Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate ClusterClick to copy article linkArticle link copied!
- Jenifer S. CalvoJenifer S. CalvoDepartment of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, United StatesMore by Jenifer S. Calvo
- Rhiza Lyne E. VillonesRhiza Lyne E. VillonesDepartment of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, United StatesMore by Rhiza Lyne E. Villones
- Nicholas J. YorkNicholas J. YorkDepartment of Chemistry and Biochemistry, University of Alabama, 250 Hackberry Lane, Tuscaloosa, Alabama 35401, United StatesMore by Nicholas J. York
- Ewelina StefaniakEwelina StefaniakInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandMore by Ewelina Stefaniak
- Grace E. HamiltonGrace E. HamiltonDepartment of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, United StatesMore by Grace E. Hamilton
- Allison L. StellingAllison L. StellingDepartment of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, United StatesMore by Allison L. Stelling
- Wojciech BalWojciech BalInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, PolandMore by Wojciech Bal
- Brad S. PierceBrad S. PierceDepartment of Chemistry and Biochemistry, University of Alabama, 250 Hackberry Lane, Tuscaloosa, Alabama 35401, United StatesMore by Brad S. Pierce
- Gabriele Meloni*Gabriele Meloni*Email: [email protected]Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, United StatesMore by Gabriele Meloni
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.
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