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Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I) 4 -Thiolate Cluster.
- Source :
-
Journal of the American Chemical Society [J Am Chem Soc] 2022 Jan 19; Vol. 144 (2), pp. 709-722. Date of Electronic Publication: 2022 Jan 05. - Publication Year :
- 2022
-
Abstract
- The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I) <subscript>4</subscript> -Cys <subscript>5-6</subscript> cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O <subscript>2</subscript> 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 Zn <subscript>7</subscript> MT-3 and Cu(II) to form Cu(I) <subscript>4</subscript> Zn(II) <subscript>4</subscript> MT-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) <subscript>4</subscript> -Cys <subscript>5</subscript> 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) <subscript>4</subscript> -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 O <subscript>2</subscript> . We thus provide evidence that the MT-3 Cu(I) <subscript>4</subscript> -Cys <subscript>5</subscript> cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.
- Subjects :
- Electron Spin Resonance Spectroscopy
Glutathione chemistry
Humans
Metallothionein 3 genetics
Metallothionein 3 metabolism
Oxidation-Reduction
Recombinant Proteins biosynthesis
Recombinant Proteins chemistry
Recombinant Proteins isolation & purification
Spectrometry, Fluorescence
Zinc chemistry
Copper chemistry
Disulfides chemistry
Free Radicals chemistry
Metallothionein 3 chemistry
Oxygen chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1520-5126
- Volume :
- 144
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of the American Chemical Society
- Publication Type :
- Academic Journal
- Accession number :
- 34985880
- Full Text :
- https://doi.org/10.1021/jacs.1c03984