Sulfiredoxin Rate-limiting step Peroxiredoxin ATP Sulfinic acid Phosphoryl anhydride intermediate a b s t r a c t The eukaryotic sulfiredoxin (Srx) catalyzes the reduction of overoxidized typical 2-Cys peroxiredox-ins PrxSO 2 via ATP/Mg 2+-dependent phosphorylation of the sulfinic acid group, followed by formation of a PrxSO-SSrx thiolsulfinate intermediate. Using real-time kinetics of wild-type and C84A Srxs, and pH-rate profiles with ATP/Mg 2+ analogues, we show that the rate-limiting step of the reaction is associated with the chemical process of transfer of the c-phosphate of ATP to the sulfinic acid, in contrast to that described by Jönsson et al. [7]. Two pK apps of 6.2 and 7.5 were extracted from the bell-shaped pH-rate profile, corresponding to the c-phosphate of ATP, and to an acid–base catalyst, respectively.