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

• Main Authors: Daniela R. Truzzi, Simone V. Alves, Luis E. S. Netto, Ohara Augusto
• Format: Article
• Language: English
• Published: MDPI AG 2020-03-01
• Series: Antioxidants
• Subjects: peroxiredoxin 1; nitrosoglutathione; dinitrosyl iron complex; kinetics; nitrosation; nitrosylation
• Online Access: https://www.mdpi.com/2076-3921/9/4/276

Protein S-nitrosation is an important consequence of NO^●&#183;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]^&#8722;), 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 &#177; 0.4 M^&#8722;1. s^&#8722;1) and of Prx1 Cys⁸³ 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 &#177; 0.4 M^&#8722;1. s^&#8722;1). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys⁵²NO denitrosation of <inline-formula> <math display="inline"> <semantics> <mrow> <msubsup> <mi>k</mi> <mrow> <mo>&#8722;</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 &#177; 0.3 M^&#8722;1. s^&#8722;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 (<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 &#177; 0.4 M^&#8722;1. s^&#8722;1) to produce DNIC-Prx1 ([Fe(NO)₂(GS)(Cys⁵²-Prx1)]^&#8722;). 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.