Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 292, Issue 52, Pages 21558-21567Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M117.817452
Keywords
copper; metal homeostasis; peptides; spectroscopy; thermodynamics; phosphorescence; potentiometry
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Funding
- National Institutes of Health [GM067169]
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The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biologically relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu-4(GS)(6)] cluster that is sufficiently stable to limit the effective free aquacopper(I) concentration to the sub-femtomolar regime. Combined spectrophotometric-potentiometric titrations at biologically realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity standards and corroborated by low-temperature phosphorescence studies, established cooperative assembly of [Cu-4(GS)(6)] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equilibrium system sets a firm upper limit on the thermodynamic availability of intracellular copper that is 3 orders of magnitude lower than previously estimated. Taking into account their ability to catalyze the production of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiological conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity determinations of Cu(I) chaperones.
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