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Made available in DSpace on 2020-12-12T02:15:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-20 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidad de la República Uruguay Sulfenic acids are the primary product of thiol oxidation by hydrogen peroxide and other oxidants. Several aspects of sulfenic acid formation through thiol oxidation were established recently. In contrast, the reduction of sulfenic acids is still scarcely investigated. Here, we characterized the kinetics of the reduction of sulfenic acids by ascorbate in several proteins. Initially, we described the crystal structure of our model protein (Tsa2-C170S). There are other Tsa2 structures in distinct redox states in public databases and all of them are decamers, with the peroxidatic cysteine very accessible to reductants, convenient features to investigate kinetics. We determined that the reaction between Tsa2-C170S-Cys-SOH and ascorbate proceeded with a rate constant of 1.40 ± 0.08 × 103 M−1 s−1 through a competition assay developed here, employing 2,6–dichlorophenol-indophenol (DCPIP). A series of peroxiredoxin enzymes (Prx6 sub family) were also analyzed by this competition assay and we observed that the reduction of sulfenic acids by ascorbate was in the 0.4–2.2 × 103 M−1 s−1 range. We also evaluated the same reaction on glyceraldehyde 3-phosphate dehydrogenase and papain, as the reduction of their sulfenic acids by ascorbate were reported previously. Once again, the rate constants are in the 0.4–2.2 × 103 M−1 s−1 range. We also analyzed the reduction of Tsa2-C170S-SOH by ascorbate by a second, independent method, following hydrogen peroxide reduction through a specific electrode (ISO-HPO-2, World Precision Instruments) and employing a bi-substrate, steady state approach. The kcat/KMAsc was 7.4 ± 0.07 × 103 M−1 s−1, which was in the same order of magnitude as the value obtained by the DCPIP competition assay. In conclusion, our data indicates that reduction of sulfenic acid in various proteins proceed at moderate rate and probably this reaction is more relevant in biological systems where ascorbate concentrations are high. Departamento de Genética e Biologia Evolutiva Instituto de Biociências Universidade de São Paulo Laboratorio de Fisicoquímica Biológica Instituto de Química Biológica Facultad de Ciencias Universidad de La República, Iguá 4225 Centro de Investigaciones Biomédicas (CEINBIO) Universidad de La República Instituto de Biociências UNESP Campus Do Litoral Paulista, São Vicente Brazilian Biorenewables National Laboratory National Center for Research in Energy and Materials Instituto de Biociências UNESP Campus Do Litoral Paulista, São Vicente FAPESP: 2013/07937-8 Universidad de la República Uruguay: 54/2013