Abstract
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 ( = 15.4 ± 0.4 M−1. s−1) and of Prx1 Cys83 residue ( = 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 Prx1Cys52NO denitrosation of = 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 ( = 7.0 ± 0.4 M−1. s−1) to produce DNIC-Prx1 ([Fe(NO)2(GS)(Cys52-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.
Highlights
Protein S-nitrosation (P-CysNO), extensively named S-nitrosylation, is a post-translational modification involving the covalent addition of a nitrosonium (NO+ ) to the sulfur atom of a deprotonated protein cysteine residue (P-CysS− )
Our study shows that the Prx1 peroxidatic Cys is the more reactive residue toward S-nitrosation by GSNO and toward formation of dinitrosyl-iron complexes (DNICs)-Prx1 by Dinitrosyl Iron Complex of GSH (DNIC-GS)
The Prx1 reactions with both GSNO and DNIC-GS were not rapid and, unlikely to be relevant in cells because of the many competitive reactions that occur under physiological conditions
Summary
Protein S-nitrosation (P-CysNO), extensively named S-nitrosylation, is a post-translational modification involving the covalent addition of a nitrosonium (NO+ ) to the sulfur atom of a deprotonated protein cysteine residue (P-CysS− ). Radical reactions are usually rapid but have low specificity, whereas transnitrosation
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