Water-soluble triarylphosphines as biomarkers for protein S-nitrosation

Abstract

S-Nitrosothiols (RSNOs) represent an important class of post-translational modifications that preserve and amplify the actions of nitric oxide and regulate enzyme activity. Several regulatory proteins are now verified targets of cellular S-nitrosation, and the direct detection of S-nitrosated residues in proteins has become essential to better understand RSNO-mediated signaling. Current RSNO detection depends on indirect assays that limit their overall specificity and reliability. Herein, we report the reaction of S-nitrosated cysteine, glutathione, and a mutated C165S alkyl hydroperoxide reductase with the water-soluble phosphine tris(4,6-dimethyl-3-sulfonatophenyl)phosphine trisodium salt hydrate (TXPTS). A combination of NMR and MS techniques reveals that these reactions produce covalent S-alkylphosphonium ion adducts (with S-P(+) connectivity), TXPTS oxide, and a TXPTS-derived aza-ylide. Mechanistically, this reaction may proceed through an S-substituted aza-ylide or the direct displacement of nitroxyl from the RSNO group. This work provides a new means for detecting and quantifying S-nitrosated species in solution and suggests that phosphines may be useful tools for understanding the complex physiological roles of S-nitrosation and its implications in cell signaling and homeostasis.

Figure 1

³¹P NMR time course experiment of the GSNO and TXPTS reaction. ¹⁵N-GSNO (1 eq.) and TXPTS (2 eq.) were incubated in a HEPES/DTPA buffered solution (pH 7.1) with 10 % D₂O and the reaction was monitored using ³¹P NMR with 1 scan every 20 min. The peaks at 47.2, 39.7, and 34.5 ppm correspond to 5, 3, and 4, respectively and TXPTS starting material at −28.8 ppm. All peaks were referenced to a 3% H₃PO₄ (δ = 0 ppm) external standard which was omitted for clarity.

Figure 2

2-D NMR of the GSNO and TXPTS reaction. a) ¹H-³¹P COSY of the GSNO/TXPTS reaction mixture in D₂O. b) ¹H-¹³C HMQC of the GSNO/TXPTS reaction mixture in D₂O

Figure 3

ESI-TOF-MS data showing TXPTS covalently labeling the S-nitrosated mutated peroxiredoxin (C165S AhpC-SNO). The exact mass of the covalent adduct is 21184.69, MW of the AhpC-SNO is 20629.55. The MW 20719.14 corresponds to the mixed disulfide that forms between AhpC-SNO and Cys-SNO (AhpC-S-S-Cys). (left) C165S AhpC-SNO with 25-fold excess TXPTS over 42 hours (right) control showing C165S AhpC-SNO in the absence of TXPTS under the same conditions

Scheme 1

Reactions of triarylphosphines with S-nitrosothiols in organic and aqueous systems

Scheme 2

Reaction of TXPTS with S-nitrosoglutathione at pH 7.

Scheme 3

Reaction of TXPTS with S-nitrosocysteine at pH 7.

Scheme 4

Proposed mechanism for the formation of the S-N=P type aza-ylide (1)

Scheme 5

Two proposed mechanisms for the formation of an SNO-derived adduct from TXPTS.