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

摘要

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) (GS) ] ), 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 . s ) and of Prx1 Cys residue ( = 1.7 ± 0.4 M . s ). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys NO denitrosation of = 14.4 ± 0.3 M . s . 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 . s ) to produce DNIC-Prx1 ([Fe(NO) (GS)(Cys -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.