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.
Pérez-Ruiz
Juan Manuel
JM
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla y CSIC, Avda Américo Vespucio 49, 41092-Sevilla, Spain.
González
Maricruz
M
Spínola
María Cristina
MC
Sandalio
Luisa María
LM
Cejudo
Francisco Javier
FJ
D017360:Arabidopsis / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant; D008027:Light; D009243:NAD / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D054464:Peroxiredoxins / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry; D011487:Protein Conformation; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000378:metabolism; D013879:Thioredoxins
D010940:Plant Proteins; D009243:NAD; D013879:Thioredoxins; D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase