The Mitochondrial NADH Pool is Involved in Hydrogen Sulfide Signaling and Stimulation of Aerobic Glycolysis

Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain at the level of complex III. Studies with HS are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added HS to cultured cells is unknown. In this study, we first examined the half-life of exogenously added HS to human colonic epithelial cells. In plate cultures, HS disappeared with a t/ of 3 to 4 min at 37 °C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of HS was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits HS to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of HS to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and, therefore, cellular energy metabolism, particularly during cell proliferation.