The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis

Abstract

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 H2S 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 H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 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 H2S 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 H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S 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. 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 H2S 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 H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 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 H2S 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 H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S 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. In contrast to most quiescent cells that rely on the efficiency of oxidative phosphorylation to harvest energy from glucose catabolism, rapidly dividing cells dial up glycolytic flux in a process referred to as the Warburg effect (1Warburg O. Posener K. Negelein E. On the metabolism of carcinoma cells.Biochem. Z. 1924; 152: 309-344Google Scholar). This metabolic switching is cued in part by growth factors and transforms mitochondria into central hubs for macromolecule precursor synthesis (2Ward P.S. Thompson C.B. Metabolic reprogramming: A cancer hallmark even Warburg did not anticipate.Cancer Cell. 2012; 21: 297-308Abstract Full Text Full Text PDF PubMed Scopus (1869) Google Scholar). In this metabolically reprogrammed setting, mitochondrial ATP generation becomes secondary to its role in anabolic precursor synthesis. The regulatory levers that dial down flux through the electron transport chain (ETC), however, continue to be elucidated. H2S is a respiratory poison that targets complex IV (3Nicholls P. Kim J.K. Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system.Can J. Biochem. 1982; 60: 613-623Crossref PubMed Google Scholar). It is also an endogenously synthesized metabolite and a product of at least three enzymes in the sulfur metabolic network (4Banerjee R. Catalytic promiscuity and heme-dependent redox regulation of H2S synthesis.Curr. Opin. Chem. Biol. 2017; 37: 115-121Crossref PubMed Scopus (34) Google Scholar, 5Singh S. Banerjee R. PLP-dependent H2S biogenesis.Biochim. Biophys. Acta. 2011; 1814: 1518-1527Crossref PubMed Scopus (119) Google Scholar). Two, cystathionine β-synthase and γ-cystathionase, are components of the transsulfuration pathway, which convert homocysteine to cysteine via cystathionine (6Chiku T. Padovani D. Zhu W. Singh S. Vitvitsky V. Banerjee R. H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.J. Biol. Chem. 2009; 284: 11601-11612Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 7Singh S. Padovani D. Leslie R.A. Chiku T. Banerjee R. Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.J. Biol. Chem. 2009; 284: 22457-22466Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). The third enzyme, mercaptopyruvate sulfurtransferase, resides in the catabolic arm of sulfur metabolism and converts 3-mercaptopyruvate to an enzyme-bound persulfide. The latter is transferred to a low-molecule-weight acceptor (e.g., cysteine) or to a protein thiol (e.g., on thioredoxin) from where H2S is subsequently eliminated (8Yadav P.K. Yamada K. Chiku T. Koutmos M. Banerjee R. Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.J. Biol. Chem. 2013; 288: 20002-20013Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 9Yadav P.K. Vitvitsky V. Carballal S. Seravalli J. Banerjee R. Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations.J. Biol. Chem. 2020; 295: 6299-6311Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Alternatively, the persulfide can potentially undergo further transsulfuration via transfer to other acceptors. Quantitatively, the output of H2S versus cysteine from the transsulfuration pathway is unknown, and there is limited insight into how these competing catalytic activities are regulated (10Kabil O. Yadav V. Banerjee R. Heme-dependent metabolite switching regulates H2S synthesis in response to ER stress.J. Biol. Chem. 2016; 291: 16418-16423Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A dedicated sulfide oxidation pathway in the mitochondrion converts H2S to the largely innocuous products, thiosulfate and sulfate (Fig. 1) (11Hildebrandt T.M. Grieshaber M.K. Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria.FEBS J. 2008; 275: 3352-3361Crossref PubMed Scopus (358) Google Scholar). The oxidation pathway begins with the conversion of H2S to a persulfide in a reaction catalyzed by the inner mitochondrial membrane protein, sulfide quinone oxidoreductase (SQOR) (12Landry A.P. Moon S. Kim H. Yadav P.K. Guha A. Cho U.S. Banerjee R. A catalytic in human sulfide quinone oxidoreductase A persulfide synthesis and Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Yadav P.K. Vitvitsky V. M. Banerjee R. of the human mitochondrial sulfide oxidation Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The from this reaction are transferred via to the and at the level of complex A.P. Banerjee R. Hydrogen sulfide oxidation by sulfide quinone PubMed Scopus Google Scholar). The persulfide product of the reaction M. Yadav P.K. Vitvitsky V. M. Banerjee R. of the human mitochondrial sulfide oxidation Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.P. Banerjee R. H2S oxidation by human sulfide quinone Biol. Chem. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, Yadav P.K. Banerjee R. kinetic analysis of sulfide oxidation catalyzed by human sulfide quinone Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is oxidized by O. Banerjee R. of in human persulfide involved in H2S Biol. Chem. 2012; Full Text Full Text PDF PubMed Scopus Google to which is oxidized to sulfate by oxidase or to thiosulfate by thiosulfate sulfurtransferase M. A. Banerjee R. of human in and sulfur transfer Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). from the oxidase reaction the at the level of complex IV via cytochrome c S. in mitochondria of and Biol. Chem. Full Text PDF PubMed Google Scholar). The of endogenously H2S is by the of the sulfide oxidation on the human protein the sulfide oxidation pathway enzymes are being to epithelial as might be for a pathway for with The mitochondrial of this pathway, and its with the at its role is to H2S or potentially other H2S is redox It can with oxidized cysteine thiols (e.g., or to or undergo oxidative metabolism to sulfur J. Banerjee R. of H2S through PubMed Scopus Google Scholar, O. Banerjee R. H2S and its role in redox Biophys. Acta. PubMed Scopus Google Scholar, M. Banerjee R. of sulfur for by sulfide oxidation Chem. Biol. PubMed Scopus Google Scholar). of the in the of H2S redox on protein Kim S. W. R. H2S through protein 2009; PubMed Scopus Google a oxidative of which on of in D. M. M. O. Banerjee R. protein metabolic during the PubMed Scopus Google Scholar, E. A. K. K. D. P. A novel persulfide protein and of and 2016; PubMed Scopus Google Scholar). and that is with the regulation of a protein in a cellular to be It is that protein is a or oxidation R. Banerjee R. of the redox and thiol by Biochem. Biol. PubMed Scopus Google Scholar). of a metabolite that as a respiratory poison a its effect on complex IV the cellular effect of H2S to It is to H2S in the of redox metabolic that from the R. Banerjee R. of the redox and thiol by Biochem. Biol. PubMed Scopus Google Scholar). Studies the of H2S in cells its as a (e.g., or as a (e.g., M. M. P.K. a novel in the Biol. 2009; PubMed Scopus Google of H2S with the of epithelial cells as that are to high of H2S from metabolism to from to of in of the human Google Scholar, K. and to in Biol. PubMed Google Scholar). The of H2S to (e.g., for to T. P.K. sulfide as a novel by cancer glycolysis and J. PubMed Scopus Google Scholar). we observed the effect of H2S on and epithelial cells by with or M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is the t1/2 of H2S cell culture which is an not to its but also to its to with and other oxidized thiols in the culture human carcinoma cell and that levels of and with a epithelial cell M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). also demonstrated that H2S a metabolic the and other H2S glycolysis, the this effect that it was of its with the T. P.K. sulfide as a novel by cancer glycolysis and J. PubMed Scopus Google Scholar). In this study, we that the of H2S is rapidly from culture and to its a fraction is trapped as via reaction with and oxidized protein that H2S energy metabolism and glucose and and that these are sensitive to the mitochondrial but not cytoplasmic NADH pool. Finally, we demonstrated that regulates these cellular a for electron flux and energy metabolism The sulfide and are as of sulfide in cell culture H2S is and the of abiotic loss culture which is to cellular is largely unknown. examined the of sulfide loss plate and suspension culture that are in as a to its on energy to at 37 °C in its (Fig. with a t1/2 of in cell culture with the sulfide decreased to min and decreased with a t1/2 of min (Fig. the sulfide was at to in the for which was of or not cells which can with H2S to cysteine and cysteine persulfide and, other sulfane sulfur species. cysteine and sulfane sulfur observed In the of the cysteine was at in 4 it decreased to in culture the (Fig. in the of cellular metabolism also to a in cysteine with with cultured cells to The sulfane sulfur in the culture decreased to in 4 (Fig. In the of the sulfane sulfur levels also of and decreased to in 4 The of the sulfane sulfur is that it a of and protein in sulfur was not observed in in the of and cells. with In with culture with sulfide was in and the of its in the of cells with a (Fig. sulfide was by the of the oxidation thiosulfate (Fig. The sulfide and thiosulfate that thiosulfate was the oxidation product of cells The effect of H2S on the of glucose was examined in suspension in with a enhanced glucose and production in response to H2S (Fig. A and of aerobic glycolysis was a response to other cell A to of glucose and production was observed at sulfide in other cell and as (Fig. and further of glycolysis was observed this sulfide as In contrast, the cells did not aerobic glycolysis in response to sulfide (Fig. and at a sulfide a of glycolysis was observed in cells (Fig. of glycolysis the of to glucose to the of in and cells (Fig. In and the was even in the of sulfide enhanced the rate of glucose oxidation in the cell it the glucose and in and where the for further from glucose to examined how enzymes in the mitochondrial sulfide oxidation pathway sulfide and the response to enhanced aerobic glycolysis. the first pathway enzymes down in as M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and (Fig. H2S by and cells sulfide and thiosulfate production (Fig. and cells an sensitivity to and of aerobic glycolysis was observed at H2S (Fig. In contrast, a in the rate of aerobic glycolysis was observed at sulfide in and cells. observed in the (Fig. H2S the glycolytic rate and the of glucose in a cell A. S. A. Singh K. M. is for of by PubMed Scopus Google we examined it the pentose phosphate pathway the M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we of sulfide by 3 to for its from cell culture The effect of sulfide on central metabolism was by the of from or (Fig. of in from the oxidative pentose phosphate pathway and the TCA via the TCA cycle. H2S did not the of from or in and (Fig. and was from versus glucose in cells and in that the oxidative pentose phosphate pathway the TCA is the of in these cell the role of the mitochondrial versus cytoplasmic redox on sulfide-stimulated aerobic glycolysis, cells cytoplasmic or mitochondrial the NADH oxidase from V. J. Z. of mitochondrial electron transport chain by of the 2016; PubMed Google (Fig. In the an oxidative in a (Fig. or mitochondria a complex to a in cytochrome E. is an of in Full Text Full Text PDF PubMed Scopus Google (Fig. In these the mitochondrial and are to be to a stimulated aerobic glycolysis in as as in cells cytoplasmic (Fig. that sulfide is not via the cytoplasmic pool. In contrast, cells mito-LbNOX a in the glucose which was stimulated by H2S (Fig. that mito-LbNOX cells activate aerobic glycolysis even in the of H2S to for mitochondrial ATP synthesis to electron flux through complex we examined the effect of H2S on glycolysis in mito-LbNOX cells in the of aerobic glycolysis a of H2S a sensitivity of these cells to H2S for a in cytoplasmic energy metabolism (Fig. A and The mito-LbNOX cells H2S rapidly or cytoplasmic which was by an production of thiosulfate (Fig. and are with for the in mito-LbNOX cells in which the mitochondrial NADH is In with the a glycolysis and a production to glucose even in the of that of glucose the cell was to of (Fig. was not further stimulated by H2S in these cells. with of H2S and a of thiosulfate (Fig. The is a for electron the cytoplasmic and mitochondrial (Fig. The role of this in sulfide from the mitochondrion to the was examined by down and in cells (Fig. A and sulfide-stimulated aerobic glycolysis with cells (Fig. and we examined sulfide phosphorylation of and from the TCA cycle. was min of sulfide phosphorylation in min (Fig. and and was for at least min A in phosphorylation levels was not observed in or cells (Fig. that this is not a for sulfide from the mitochondrion to the IV by sulfide is to ATP synthesis via oxidative phosphorylation and decreased ATP to in and cells and (Fig. A in was observed in the cell In with the cells ATP and which did not to sulfide (Fig. levels low in most cell and not be to the high with sulfide in a in the in cell (Fig. of the in the H2S is the sulfide that are to cellular and the of on the of exogenously sulfide from of to sulfide are in cell culture with high sulfide The using high sulfide in the of a of cellular of that in the in sulfide a in Biophys. Acta. 2009; PubMed Scopus Google In however, there a that H2S in mammalian cells are in the low J. A. sulfide are of J. 2008; 295: PubMed Scopus Google Scholar, V. O. Banerjee R. for sulfide for regulation of its 2012; PubMed Scopus (119) Google high to sulfide from metabolism that is to be in the of to of in of the human Google Scholar, K. and to in Biol. PubMed Google Scholar). a for sulfide we examined the of exogenously H2S in cell culture and cell suspension that are for metabolic that H2S rapidly cell culture 37 with a t1/2 of min of or not cells are (Fig. This abiotic loss is to the of H2S as it is from at an a small fraction of H2S is trapped in a sulfane sulfur that is a of persulfide and species. The trapped sulfane sulfur of the added sulfide at to at 4 and be a of low sulfide to cells in culture via sulfur transfer as by In suspension culture in with a culture the rate of abiotic sulfide loss is and is in the of metabolism (Fig. metabolism in cell suspension was sensitive to the of the first in the sulfide oxidation pathway (Fig. but not to sulfide was to thiosulfate (Fig. that thiosulfate as a for oxidative sulfide metabolism these The sensitivity of aerobic glycolysis to but not to (Fig. is with the of cells to complex IV M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of glucose was observed in which are on growth glucose as as in which are not (Fig. The however, of sulfide to a metabolic response with the cells and The by which aerobic glycolysis is up in the in response to sulfide metabolism in the mitochondrion is not to the Warburg effect decreased energy loss of (e.g., by in complex of mitochondrial and of the in glycolysis Thompson C.B. the Warburg The metabolic of cell 2009; PubMed Scopus Google Scholar). the potential contributions of of these to The for the glucose is of glucose is to of can be from other as which was not in the in suspension however, that sulfide did not the flux of glucose through the oxidative pentose phosphate pathway or through the oxidative TCA (Fig. in cell in which the glucose was the of glucose and production enhanced (Fig. low concentrations, sulfide as a the at the level of complex (Fig. concentrations, sulfide complex which is to to membrane and, to decreased ATP synthesis by complex V. with this a in ATP and an in was observed in response to sulfide in and cells (Fig. In contrast, in cells in which the is ATP levels and sulfide did not affect the ATP levels in cells are to a response by glycolysis. The glycolytic flux is by up a redox which of This is in the of by which is to the of the a of the (Fig. in cells decreased of into the the that we in cells M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of effect of or on aerobic glycolysis (Fig. is also with a of the by a on the mitochondrial but not the cytoplasmic for by that the is in and an in the which was further in cells M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the genetically encoded tool V. J. Z. of mitochondrial electron transport chain by of the 2016; PubMed Google Scholar, V. H. Z. A genetically encoded tool for of in Chem. Biol. 2017; PubMed Scopus Google we the in a a role for the mitochondrial NADH by a blunted response to sulfide of aerobic glycolysis in cells mito-LbNOX (Fig. In mito-LbNOX cells in which there is for the from complex sulfide was forming thiosulfate (Fig. In we demonstrated that the dual of mitochondrial and energy metabolism by sulfide its with the The in the mitochondrial is an in the sulfide pathway, which leads to enhanced aerobic glycolysis. The of sulfide to in metabolic by tuning the might be for of and which energy metabolism from oxidative phosphorylation to aerobic glycolysis to the energy and anabolic of cell (2Ward P.S. Thompson C.B. Metabolic reprogramming: A cancer hallmark even Warburg did not anticipate.Cancer Cell. 2012; 21: 297-308Abstract Full Text Full Text PDF PubMed Scopus (1869) Google Scholar). culture inhibitor for mammalian and from was from and from culture with and with and from cancer cell and from The human and a from and the human cell was from of and cells cultured in with and and cultured in the that was also cells cultured in with The of and down cell was M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and these cells cultured as for that the culture was with the of in cell culture at 37 °C a cells cultured in a of and with human growth and in a with an and at 37 was in cells as for M. Vitvitsky V. T. E. E. Banerjee R. Hydrogen sulfide mitochondrial and metabolic in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the with was from and from with a was as a was not observed with with there was (Fig. cells cultured as for cells that the cell culture The and mito-LbNOX a from of and mito-LbNOX by the of cell the cytoplasmic or as cells in a plate with of with and The cells for at 37 °C in a with a with the in the was with and was for an the cells using It to 3 to cells for during which with and was the of an cells for with to was with the cells with and in with inhibitor to mammalian cell and three and for min at at 4 The was and protein was using the on a and transferred to a was with a an was to for its at 4 °C with at a of secondary was at a of The using and using a and from of of cells was as with The cells cultured in with and or was by cells with for or was in cells with analysis was as for and at a of secondary was at a of cells at a of cells plate 4 of culture and at 37 The the was with and was added from a in culture but cells as The of abiotic sulfide was in and The and with and plate was to a In with cell culture as of cell culture or at the and cysteine analysis with of and on and at °C further for sulfane sulfur analysis on and at °C further The culture was from in and cells with and with cells in cell culture and at for min at 4 The was and the cell was in of from and by glucose and The suspension was at for min at 4 and the cell was in a the was The cell was in to a to cell suspension and at 37 °C with at was added from a to the The cell suspension to of the and the was with at the with of and on and thiosulfate levels in the by as and cysteine analysis the with as The and of in was and the was in the at °C for was the was at for min at 4 and the was at °C with the from The on a with and as V. 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