Abstract
Thioredoxins (Trxs) are ubiquitous enzymes catalyzing the reduction of disulfide bonds, thanks to a CXXC active site. Among their substrates, 2-Cys methionine sulfoxide reductases B (2-Cys MSRBs) reduce the R diastereoisomer of methionine sulfoxide (MetSO) and possess two redox-active Cys as follows: a catalytic Cys reducing MetSO and a resolving one, involved in disulfide bridge formation. The other MSRB type, 1-Cys MSRBs, possesses only the catalytic Cys, and their regeneration mechanisms by Trxs remain unclear. The plant plastidial Trx CDSP32 is able to provide 1-Cys MSRB with electrons. CDSP32 includes two Trx modules with one potential active site (219)CGPC(222) and three extra Cys. Here, we investigated the redox properties of recombinant Arabidopsis CDSP32 and delineated the biochemical mechanisms of MSRB regeneration by CDSP32. Free thiol titration and 4-acetamido-4'-maleimidyldistilbene-2,2'-disulfonic acid alkylation assays indicated that the Trx possesses only two redox-active Cys, very likely the Cys(219) and Cys(222). Protein electrophoresis analyses coupled to mass spectrometry revealed that CDSP32 forms a heterodimeric complex with MSRB1 via reduction of the sulfenic acid formed on MSRB1 catalytic Cys after MetSO reduction. MSR activity assays using variable CDSP32 amounts revealed that MSRB1 reduction proceeds with a 1:1 stoichiometry, and redox titrations indicated that CDSP32 and MSRB1 possess midpoints potentials of -337 and -328 mV at pH 7.9, respectively, indicating that regeneration of MSRB1 activity by the Trx through sulfenic acid reduction is thermodynamically feasible in physiological conditions.
Highlights
Ubiquitous protein-disulfide reductases that supply the reducing power needed to break disulfide bonds in physiological partners [1, 2]
Affinity chromatography and co-immunoprecipitation assays identified plastidial 2-Cys Prxs and 1-Cys MSRB1 as potential targets of CDSP32 [28, 29]. This Trx is able to regenerate the activity of plant and mammalian 1-Cys MSRBs without addition of GSH or any other thiol compound [30], raising the hypothesis that regeneration might proceed through an alternative mechanism, distinct from the glutathionylation/deglutathionylation process used by Grxs [21]
The aim of this study was to delineate the mechanism used by the CDSP32 Trx in the regeneration of Arabidopsis thaliana plastidial 1-Cys MSRB1 activity
Summary
Ubiquitous protein-disulfide reductases that supply the reducing power needed to break disulfide bonds in physiological partners [1, 2]. Since the discovery of ribonucleotide reductase as a substrate for Trxs [6] and Grxs [7], both thiol oxidoreductases have been shown to regulate enzymes involved in numerous metabolic pathways, such as apoptosis [8], protein folding [9], or carbon assimilation in photosynthetic organisms [10, 11] Another important function of Trxs and Grxs is their role in the protection against oxidative stress through the regeneration of the activity of peroxiredoxins (Prxs) [12, 13] and methionine sulfoxide reductases (MSRs) (14 –16). Affinity chromatography and co-immunoprecipitation assays identified plastidial 2-Cys Prxs and 1-Cys MSRB1 as potential targets of CDSP32 [28, 29] Very interestingly, this Trx is able to regenerate the activity of plant and mammalian 1-Cys MSRBs without addition of GSH or any other thiol compound [30], raising the hypothesis that regeneration might proceed through an alternative mechanism, distinct from the glutathionylation/deglutathionylation process used by Grxs [21]. Our data show that the Trx, unlike Grxs, is able to directly reduce the sulfenic acid formed on the catalytic Cys of MSRB1 after MetSO reduction
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