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Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d 10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn 2+ and Cu + binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn 2+ binding was measured by chelation titrations of Zn 7 MT-3, while Cu + binding was measured by Zn 2+ displacement from Zn 7 MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant ( K ) and the change in enthalpy (Δ H ) and entropy (Δ S ) for these metal ions binding to MT-3. Zn 2+ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn 7 MT-2 with Cu + revealed that both MT isoforms have similar Cu + affinities and binding thermodynamics, indicating that Δ H and Δ S are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu 4 + –thiolate clusters when Cu + displaces Zn 2+ under physiological conditions. Comparison of the Zn 2+ and Cu + binding thermodynamics reveal that enthalpically-favoured Cu + , which forms Cu 4 + –thiolate clusters, displaces the entropically-favoured Zn 2+ . These results provide a detailed thermodynamic analysis of d 10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper. 

The toxicity of lead, one of the most ubiquitous toxic metals, is well known. Some of its pathological effects are related to its preference for the sulfhydryl groups of proteins. Metallothioneins (MT) are a particular family of metalloproteins characterized by their high Cys content that, among other functions, are linked to the detoxification of heavy metals. In mammals, 4 MT isoforms have been found. The MT3 isoform, also called “neuronal growth inhibitory factor”, is mainly synthesized in the brain and contains several structural differences that may contribute to important functional differences between it and other MT isoforms. The abilities of recombinant MT3 and its individual αMT3 and βMT3 fragments to bind Pb( ii ) have been investigated here, under different pH conditions, by means of spectroscopy, mass spectrometry and isothermal titration calorimetry. The results obtained show that the binding of Pb( ii ) to the intact MT3 protein is relatively unaffected by pH, while the individual domains interact with Pb( ii ) in a pH-sensitive manner. The mass spectrometry data reveal the evolution with time of the initially formed Pb-MT complexes. In the case of the full length protein, Pb( ii ) remains bound for a long period of time. With the isolated fragments, the lead is eventually released. The Pb-species formed depend on the amount of Pb( ii ) present in solution. The thermodynamic data recorded, as measured by ITC, for the replacement of Zn( ii ) by Pb( ii ) in reactions with Zn-MT3, Zn-αMT3 and Zn-βMT3 are all similar, and in all cases, the displacement of Zn( ii ) by Pb( ii ) is thermodynamically favorable. Zn-Replete and Pb-replete MT3 have distinctive circular dichroism spectra, suggestive of structural differences with different metallation status.