| 20236937 |
Tarrago L, Laugier E, Zaffagnini M, Marchand CH, Le Marechal P, Lemaire SD, Rey P: The plant thioredoxin CDSP32 regenerates 1-CYS methionine sulfoxide reductase B activity through the direct reduction of sulfenic acid. J Biol Chem. 2010 Mar 17.
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: 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 219CGPC222 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 AMS 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 mV 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. |
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