| Summary: | Protein S-nitrosation is an important consequence of NO<sup>●</sup>·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)<sub>2</sub>(GS)<sub>2</sub>]<sup>−</sup>), 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 (<inline-formula> <math display="inline"> <semantics> <mrow> <msubsup> <mi>k</mi> <mrow> <mo>+</mo> <mi>N</mi> <mi>O</mi> </mrow> <mrow> <mi>C</mi> <mi>y</mi> <mi>s</mi> <mn>52</mn> </mrow> </msubsup> </mrow> </semantics> </math> </inline-formula> = 15.4 ± 0.4 M<sup>−1</sup>. s<sup>−1</sup>) and of Prx1 Cys<sup>83</sup> residue (<inline-formula> <math display="inline"> <semantics> <mrow> <msubsup> <mi>k</mi> <mrow> <mo>+</mo> <mi>N</mi> <mi>O</mi> </mrow> <mrow> <mi>C</mi> <mi>y</mi> <mi>s</mi> <mn>83</mn> </mrow> </msubsup> </mrow> </semantics> </math> </inline-formula> = 1.7 ± 0.4 M<sup>−1</sup>. s<sup>−1</sup>). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys<sup>52</sup>NO denitrosation of <inline-formula> <math display="inline"> <semantics> <mrow> <msubsup> <mi>k</mi> <mrow> <mo>−</mo> <mi>N</mi> <mi>O</mi> </mrow> <mrow> <mi>C</mi> <mi>y</mi> <mi>s</mi> <mn>52</mn> </mrow> </msubsup> </mrow> </semantics> </math> </inline-formula> = 14.4 ± 0.3 M<sup>−1</sup>. s<sup>−1</sup>. 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 (<inline-formula> <math display="inline"> <semantics> <mrow> <msubsup> <mi>k</mi> <mrow> <mi>D</mi> <mi>N</mi> <mi>I</mi> <mi>C</mi> </mrow> <mrow> <mi>C</mi> <mi>y</mi> <mi>s</mi> <mn>52</mn> </mrow> </msubsup> </mrow> </semantics> </math> </inline-formula> = 7.0 ± 0.4 M<sup>−1</sup>. s<sup>−1</sup>) to produce DNIC-Prx1 ([Fe(NO)<sub>2</sub>(GS)(Cys<sup>52</sup>-Prx1)]<sup>−</sup>). 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.
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