ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin.
2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.
Aran
Martin
M
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
Caporaletti
Daniel
D
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
Senn
Alejandro M
AM
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
Tellez de Iñon
María T
MT
 INGEBI-CONICET, Buenos Aires, Argentina.
Girotti
María R
MR
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
Llera
Andrea S
AS
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
Wolosiuk
Ricardo A
RA
 Instituto Leloir, IIBBA-CONICET, Universidad de Buenos Aires, Argentina.
D000255:Adenosine Triphosphate / Q000502:physiology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D029741:Brassica rapa / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000502:physiology; D008969:Molecular Sequence Data; D054464:Peroxiredoxins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010940:Plant Proteins; D000255:Adenosine Triphosphate; C504194:2-cys peroxiredoxin, human; D054464:Peroxiredoxins; D008274:Magnesium; D003545:Cysteine