The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

NADPH thioredoxin reductase C （NTRC） is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase （NTR） and thioredoxin （TRX） activities for the efficient reduction of 2-Cys peroxiredoxin （2-Cys PRX）. Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G- V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.

2-Cys Peroxiredoxins Participate in the Oxidation of Chloroplast Enzymes in the Dark

Most redox-regulated chloroplast enzymes are reduced during the day and oxidized during the night.While the reduction mechanism of light-dependent enzymes is well known,the mechanism mediating their oxidation in the dark remains unknown.The thiol-dependent peroxidases,2-Cys peroxiredoxins (Prxs),play a key role in light-dependent reduction of chloroplast enzymes.Prxs transfer reducing equivalents of thiols to hydrogen peroxide,suggesting the participation of these peroxidases in enzyme oxidation in the dark.Here,we have addressed this issue by analyzing the redox state of well-known redox-regulated chloroplast enzymes in response to darkness in Arabidopsis thaliana mutants deficient in chloroplastlocalized Prxs (2-Cys Prxs A and B,Prx ⅡE,and Prx Q).Mutant plants lacking 2-Cys Prxs A and B,and plants overexpressing NADPH-dependent thioredoxin (Trx) reductase C showed delayed oxidation of chloroplast enzymes in the dark.In contrast,the deficiencies of Prx ⅡE or Prx Q exerted no effect.In vitro assays allowed the reconstitution of the pathway of reducing equivalents from reduced fructose 1,6-bisphosphatase to hydrogen peroxide mediated by Trxs and 2-Cys Prxs.Taken together,these results suggest that 2-Cys Prxs participate in the short-term oxidation of chloroplast enzymes in the dark.