Enzymes in the sulfur network generate the signaling molecule, hydrogen sulfide (H2S), from the amino acids cysteine and homocysteine. Since it is toxic at elevated concentrations, cells are equipped to clear H2S. A canonical sulfide oxidation pathway operates in mitochondria, converting H2S to thiosulfate and sulfate. We have recently discovered the ability of ferric hemoglobin to oxidize sulfide to thiosulfate and iron-bound hydropolysulfides. In this study, we report that myoglobin exhibits a similar capacity for sulfide oxidation. We have trapped and characterized iron-bound sulfur intermediates using cryo-mass spectrometry and X-ray absorption spectroscopy. Further support for the postulated intermediates in the chemically challenging conversion of H2S to thiosulfate and iron-bound catenated sulfur products is provided by EPR and resonance Raman spectroscopy in addition to density functional theory computational results. We speculate that the unusual sensitivity of skeletal muscle cytochrome c oxidase to sulfide poisoning in ethylmalonic encephalopathy, resulting from the deficiency in a mitochondrial sulfide oxidation enzyme, might be due to the concentration of H2S by myoglobin in this tissue.

Unlike most other tissues, the colon epithelium is exposed to high levels of H 2 S derived from gut microbial metabolism. H 2 S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H 2 S exposure as they harbor an efficient mitochondrial H 2 S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H 2 S poisoning. H 2 S at concentrations <20 M stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H 2 S concentrations as low as 5 M poisoned cells. Mitochondrial H 2 S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD ؉ /NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H 2 S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H 2 S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H 2 S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming

Hydrogen sulfide (HS) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by HS has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by HS exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of HS oxidized. HS increases O consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent HS oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.

Synthesis of H2S by gut microbiota leads to routine colonic cells exposure to this respiratory toxin. Herein we studied the effect of H2S on metabolism and proliferation in human colonic cells in culture at sulfide concentrations up to 300 μM, which represents a physiologically relevant exposure for these cells. H2S caused a marked activation of glycolysis and inhibition of proliferation. Between 5–20 μM H2S the oxygen consumption rate (OCR) was activated, while OCR was inhibited at higher concentrations. A high‐capacity mitochondrial sulfide oxidation pathway housed in colonocytes supports O2‐dependent H2S clearance and the absence of the committing enzyme (sulfide quinone reductase) in the pathway leads to inhibition of OCR even at the lowest H2S concentration that was tested. H2S triggers persulfidation of numerous protein targets that are enriched in energy metabolism pathways. The antiproliferative effect of H2S exposure results from electron acceptor insufficiency that ensues from H2S oxidation activity and inhibition of respiration. Under these conditions, reductive carboxylation of a‐ketoglutarate is enhanced and exogenous uridine and aspartate alleviated H2S‐imposed growth restriction. Overexpression of the sulfide oxidation enzymes in colon cancer tissue and cells confers a growth advantage during H2S exposure compared to non‐malignant cells. These results predict that the stress response in colonocytes triggered by microbial H2S exposure involves metabolic reprogramming to recycle electron acceptors.Support or Funding InformationNIH (GM112455 to R.B.), AACR NextGen Grant for Transformative Cancer Research (17‐20‐01‐LYSS) to C.A.L., UMCCC Core Grant (P30 CA046592) to R.B. and C.A.L.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.