Fig. 2: Cyanide is enzymatically generated by lysosomal peroxidases.

a, The cyanide signal in HepG2 cells partially colocalizes with lysosomes (confocal microscopy using Chemosensor P). Images shown are representative of n = 3 biological replicates per group. b,c, Cyanide generation in lysosomal and cytosolic fractions (Lyso and Cyto, respectively) obtained from mouse liver or HepG2 cells ± 10 mM glycine (Gly; ECh method; at least n = 5 per group, biological replicates; b) or from isolated intact versus disrupted lysosomes (ECh method; at least n = 5 per group, biological replicates; c) as visually confirmed by electron microscopy. d, Cyanide generation in isolated lysosomes after treatment with 1 µM bafilomycin (Baf), 30 µM hydroxychloroquine (Hcq) or 150 mM glycylglycine dipeptide (Gly-Gly; ECh method; n = 5 per group, biological replicates). e, Cyanide detection in HepG2 cells with 0.1–10 µM phloroglucinol (Phl; ECh method; at least n = 5 per group, biological replicates). f, Cyanide detection from isolated lysosomes obtained from mouse liver homogenates incubated with 0.4 mM glycine in the absence or presence of 10 µM Phl (left) or HepG2 cells incubated with 0.4 mM or 10 mM glycine in the absence or presence of 10 µM Phl (ECh method; at least n = 5 per group, biological replicates). g, MPO and PXDN expression in lysosomal (L) and cytosolic (C) fractions of HepG2 cells (n = 5 biological replicates for MPO and n = 4 biological replicates for PXDN). h, Confocal microscopy of MPO localization in lysosomes. Nuclei were chemically stained using DAPI, while lysosomes and MPO were immunohistochemically detected using LAMP1 and MPO antibodies, respectively. Images shown are representative of n = 3 biological replicates per group. i, MPO or PXDN catalyse cyanide generation at pH 4.5 (enzyme was incubated with various combinations of 1 mM glycine, 1 mM H₂O₂ and 150 mM NaCl; ECh method; at least n = 5 per group, technical replicates). j, Determination of an optimal pH for cyanide generation using equimolar concentrations of HOCl and glycine (optimum pH 4.5; ECh method; n = 9 per group, technical replicates). k, Cyanide production in liver and spleen homogenates of WT, PXDN^+/− and MPO^−/− male mice under baseline conditions and after the addition of 10 mM glycine (Gly; ECh method; at least n = 4 per group, biological replicates). l, Detection of cyanide in HepG2 cells treated with 1–100 µM MPO inhibitor AZD-5904 (ECh method; at least n = 5 per group, biological replicates). m, Detection of cyanide in HEK293T cells overexpressing (OE) MPO or PXDN in the absence or presence of an additional 10 mM glycine (ECh method; at least n = 5 per group, biological replicates). n, Impact of overexpression of human rhodanese (OE-TST, cells from two different passages) or its downregulation (shTST) in HepG2 cells (as confirmed by western blots) on cellular capacity to degrade exogenously administered 100 µM KCN in the absence or presence of 1 mM sodium thiosulfate (ECh method; n = 5 per group, biological replicates). o, Overexpression of human rhodanese (OE-TST) or its downregulation (shTST) in HepG2 cells resulted in the reduction or accumulation, respectively, of endogenous cyanide in HepG2 cells (ECh method; n = 7 per group, biological replicates). Data in b–g, i, j–m and o are expressed as the mean ± s.e.m. Data in b–f, i and k–o were analysed with a two-way ANOVA followed by Bonferroni’s multiple-comparisons test. Data in c and g were analysed with a two-sided Student’s t-test. *P < 0.05 and **P < 0.01 indicate significant differences. p, Our proposed scheme of lysosomal cyanide generation. In lysosomes, at pH 4.5, glycine undergoes a two-step chlorination reaction in the presence of peroxidase-derived HOCl. The subsequent hydrolysis of N,N-dichloroglycine leads to the formation of an unstable nitrile derivative intermediate, which spontaneously decomposes to carbon dioxide (CO₂) and hydrogen cyanide (HCN). HCN, in turn, is converted to SCN⁻ and CO₂ via rhodanese/TST using thiosulfate in the extra-lysosomal cell compartment.

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