Evidence that glutathione and the glutathione system efficiently recycle 1-cys sulfiredoxin in vivo.
OBJECTIVE
Typical 2-Cys peroxiredoxins (2-Cys Prxs) are Cys peroxidases that undergo inactivation by hyperoxidation of the catalytic Cys, a modification reversed by ATP-dependent reduction by sulfiredoxin (Srx). Such an attribute is thought to provide regulation of 2-Cys Prxs functions. The initial steps of the Srx catalytic mechanism lead to a Prx/Srx thiolsulfinate intermediate that must be reduced to regenerate Srx. In Saccharomyces cerevisiae Srx, the thiolsulfinate is resolved by an extra Cys (Cys48) that is absent in mammalian, plant, and cyanobacteria Srxs (1-Cys Srxs). We have addressed the mechanism of reduction of 1-Cys Srxs using S. cerevisiae Srx mutants lacking Cys48 as a model.


RESULTS
We have tested the recycling of Srx by glutathione (GSH) by a combination of in vitro steady-state and single-turnover kinetic analyses, using enzymatic coupled assays, Prx fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reverse-phase chromatography coupled to mass spectrometry. We demonstrate that GSH reacts directly with the thiolsulfinate intermediate, by following saturation kinetics with an apparent dissociation constant of 34 μM, while producing S-glutathionylated Srx as a catalytic intermediate which is efficiently reduced by the glutaredoxin/glutathione reductase system. Total cellular depletion of GSH impacted the recycling of Srx, confirming in vivo that GSH is the physiologic reducer of 1-Cys Srx.


METHODS
Our study suggests that GSH binds to the thiolsulfinate complex, thus allowing non-rate limiting reduction. Such a structural recognition of GSH enables an efficient catalytic reduction, even at very low GSH cellular levels.


CONCLUSIONS
This study provides both in vitro and in vivo evidence of the role of GSH as the primary reducer of 1-Cys Srxs.
1 UMR 7365 CNRS-Université de Lorraine IMoPA , Vandœuvre-lès-Nancy Cedex, France .
Samia
S
Boukhenouna
Hortense
H
Mazon
Guy
G
Branlant
Christophe
C
Jacob
Michel B
MB
Toledano
Sophie
S
Rahuel-Clermont
D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C479426:SRX1 protein, S cerevisiae; D005978:Glutathione