Syracuse

Background: Peroxiredoxin 2 (Prx2) reduces peroxides through a cysteine-dependent mechanism and is susceptible to overoxidation of its reactive cysteine during catalysis. Results: Nitration rendered a more active peroxidase, less sensitive to overoxidation. Conclusion: Nitration of Prx2 favors disulfide bond formation over overoxidation. Significance: Understanding the mechanisms by which post-translational modifications modify Prx2 functionality in vitro is crucial to evaluate potential in vivo consequences for redox signaling.

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H 2 O 2 -induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H 2 O 2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.

Peroxiredoxins (Prx, 4 EC 1.11.1.15) are a group of enzymes that efficiently reduce peroxides based on a critical cysteine residue (peroxidatic cysteine, C P ). The first step in catalysis is the reaction of C P with H 2 O 2 to form a sulfenic acid derivative (C P -SOH) which, in 2-cysteine Prx (2-Cys Prx), reacts with another cysteine residue (the resolving cysteine, C R ) to form a disulfide that is then reduced by the thioredoxin/thioredoxin reductase/NADPH system (Fig. 1) (1-4). In typical 2-Cys Prx or Prx1 subfamily (Prx 1-4 in mammals (5)), the reaction occurs between the C P -SOH of one subunit with the C R -SH of another subunit; thus, each homodimer contains two active sites (3). In addition, for the intermolecular disulfide to be formed, a conformational change is needed to approach the C P -SOH from one subunit toward the C R from the other subunit. This rearrangement involves the transition from the so-called fully folded (FF) form, in which the C P and the C R are approximately 14 Å apart, to a locally unfolded (LU) conformation (Fig. 1) (2, 7, 8).The sulfenic acid intermediate can react with the C R -SH forming a disulfide, or with a second molecule of H 2 O 2 to form sulfinic acid (overoxidation), inactivating the peroxidase activ-