Resumen
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 (k+NOCys52" role="presentation">????????52+????k+NOCys52
k
+
N
O
C
y
s
52
= 15.4 ± 0.4 M-1. s-1) and of Prx1 Cys83 residue (k+NOCys83" role="presentation">????????83+????k+NOCys83
k
+
N
O
C
y
s
83
= 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 k−NOCys52" role="presentation">????????52-????k-NOCys52
k
-
N
O
C
y
s
52
= 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 (kDNICCys52" role="presentation">????????52????????kDNICCys52
k
D
N
I
C
C
y
s
52
= 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.