title: Disulfide biochemistry in 2-cys peroxiredoxin: requirement of Glu50 and Arg146 for the reduction of yeast Tsa1 by thioredoxin.
abstract: 2-Cys peroxiredoxin (Prx) enzymes are ubiquitously distributed peroxidases that make use of a peroxidatic cysteine (Cys(P)) to decompose hydroperoxides. A disulfide bond is generated as a consequence of the partial unfolding of the α-helix that contains Cys(P). Therefore, during its catalytic cycle, 2-Cys Prx alternates between two states, locally unfolded and fully folded. Tsa1 (thiol-specific antioxidant protein 1 from yeast) is by far the most abundant Cys-based peroxidase in Saccharomyces cerevisiae. In this work, we present the crystallographic structure at 2.8Å resolution of Tsa1(C47S) in the decameric form [(α(2))(5)] with a DTT molecule bound to the active site, representing one of the few available reports of a 2-Cys Prx (AhpC-Prx1 subfamily) (AhpC, alkyl hydroperoxide reductase subunit C) structure that incorporates a ligand. The analysis of the Tsa1(C47S) structure indicated that Glu50 and Arg146 participate in the stabilization of the Cys(P) α-helix. As a consequence, we raised the hypothesis that Glu50 and Arg146 might be relevant to the Cys(P) reactivity. Therefore, Tsa1(E50A) and Tsa1(R146Q) mutants were generated and were still able to decompose hydrogen peroxide, presenting a second-order rate constant in the range of 10(6)M(-1)s(-1). Remarkably, although Tsa1(E50A) and Tsa1(R146Q) were efficiently reduced by the low-molecular-weight reductant DTT, these mutants displayed only marginal thioredoxin (Trx)-dependent peroxidase activity, indicating that Glu50 and Arg146 are important for the Tsa1-Trx interaction. These results may impact the comprehension of downstream events of signaling pathways that are triggered by the oxidation of critical Cys residues, such as Trx.
authors.affiliation: Departamento de Biologia, Universidade Estadual Paulista Júlio de Mesquita Filho, Campus do Litoral Paulista São Vicente, São Paulo, Brazil.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Tairum
authors.forename: Marcos A
authors.initials: MA
authors.lastname: de Oliveira
authors.forename: Bruno B
authors.initials: BB
authors.lastname: Horta
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Zara
authors.forename: Luis E S
authors.initials: LE
authors.lastname: Netto
mesh_terms: D001120:Arginine / Q000378:metabolism*; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D054464:Peroxiredoxins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D004220:Disulfides; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D013879:Thioredoxins; D001120:Arginine; D010544:Peroxidases; C496253:Tsa1 protein, S cerevisiae; C504194:2-cys peroxiredoxin, human; D054464:Peroxiredoxins