Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)₄-Thiolate Cluster

Abstract

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu­(II) to Cu­(I) and form a polynuclear Cu­(I)4-Cys5–6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu­(II) to form Cu­(I)4Zn­(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430–450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu­(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu­(II) leads to the formation of a redox-inert Cu­(I)4-Cys5 cluster with short Cu–Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu­(II) reduction, generating the final Cu­(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu­(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu­(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+supplants entropically favoured Zn2+to form Cu4+clusters under physiological conditions

“…The presence of an unusually air stable Cu 4 + –thiolate cluster in the β-domain of MT-3 is thought to be important in its ability to detoxify Cu 2+ in vivo . 82 The Cu + clusters formed in the samples of this study differ in important ways from those formed in the reaction of MT-3 and Cu 2+ , as the binding of Cu + does not require the concomitant oxidation of MT-3. However, the resulting Cu + 4 –thiolate cluster, which is thought to form an intriguing adamantane-like shape, 120 must be similar, whether formed with Cu 2+ in vivo or by binding Cu + in vitro , given the similarities in their luminescence spectra.…”

“…However, the resulting Cu + 4 –thiolate cluster, which is thought to form an intriguing adamantane-like shape, 120 must be similar, whether formed with Cu 2+ in vivo or by binding Cu + in vitro , given the similarities in their luminescence spectra. 82 The approximately ten-fold difference in Cu + affinity between MT-3 and MT-2 in scavenging reactions, where Cu 2+ is reduced and bound by the protein, as measured by Calvo et al , 77 likely reflects the formation of intramolecular disulfide bonds and is the subject of ongoing studies.…”

“…, Cu 6 + –thiolate clusters in MTs). 82,115–117 Low temperature (77 K) luminescence measurements for both MT-3 and MT-2 samples prepared by Cu + addition to Zn 7 MT in excess GSH show emission features at 425 and 575 nm (Fig. 5), with the emission peak at 425 nm diagnostic of Cu 4 + –thiolate cluster formation.…”

“…We also provide evidence that Cu + forms Cu 4 + –thiolate clusters that are structurally similar to those found in the β-domain of Cu 4 + Zn 3–4 2+ MT-3 isolated from neuronal tissue. 78,82,83 The thermodynamics of the competition between these two metal ions for MT-3 and domain–domain interaction in metal binding by MT, determined here, provide new insight on the molecular basis for the biological roles of these proteins.…”

“…61,80,81 The driving force for this "metal swap" is presumed to be the high affinity of MT-3 for Cu + , coupled with its ability to reduce Cu 2+ to Cu + via intramolecular disulde bond formation. 82 MT-3 may also scavenge Cu + in the reducing environment of the cell and help to maintain the low levels of free copper in neuronal cells.…”