The Peroxidatic Thiol of Peroxiredoxin 1 is Nitrosated by Nitrosoglutathione but Coordinates to the Dinitrosyl Iron Complex of Glutathione

Protein S-nitrosation is an important consequence of NO(●)·metabolism with implications in physiology and pathology. The mechanisms responsible for S-nitrosation in vivo remain debatable and kinetic data on protein S-nitrosation by different agents are limited. 2-Cys peroxiredoxins, in particular Prx1 and Prx2, were detected as being S-nitrosated in multiple mammalian cells under a variety of conditions. Here, we investigated the kinetics of Prx1 S-nitrosation by nitrosoglutathione (GSNO), a recognized biological nitrosating agent, and by the dinitrosyl-iron complex of glutathione (DNIC-GS; [Fe(NO)(2)(GS)(2)](−)), a hypothetical nitrosating agent. Kinetics studies following the intrinsic fluorescence of Prx1 and its mutants (C83SC173S and C52S) were complemented by product analysis; all experiments were performed at pH 7.4 and 25 ℃. The results show GSNO-mediated nitrosation of Prx1 peroxidatic residue ([Formula: see text] = 15.4 ± 0.4 M(−1). s(−1)) and of Prx1 Cys(83) residue ([Formula: see text] = 1.7 ± 0.4 M(−1). s(−1)). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys(52)NO denitrosation of [Formula: see text] = 14.4 ± 0.3 M(−1). s(−1). In contrast, the reaction of DNIC-GS with Prx1 did not nitrosate the enzyme but formed DNIC-Prx1 complexes. The peroxidatic Prx1 Cys was identified as the residue that more rapidly replaces the GS ligand from DNIC-GS ([Formula: see text] = 7.0 ± 0.4 M(−1). s(−1)) to produce DNIC-Prx1 ([Fe(NO)(2)(GS)(Cys(52)-Prx1)](−)). Altogether, the data showed that in addition to S-nitrosation, the Prx1 peroxidatic residue can replace the GS ligand from DNIC-GS, forming stable DNIC-Prx1, and both modifications disrupt important redox switches.