Fig 2: Expression levels of proteins involved in H2S metabolism. A. Expression levels of proteins involved in cysteine catabolism (SO, SQR, CBS, CSE, TST, GOT1, MPST, GOT2 and ETHE1) in WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells. Tubulin was used as a loading control. B. Quantification of expression changes of SQR in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.C. Quantification of expression changes of ETHE1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Quantification of expression changes of CSE in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. E. Quantification of expression changes of CBS in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. F. Quantification of expression changes of GOT1 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. G. Quantification of expression changes of GOT2 in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. H. Quantification of expression changes of MPST (cytosolic and mitochondrial isoforms) in relation to tubulin and WT samples (n = 4). Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.

Fig 3: Alterations in cysteine catabolism followingsulfite oxidase (SO) deficiency. Cysteine catabolism in WT and SO-deficient cells. Steps and enzymes that localize to mitochondria are depicted by the light blue background. The area outside refers to the cytosol. Cytosolic glutamate oxaloacetate transaminase 1 (GOT1) is responsible for cellular sulfite production, whereas mitochondrial GOT (GOT2), plays an important role in H2S production in SO deficiency. In absence of SO, sulfite (SO32-) and hydrogen sulfide (H2S) accumulate. SO deficiency furthermore further impacts cellular cysteine catabolism resulting in lower intracellular cysteine levels and downregulation of many involved enzymes (depicted in gray letters). Moderate increase in GOT2 and strong induction of sulfide:quinone oxidoreductase (SQR) suggest an increased flux of sulfur via the GOT2-MPST-SQR axis. Abbreviations: CBS, cystathionine ß-synthase; CSE, cystathionine ?-lyase; CDO, cysteine dioxygenase; GOT, glutamate oxaloacetate transaminase; MPST, mercaptopyruvate sulfurtransferase; SQR, sulfide:quinone oxidoreductase; ETHE1, persulfide dioxygenase; TST, thiosulfate sulfurtransferase.

Fig 4: Contribution of GOT1 and GOT2 to cellular sulfite production. A. Western Blot of HEK293 lysates confirming knockout of SO, GOT1 and GOT2 (n = 3). B. Sulfite standard curve used for the determination of sulfite concentration from cellular extracts (n = 3). C. Sulfite measurement from cell extracts of WT and SUOX-/- cells with and without addition of purified SO (n = 4). Sulfite levels were normalized to cellular protein concentration. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. D. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells (n = 4). Sulfite levels were normalized to cellular protein concentration. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. E. Relative (isoform-unspecific) GOT activity determined from extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells (n = 3). Values were adjusted to cellular protein concentration and normalized to WT. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. pvalue: *** <0.001; ** <0.01; * <0.05; ns > 0.05. F. Relative cell proliferation of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells determined by MTT assay (n = 3). Values were adjusted to cellular protein concentration and normalized to WT. Error bars indicate standard deviation. One-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.

Fig 5: Dependence of sulfite production on cellular cysteine- and CDO levels. A. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells after incubation in different cysteine concentrations (n = 4). Sulfite levels were adjusted to cellular protein concentration. Values were normalized to WT. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05. B. Sulfite measurement from cell extracts of WT, SUOX-/-, GOT1-/-/SUOX-/- and GOT2-/-/SUOX-/- cells following overexpression of CDO (n = 4). Sulfite levels were adjusted to cellular protein concentration. Values were normalized to WT. Error bars indicate standard deviation. Two-way ANOVA with Tukey's post-hoc test for pairwise comparisons was performed as indicated. p value: *** <0.001; ** <0.01; * <0.05; ns > 0.05.