Plant thioredoxin CDSP32 regenerates 1-cys methionine sulfoxide reductase B activity through the direct reduction of sulfenic acid.
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.
Commissariat à l'Energie Atomique et aux Energies Alternative, (Cadarache), Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Laboratoire d'Ecophysiologie Moléculaire des Plantes, Unité Mixte de Recherche 6191 Commissariat à l'Energie Atomique/CNRS/Université Aix-Marseille II, 13108 Saint-Paul-lez-Durance Cedex.
Lionel
L
Tarrago
Commissariat à l'Energie Atomique et aux Energies Alternative, (Cadarache), Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Laboratoire d'Ecophysiologie Moléculaire des Plantes, Unité Mixte de Recherche 6191 Commissariat à l'Energie Atomique/CNRS/Université Aix-Marseille II, 13108 Saint-Paul-lez-Durance Cedex.
Edith
E
Laugier
Institut de Biotechnologie des Plantes, Unité Mixte de Recherche 8618.
Mirko
M
Zaffagnini
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 8619, CNRS, Université Paris-Sud, 91405 Orsay Cedex, France.
Christophe H
CH
Marchand
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Unité Mixte de Recherche 8619, CNRS, Université Paris-Sud, 91405 Orsay Cedex, France.
Pierre
P
Le Maréchal
Institut de Biotechnologie des Plantes, Unité Mixte de Recherche 8618.
Stéphane D
SD
Lemaire
Commissariat à l'Energie Atomique et aux Energies Alternative, (Cadarache), Direction des Sciences du Vivant, Institut de Biologie Environnementale et Biotechnologie, Laboratoire d'Ecophysiologie Moléculaire des Plantes, Unité Mixte de Recherche 6191 Commissariat à l'Energie Atomique/CNRS/Université Aix-Marseille II, 13108 Saint-Paul-lez-Durance Cedex. Electronic address: pascal.rey@cea.fr.
Pascal
P
Rey
D017360:Arabidopsis / Q000201:enzymology*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013434:Sulfenic Acids / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D013434:Sulfenic Acids; D013879:Thioredoxins