Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2

Abstract

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 + 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 + 0.08 s -1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions..

Peroxiredoxin AhpC1 protects Pseudomonas aeruginosa against the inflammatory oxidative burst and confers virulence

“…8 D) concentrations gave a second-order rate constant of 2.3 ± 0.09 × 10 6 and 1.5 ± 0.07 × 10 8 M −1 s −1 , respectively. These values are much in agreement with those calculated by the reaction of urate hydroperoxide with human 2-Cys Prx2 [ 49 ] and by H 2 O 2 with human 2-Cys Prx2 and AhpC from Salmonella typhimurium [ 50 , [63] , [64] , [65] ]. AhpC also seems to react faster with HOCl, however, we could not determine the rate constant by fluorescence since HOCl also oxidizes the tryptophan residues altering protein fluorescence (data not shown).…”

“…AhpC rapidly reduces urate hydroperoxide and H 2 O 2 . The rate constants obtained in this study were much similar to that found for the reduction of urate hydroperoxide and H 2 O 2 by the human 2-Cys peroxiredoxin Prx2 and the Salmonella typhimurium AhpC [ 49 , 50 , 64 , 65 ]. We could not calculate the rate constant for the reaction of AhpC with HOCl since the latter also oxidizes tryptophan, interfering in the intrinsic protein fluorescence.…”

Modifying the resolving cysteine affects the structure and hydrogen peroxide reactivity of peroxiredoxin 2

“…With a 2-fold excess of H 2 O 2 recovery was slower for C172S than for the WT (Fig. 2B), but as expected for hyperoxidation and described in detail in (17), the rate increased with increasing H 2 O 2 concentration. In contrast, the C172D (Fig.…”

“…2A). This recovery is associated with disulfide formation, plus at higher H 2 O 2 concentrations, hyperoxidation (16,17). The C R mutants cannot form disulfides, and the recovery phase is not expected unless there is hyperoxidation.…”

Hydrogen peroxide signaling via its transformation to a stereospecific alkyl hydroperoxide that escapes reductive inactivation

“…the concentrations tested only trans-WOOH gave rise to hyperoxidized Prx2. It is not clear whether this can be explained simply by the different extent of oxidation, or whether it supports a mechanism of intra-dimer cooperativity, whereby the oxidation by trans-but not cis-WOOH of the first peroxidatic thiol to sulfenic acid in the Prx2 homodimer enhances the rate of oxidation of the second peroxidatic site 58 .…”