Human metallothioneins (MTs) are small proteins (6-7 kDa; 61-68 amino acids) that bind Zn (II) and Cu(I) with high affinity (KD, Zn(II) = 10-10/-11 M; KD, Cu(I) =10-19/-21 M) via an array of 20 conserved cysteines. Apo MTs are unstructured, and a characteristic fold develops upon metal coordination in two separate domains. Zn7MTs feature a Zn3Cys9 cluster in the N-terminal β-domain and a Zn4Cys11 cluster in the C-terminal α-domain, with each Zn(II) tetrahedrally coordinated by terminal and µ2-bridging thiolates. Contrarily Cu(I)4/ Cu(I)6-thiolate clusters can be formed in each domain by Cu(I) digonal/trigonal coordination, with the β-domain showing a higher Cu(I)-binding stability and cooperative formation of a Cu(I)4CysS5-9 cluster. However, MTs are also secreted from cells where the oxidizing extracellular environment promotes copper redox-cycling, disulfide formation and cluster collapse. Peculiarly, in the latter case the Cu(I)4-thiolate cluster in MT’s β-domain is inert to oxidation in the presence of O2. Among all mammalian MT isoforms, MT-3 shows the highest Cu(I)-thionein character and it is purified as Cu(I)4Zn3-4(II) species when isolated form native sources (brain), suggesting that it might play important functions as a copper buffering and storage metalloprotein. By combined biophysical and spectroscopic approaches we addressed the structural features underlying the unique copper-thionein character in MT-3 and its unique reactivity. We conducted electronic absorption, low temperature luminescence, Raman and EPR characterization to investigate the chemistry and pathway of assembly of the Cu(I)4-thiolate cluster in Cu(I)4Zn(II)4MT-3 upon reaction between Zn7MT-3 and Cu(II). The analysis revealed that the Cu(I)4-cluster assembly process involves the formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates that are inert to O2. A controlled electron transfer process underlies the Cu(I)4-thiolate cluster assembly, mediated by the formation of “metal-stabilized” radicals. This work, combined with previous mutational analysis, also reveal how non-coordinating residues are instrumental for outer-sphere control of Cu(I) selectivity bias in MT-3 metal-clusters versus other MT isoforms and establish the basis to understand how metal selectivity bias is governed in MTs.

Copper(I) selectivity bias, cluster assembly, and reactivity in metallothionein-3 metal-thiolate clusters

ACS Spring 2022

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