Cystathionine γ-lyase (CSE) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the final step of the transsulfuration pathway, converting cystathionine into cysteine. Additionally, CSE is also essential for the formation of cysteine hydropolysulfide (Cys-S-(S)n-H), known as supersulfides, by metabolizing cystine under pathological conditions. We previously reported that, during cystine metabolism, CSE undergoes self-inactivation through polysulfidation at the Cys136 residue. Here, contrary to the anticipated role of L-S-nitrosocysteine (L-CysNO) as a nitric oxide (NO) donor, we demonstrate that it serves as a substrate for CSE and that its metabolites inhibit the activity of the enzyme during L-CysNO metabolism. The in vitro incubation of CSE—but not the Cys136/171Val mutant—with L-CysNO resulted in the dose-dependent inhibition of supersulfide production, which was not reversed by the reducing agents. Notably, CSE activity remained unchanged upon preincubation with other NO donors, such as S-nitrosoglutathione or D-CysNO, but was inhibited when coincubated with cysteine. Furthermore, when PLP was removed from the CSE/L-CysNO premix, L-CysNO no longer inhibited CSE activity, suggesting that CSE metabolizes L-CysNO and that its metabolites contribute to enzyme inactivation. Indeed, we identified thionitrous acid and pyruvate as the primary CSE/L-CysNO reaction products. Thus, we establish L-CysNO as a CSE substrate and demonstrate that its metabolites act as enzyme inhibitors through a novel irreversible modification at the Cys136/171 residues.

Cystathionine γ-lyase (CSE) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. Additionally, CSE also plays a crucial role in the production of cysteine hydropolysulfide (Cys-S-(S)n-H), known as supersulfides, by metabolizing cystine under pathological conditions. We previously reported that, during cystine metabolism, CSE undergoes self-inactivation through polysulfidation at the Cys136 residue. Here, contrary to the anticipated role of L-S-nitrosocysteine (L-CysNO) as a nitric oxide (NO) donor, we demonstrate that it serves as a substrate for CSE and that its metabolites inhibit its activity. The in vitro incubation of CSE—but not the Cys136/171Val mutant—with L-CysNO resulted in the dose-dependent inhibition of supersulfide production, which was not reversed by the reducing agents. Notably, CSE activity remained unchanged upon pre-incubation with other NO donors, such as S-nitrosoglutathione or D-CysNO, but was inhibited when co-incubated with cysteine. Furthermore, when PLP was removed from the CSE/L-CysNO premix, L-CysNO no longer inhibited CSE activity, suggesting that CSE metabolizes L-CysNO and that its metabolites contribute to enzyme inactivation. Indeed, we identified thionitrous acid and pyruvate as the primary CSE/L-CysNO reaction products. Thus, we establish L-CysNO as a CSE substrate and demonstrate that its metabolites act as enzyme inhibitors through a novel irreversible modification at the Cys136/171 residues.