Evidence for the formation of a covalent thiosulfinate intermediate with peroxiredoxin in the catalytic mechanism of sulfiredoxin.
The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of approximately 2 min(-1), for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.
Roussel
Xavier
X
Unité Mixte de Recherche, CNRS-UHP 7567, Maturation des Acide Ribonucléique et Enzymologie Moléculaire, Nancy Université, Faculté des Sciences, Bld. des Aiguillettes, BP 239, Vandoeuvre-lès-Nancy 54506, France.
Béchade
Guillaume
G
Kriznik
Alexandre
A
Van Dorsselaer
Alain
A
Sanglier-Cianferani
Sarah
S
Branlant
Guy
G
Rahuel-Clermont
Sophie
S
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D003545:Cysteine / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D009211:Myoglobin / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000378:metabolism*; D054464:Peroxiredoxins / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013441:Sulfinic Acids / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism
D029968:Escherichia coli Proteins; D009211:Myoglobin; D017382:Reactive Oxygen Species; D013441:Sulfinic Acids; C100040:thiosulfinic acids; D013879:Thioredoxins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D054464:Peroxiredoxins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C080499:sulfide oxidase; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine