Mechanistic insights into uptake, transfer and exchange of metal ions by the three‐metal clusters of a metalloprotein

Metallothioneins (MTs) are small proteins that coordinate d-block metal ions in sulfur-metal clusters to control metal ion concentrations within the cell. Here we study metal cluster formation in the MT of the periwinkle Littorina littorea (LlMT) by nuclear magnetic resonance (NMR). We demonstrate that the three Cd²⁺ ions in each domain are taken up highly cooperatively, that is, in an all-or-none fashion, with a four- to six-fold higher affinity for the C-terminal domain. During the transfer of metal ions from Cd²⁺-loaded MT to apo MT, Cd²⁺ is most efficiently transferred from the metalated protein to the apo C-terminal domain. This behavior might be connected to unique structural motifs in the C-terminal domain, such as two double-CXC motifs and an increased proportion of positively charged residues. In Cd²⁺/Zn²⁺ metal exchange experiments, the N-terminal domain displayed the most efficient inter-molecular metal exchange. Amide hydrogen exchange reveals fewer protected amides for the N-terminal domain, suggesting the structure might more easily "open up" to facilitate metal exchange. Experiments with a physical separation of donor and acceptor species demonstrate that metal exchange and transfer require protein-protein contacts. These findings provide insights into the mechanism of metal uptake and metal transfer, which are important processes during metal detoxification in snail MTs.