity of Cu(I) ions for MT4 was assessed using copper chelators (BCA, BCS). Spectroscopic research indicated the binding of Zn(II), Cu(I), and Cu(II) ions to MT4 and the formation of mixed copper-zinc complexes with metallothionein-4. The formation of mixed complexes indicates the displacement of Zn(II) from MT4 and the binding of Cu(I)/Cu(II) ions with varying affinity. This allowed the determination of the affinity of copper ions to MT4. Binding of Cu(I) to MT4 results in the formation of Cu6Cys9 and Cu7Cys11 clusters in the β and α domains, respectively. Due to the binding of Cu(I)/Cu(II) and the formation of copper-zinc complexes, metallothionein-4 plays a crucial role in understanding and determining its function in copper and zinc metabolism within cells.
This research was supported by the National Science Centre of Poland (Opus grant no. 2021/43/B/NZ1/02961 to AK).