Fig 2: Sulfenic acid modification of the residue Cys255 is crucial for oxidation sensing. (a,b) Recombinant wild-type or mutant CSE was treated with 10 mM DTT. The samples were then ultrafiltered to remove DTT and exposed to air oxygen for 1 h at room temperature by gently pipetting up and down. CSE activity was measured using 20 mM L-cysteine (a) or 10 mM S-methylcysteine (b) as substrates. The data points and errors are the means ± SD (n = 4). An unpaired, two-tailed Student’s t test was used to determine the significance of differences between two group means. (c) LC-MS/MS analysis indicates the formation of the dimedone adduct (+138.068 Da) of sulfenic acid in Cys255.

Fig 3: The fluorescence microscope images of H2S signaling in HA-VSMCs treated with VEGF. (a) Untreated cells imaged as described in methods and were used as a control group. (b) Cells were incubated with VEGF at a final concentration of 50 ng/mL for 30 min at 37 °C and then imaged. (c,d) Brightfield images corresponding to (a,b) (Scale bar, 250 µm). (e,f) Cells were preincubated with 2 mM PAG for 12 h to inhibit CSE activity. Subsequently, the cells were incubated with PBS (e) or 50 ng/mL VEGF (f) for 30 min, washed and then imaged. (g,h) Brightfield images corresponding to (e,f) (Scale bar, 250 µm). (i) Quantification of the fluorescence microscope images of H2S signaling in HA-VSMCs, with data from (a,b,e) and (f) for comparison. The graph represents the relative fluorescence intensity compared with the relative fluorescence intensity of the untreated cells (a) and shows the means ± SD (n = 6). (j) Cells were exposed to 50 ng/mL VEGF for 30 min at 37 °C. The cell lysates were resolved on SDS-PAGE and immunoblotted for CBS and CSE. Untreated cells were used as a control group.

Fig 4: The redox state of CXXC motif in CSE affects its activity. (a,b) Recombinant wild-type CSE was incubated with 10 mM DTT, 5 mM oxidized DTT (DTToxi), or 400 µM H2O2 for 1 h at room temperature. The samples were then ultrafiltered to remove DTT, DTToxi or H2O2. CSE activity was measured using 20 mM L-cysteine (a) or 10 mM S-methylcysteine (b) as substrates. The data points and errors are the means ± SD (n = 4). An unpaired, two-tailed Student’s t test was used to determine the significance of differences between two group means. Reduced (DTT): CSE treated with DTT; Oxidized (DTToxi) and Oxidized (H2O2): CSE treated with DTToxi or H2O2; Reoxidized (H2O2) and Reoxidized (Air): reduced CSE was reoxidized by exposure to H2O2 or air oxygen. The specific activity of the reduced CSE using S-methylcysteine as a substrate (using DTNB detection) was 15.3 ± 1.22 nmol methanethiol mg-1 min-1. (c) Alignment of amino acid sequences of CSE from different species using the Unipro UGENE software. (d,e) LC-MS/MS analysis of the chymotrypsin-digested peptides containing the Cys252-Cys255 disulfide bond in CSE protein.

Fig 5: Structural comparison of the reduced and oxidized CSE during molecular dynamics (MD) simulations and molecular docking. (a) Snapshot structure of the reduced CSE at t = 0 ns (Left) and t = 100 ns (Center), along with the optimal backbone alignment of the two structures (Right). (b) Snapshot structure of the oxidized CSE at t = 0 ns (Left) and t = 200 ns (Center) along with the optimal backbone alignment of the two structures (Right). For clarity, the residues Cys252 and Cys255 in the CXXC motif are colored in red, and the PLP cofactor is colored in blue. (c,d) Comparison of the substrate entrance tunnel of the reduced CSE and the oxidized CSE after MD simulations. (e,f) Interaction between the substrate l-cysteine and the active center of the reduced and oxidized CSE using molecular docking. The blue dashed line denotes the hydrogen bond, and the red dashed line denotes the distance between the amino group of l-cysteine and the cofactor PLP.