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Thiol-disulfide exchange between glutaredoxin and glutathione
Rasmus Iversen1, Peter Anders Andersen, Kristine Steen Jensen
1Department of Biology, University of Copenhagen, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
Biochemistry
|December 9, 2009
Summary
Glutaredoxins, essential oxidoreductases, were studied to understand glutathione-protein mixed disulfide stability. A mutant yeast glutaredoxin 1 revealed an enthalpy-driven interaction with a significant entropy penalty, impacting protein thermal stability.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Glutaredoxins are thiol-disulfide oxidoreductases within the thioredoxin family, crucial for reducing glutathione-protein mixed disulfides.
- They possess a conserved CXXC active site motif, enabling the N-proximal cysteine to form mixed disulfides with glutathione or intramolecular disulfides.
Purpose of the Study:
- To investigate the stability of the glutathione-protein mixed disulfide using a mutant yeast glutaredoxin 1 (Grx1) lacking the C-proximal active site cysteine.
- To develop and validate a method for analyzing the glutaredoxin-glutathione interaction using isothermal titration calorimetry (ITC).
- To determine the pK(a) of the active site cysteine and assess the impact of glutathionylation on protein thermal stability.
Main Methods:
- Isothermal titration calorimetry (ITC) was employed to monitor the exchange reaction between reduced yeast glutaredoxin 1 (mutant) and oxidized glutathione.
- A novel algorithm was developed for ITC data analysis of this specific reaction.
- Mass spectrometry was used to verify the ITC results and determine the standard reduction potential.
- Differential scanning calorimetry (DSC) assessed the thermal stability of glutaredoxin before and after glutathionylation.
Main Results:
- The interaction between reduced yeast Grx1 and oxidized glutathione is enthalpy-driven, characterized by a significant entropy penalty.
- The developed ITC method accurately quantified the reaction, corroborated by mass spectrometry yielding a standard reduction potential of -295 mV for the mixed disulfide.
- The active site cysteine exhibited a very low pK(a) value, consistent with other glutaredoxins.
- Glutathionylation significantly altered the thermal stability of glutaredoxin, as observed through DSC.
Conclusions:
- The study elucidates the thermodynamic driving forces and stability of glutathione-protein mixed disulfides formed by glutaredoxins.
- A robust ITC-based methodology was established for analyzing these critical redox reactions.
- The findings highlight the influence of glutathionylation on glutaredoxin's structural integrity and stability.
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