Protein cysteine oxidation in redox signaling: Caveats on sulfenic acid detection and quantification

Abstract

Oxidation of critical signaling protein cysteines regulated by H₂O₂ has been considered to involve sulfenic acid (RSOH) formation. RSOH may subsequently form either a sulfenyl amide (RSNHR') with a neighboring amide, or a mixed disulfide (RSSR') with another protein cysteine or glutathione. Previous studies have claimed that RSOH can be detected as an adduct (e.g., with 5,5-dimethylcyclohexane-1,3-dione; dimedone). Here, kinetic data are discussed which indicate that few proteins can form RSOH under physiological signaling conditions. We also present experimental evidence that indicates that (1) dimedone reacts rapidly with sulfenyl amides, and more rapidly than with sulfenic acids, and (2) that disulfides can react reversibly with amides to form sulfenyl amides. As some proteins are more stable as the sulfenyl amide than as a glutathionylated species, the former may account for some of the species previously identified as the "sulfenome" - the cellular complement of reversibly-oxidized thiol proteins generated via sulfenic acids.

Keywords: Glutathione; Hydrogen peroxide; Redox signaling; Sulfenyl amide; Thiolate.

Figure 1

Proposed mechanism of involvement of Zn²⁺ in H₂O₂-mediated oxidation of Cys residues on Keap1 and regulation of protein activity via intra- and inter-molecular disulfide formation. Oxidation of Cys residues in proteins by H₂O₂ or ROOH, in which a zinc ion is bound to the Cys residues, results in Zn²⁺-hydroxyl species being the leaving group instead of water or an alcohol, and result in rapid disulfide formation. Adapted from [24,25].

Figure 2

A proposed mechanism for enzymatic glutathionylation of target signaling proteins by peroxiredoxin (Prx), glutathione S-transferase π (GST), or glutaredoxin (Grx). Similar reactions may also occur with GPx. In (A), 1- H₂O₂ oxidized Prx; 2- GSH forms a disulfide with Prx; 3- disulfide exchange occurs with the target protein thiolate. In (B), steps 2 and 3 are in reverse sequence from (A). In (C), 1- GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of GST π; 2- disulfide exchange occurs with the target protein thiolate. In (D), GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of Grx; 2- An intramolecular disulfide forms in the active site of Grx; 3- disulfide exchange occurs with the target protein thiolate; 4- disulfide exchange occurs with GSH.

Figure 2

A proposed mechanism for enzymatic glutathionylation of target signaling proteins by peroxiredoxin (Prx), glutathione S-transferase π (GST), or glutaredoxin (Grx). Similar reactions may also occur with GPx. In (A), 1- H₂O₂ oxidized Prx; 2- GSH forms a disulfide with Prx; 3- disulfide exchange occurs with the target protein thiolate. In (B), steps 2 and 3 are in reverse sequence from (A). In (C), 1- GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of GST π; 2- disulfide exchange occurs with the target protein thiolate. In (D), GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of Grx; 2- An intramolecular disulfide forms in the active site of Grx; 3- disulfide exchange occurs with the target protein thiolate; 4- disulfide exchange occurs with GSH.

Figure 2

A proposed mechanism for enzymatic glutathionylation of target signaling proteins by peroxiredoxin (Prx), glutathione S-transferase π (GST), or glutaredoxin (Grx). Similar reactions may also occur with GPx. In (A), 1- H₂O₂ oxidized Prx; 2- GSH forms a disulfide with Prx; 3- disulfide exchange occurs with the target protein thiolate. In (B), steps 2 and 3 are in reverse sequence from (A). In (C), 1- GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of GST π; 2- disulfide exchange occurs with the target protein thiolate. In (D), GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of Grx; 2- An intramolecular disulfide forms in the active site of Grx; 3- disulfide exchange occurs with the target protein thiolate; 4- disulfide exchange occurs with GSH.

Figure 2

A proposed mechanism for enzymatic glutathionylation of target signaling proteins by peroxiredoxin (Prx), glutathione S-transferase π (GST), or glutaredoxin (Grx). Similar reactions may also occur with GPx. In (A), 1- H₂O₂ oxidized Prx; 2- GSH forms a disulfide with Prx; 3- disulfide exchange occurs with the target protein thiolate. In (B), steps 2 and 3 are in reverse sequence from (A). In (C), 1- GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of GST π; 2- disulfide exchange occurs with the target protein thiolate. In (D), GSSG, which must be formed by a closely associated peroxidase undergoes disulfide exchange with the thiolate in the active site of Grx; 2- An intramolecular disulfide forms in the active site of Grx; 3- disulfide exchange occurs with the target protein thiolate; 4- disulfide exchange occurs with GSH.

Figure 3

Structures of sulfenyl amides examined.

Figure 4

Detection by mass spectrometry of dimedone adducts of sulfenyl amides arising from incubation of compound (2) with dimedone. MS spectra showing the formation of the dimedone adduct of 2-methyl-4-isothiazolin-3-one corresponding to m/z 256.101 (z = 1). The identity of this adduct has been confirmed by the MS/MS spectra obtained by CID fragmentation (insert). Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 5a

Detection by mass spectrometry of products of reaction of sulfenyl amides arising from incubation with GSH in aqueous buffer. MS spectra showing the compounds arising from (1)1,2-benzisothiazolin-3-one (m/z 152.0165, z = 1) (upper panel) and (2) 2-methyl-4-isothiazolin-3-one (m/z 116.0165, z = 1) (lower panel) after 30 minutes of incubation with equimolar reduced glutathione in aqueous buffer (75:25 v/v, ammonium bicarbonate 40 mM, pH 8.0, and CH₃CN). The main products observed are: the glutathionylated form (1) m/z 459.0988 (z =1) and (2) m/z 423.092 (z =1); the reduced form (1) m/z 154.0321 (z =1) and (2) m/z 118.0321 (z =1), the disulfide linked dimer of sulfenyl amides (1) m/z 305.039 (z =1) and the oxidized glutathione (GSSG) m/z 613.159 (z =1). The identity of the glutathionylated form was confirmed on the basis of the MS/MS spectra obtained by CID fragmentation (inserts). Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 5a

Detection by mass spectrometry of products of reaction of sulfenyl amides arising from incubation with GSH in aqueous buffer. MS spectra showing the compounds arising from (1)1,2-benzisothiazolin-3-one (m/z 152.0165, z = 1) (upper panel) and (2) 2-methyl-4-isothiazolin-3-one (m/z 116.0165, z = 1) (lower panel) after 30 minutes of incubation with equimolar reduced glutathione in aqueous buffer (75:25 v/v, ammonium bicarbonate 40 mM, pH 8.0, and CH₃CN). The main products observed are: the glutathionylated form (1) m/z 459.0988 (z =1) and (2) m/z 423.092 (z =1); the reduced form (1) m/z 154.0321 (z =1) and (2) m/z 118.0321 (z =1), the disulfide linked dimer of sulfenyl amides (1) m/z 305.039 (z =1) and the oxidized glutathione (GSSG) m/z 613.159 (z =1). The identity of the glutathionylated form was confirmed on the basis of the MS/MS spectra obtained by CID fragmentation (inserts). Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 5b

Detection by mass spectrometry of products of reaction of sulfenyl amides arising from incubation with GSH in aprotic buffer. MS spectra showing the compounds arising from (1) 1,2-benzisothiazolin-3-one (m/z 152.0165, z = 1) (upper panel) and (2) 2-methyl-4-isothiazolin-3-one (m/z 116.0165, z = 1) (lower panel) after 30 minutes of incubation with equimolar reduced glutathione in 100% CH₃CN. The main products observed are: the glutathionylated form (1) m/z 459.0988 (z =1) and (2) m/z 423.092 (z =1) and very small amount of the oxidized glutathione (GSSG) m/z 613.159 (z =1) and m/z 307.09 (z =2) as evidenced from comparison with reduced glutathione (GSH) m/z 308.091 in right inserts. No reduced forms of (1) and (2) were detected as shown in the left-hand inserts. Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 5b

Detection by mass spectrometry of products of reaction of sulfenyl amides arising from incubation with GSH in aprotic buffer. MS spectra showing the compounds arising from (1) 1,2-benzisothiazolin-3-one (m/z 152.0165, z = 1) (upper panel) and (2) 2-methyl-4-isothiazolin-3-one (m/z 116.0165, z = 1) (lower panel) after 30 minutes of incubation with equimolar reduced glutathione in 100% CH₃CN. The main products observed are: the glutathionylated form (1) m/z 459.0988 (z =1) and (2) m/z 423.092 (z =1) and very small amount of the oxidized glutathione (GSSG) m/z 613.159 (z =1) and m/z 307.09 (z =2) as evidenced from comparison with reduced glutathione (GSH) m/z 308.091 in right inserts. No reduced forms of (1) and (2) were detected as shown in the left-hand inserts. Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 6

Evaluation by mass spectrometry of reduced glutathione (GSH) levels following incubation of (1) in aqueous buffer containing an equimolar concentration of GSH. The amount of GSH has been estimated by the intensity of its monoisotopic mass (m/z 308.091, z =1) corrected for the intensity of the isobaric component present in the isotopic pattern of concurrent double charged GSSG (m/z 307.09, z =2), equivalent to 14.7 % of the monoisotopic mass intensity (insert in panel A). GSH (1 mM) was incubated 0.5 hours in aqueous buffer (75:25 v/v ammonium bicarbonate 40 mM, pH 8.0, CH₃CN) with the resulting analysis showing > 99% oxidation to GSSG, as deduced from isotopic ratio of the double charged GSSG (panel A). Meanwhile the presence of (1) 1,2-benzisothiazolin-3-one gave rise to significant concentrations of GSH that increased over 48 hours (panel B, C and table 2). Samples were analyzed by direct infusion as described in the Material and methods. Resolution 10000, accuracy 5 ppm.

Figure 7

Proposed interconversion between sulfenyl amide and glutathionylated forms.

Figure 8

Proposed mechanism for sulfenyl amide formation and reduction in proteins. Step 1 shows the reversible reaction between the sulfenyl amide- and disulfide-containing protein. Step 2 shows the reversible reaction between the glutathionylated and completely reduced-protein.