期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 144, 期 2, 页码 709-722出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.1c03984
关键词
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资金
- Robert A. Welch Foundation [AT-1935-20170325, AT-2073-20210327]
- National Institutes of Health, Institute of General Medical Sciences [R35GM128704, 2 R15 GM117511-01]
Researchers have identified and characterized transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II). These intermediates are stable and have long lifetimes, providing insights into the assembly process of the MT-3 Cu(I)(4)-Cys(5) cluster.
The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)(4)-Cys(5-6) cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O-2 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)(4)Zn(II)(4)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)(4)-Cys(5) cluster with short Cu-Cu distances (<2.8 angstrom), 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 O-2. We thus provide evidence that the MT-3 Cu(I)(4)-Cys(5) 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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