Differentiated Zn(II) binding affinities in animal, plant, and bacterial metallothioneins define their zinc buffering capacity at physiological pZn.

Metallothioneins (MTs) are small, Cys-rich proteins present in various but not all organisms, from bacteria to humans. They participate in zinc and copper metabolism, toxic metals detoxification, and protection against reactive species. Structurally, they contain one or multiple domains, capable of binding a variable number of metal ions. For experimental convenience, biochemical characterization of MTs is mainly performed on Cd(II)-loaded proteins, frequently omitting or limiting Zn(II) binding features and related functions. Here, by choosing 10 MTs with relatively well-characterized structures from animals, plants, and bacteria, we focused on poorly investigated Zn(II)-to-protein affinities, stability-structure relations, and the speciation of individual complexes. For that purpose, MTs were characterized in terms of stoichiometry, pH-dependent Zn(II) binding, and competition with chromogenic and fluorescent probes. To shed more light on protein folding and its relation with Zn(II) affinity, reactivity of variously Zn(II)-loaded MTs was studied by (5,5'-dithiobis(2-nitrobenzoic acid) oxidation in the presence of mild chelators. The results show that animal and plant MTs, despite their architectural differences, demonstrate the same affinities to Zn(II), varying from nano- to low picomolar range. Bacterial MTs bind Zn(II) more tightly but, importantly, with different affinities from low picomolar to low femtomolar range. The presence of weak, moderate, and tight zinc sites is related to the folding mechanisms and internal electrostatic interactions. Differentiated affinities of all MTs define their zinc buffering capacity required for Zn(II) donation and acceptance at various free Zn(II) concentrations (pZn levels). The data demonstrate critical roles of individual Zn(II)-depleted MT species in zinc buffering processes.