Kinetic studies of peroxiredoxin 6 from Arenicola marina: Rapid oxidation by hydrogen peroxide and peroxynitrite but lack of reduction by hydrogen sulfide

► Arenicola marina Prx6 rapidly reduced H 2O 2, peroxynitrite and t-BuOOH. ► The rate constants of H 2O 2 reduction was 1.1 × 10 7 M −1 s −1 at pH 7.4 and 25 °C. ► The rate constants of peroxynitrite reduction was 2 × 10 6 M −1 s −1 at pH 7.4 and 25 °C. ► The p K a value of the peroxidatic thiol in AmPrx6 was 5.1. ► Hydrogen sulfide, dihydrolipoic acid and GSH plus TR1 could not reduce AmPrx6. Arenicola marina lives in marine environments where hydrogen peroxide concentrations reach micromolar levels. The annelid also forms reactive species through metabolic pathways. Its antioxidant systems include a cytosolic peroxiredoxin, peroxiredoxin 6 ( AmPrx6 or AmPRDX6) that shows high homology to the mammalian 1-Cys peroxiredoxin. Previous work confirmed the peroxidase activity of AmPrx6 in the presence of dithiotreitol. Herein, we performed an in vitro kinetic characterization of the recombinant enzyme. AmPrx6 reduced hydrogen peroxide and peroxynitrite with rate constants of 1.1 × 10 7 and 2 × 10 6 M −1 s −1, respectively, at pH 7.4 and 25 °C. Reduction of tert-butyl hydroperoxide was slower. The p K a of the peroxidatic thiol of AmPrx6 was determined as 5.1 ± 0.2, indicating that it exists as thiolate, the reactive species, at physiological pH. The reductive part of the catalytic cycle was also explored. Hydrogen sulfide, present in millimolar concentrations in marine sediments where the annelid lives and that is able to reduce the mammalian 1-Cys peroxiredoxin, did not support AmPrx6 peroxidase activity. The enzyme was not reduced by other potential physiological reductants tested. Our data indicate that in this annelid, Prx6 could contribute to peroxide detoxification in the presence of a so far unidentified reducing counterpart.