Human metallothioneins (MTs) are small cysteine-rich proteins involved in the homeostasis of essential metal ions, such as Zn(II) and Cu(I).1 Among these proteins, MT3 exhibits the most pronounced copper character and was isolated from the brain as a Cu(I)4Zn(II)4MT3ox complex.2,3 However, knowledge regarding other heterometallic complexes, especially reduced ones, remains limited. Our research aimed to determine the stoichiometry, structure, and stability of the aforementioned complexes, providing a deeper understanding of the function of this crucial protein. To this end, human MT3 was produced using the E. coli bacterial expression system with the IMPACT protocol. SEC-purified MT3 underwent spectroscopic analysis under anaerobic conditions. By using Cu(I) (BCS) and Zn(II) (PAR, ZnAF-2F) chelators, the affinities of the subsequent Cu(I) and Zn(II) ions in the mixed CuxZnyMT3 complexes were determined. UV-Vis and CD titrations demonstrated the displacement of Zn(II) by Cu(I) from Zn7MT3. Even with higher Cu(I) occupancy, not all Zn(II) ions were displaced, forming mixed complexes. Both apo- and Zn(II)-saturated MT3 were able to bind the same amount of Cu(I), suggesting that Zn(II) affects the coordination of Cu(I) ions. Experiments involving chelators provided insights into the thermodynamic and structural interactions between these metal ions, demonstrating that Zn(II) and Cu(I) influence each other. The affinities of these metal ions in mixed complexes are lower than in homometallic complexes. Thus, CuxZnyMT3 complexes can still act as metal buffers for these essential metal ions in the cellular environment.
This research was supported by the National Science Centre of Poland (NCN) under the Opus grant no. 2021/43/B/NZ1/02961 (to A.K.).