Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

Sulfide Oxidation by a Noncanonical Pathway in Red Blood Cells Generates Thiosulfate and Polysulfides

A cardioprotectant at low concentrations, H₂S is a toxin at high concentrations and inhibits cytochrome c oxidase. A conundrum in H₂S homeostasis is its fate in red blood cells (RBCs), which produce H₂S but lack the canonical mitochondrial sulfide oxidation pathway for its clearance. The sheer abundance of RBCs in circulation enhances the metabolic significance of their clearance strategy for H₂S, necessary to avoid systemic toxicity. In this study, we demonstrate that H₂S generation by RBCs is catalyzed by mercaptopyruvate sulfurtransferase. Furthermore, we have discovered the locus of sulfide oxidation in RBCs and describe a new role for an old protein, hemoglobin, which in the ferric or methemoglobin state binds H₂S and oxidizes it to a mixture of thiosulfate and hydropolysulfides. Our study reveals a previously undescribed route for the biogenesis of hydropolysulfides, which are increasingly considered important for H₂S-based signaling, but their origin in mammalian cells is unknown. An NADPH/flavoprotein oxidoreductase system restores polysulfide-carrying hemoglobin derivatives to ferrous hemoglobin, thus completing the methemoglobin-dependent sulfide oxidation cycle. Methemoglobin-dependent sulfide oxidation in mammals is complex and has similarities to chemistry reported for the dissolution of iron oxides in sulfidic waters and during bioleaching of metal sulfides. The catalytic oxidation of H₂S by hemoglobin explains how RBCs maintain low steady-state H₂S levels in circulation, and suggests that additional hemeproteins might be involved in sulfide homeostasis in other tissues.     

Association of the I264T Variant in the Sulfide Quinone Reductase-Like (SQRDL) Gene with Osteoporosis in Korean Postmenopausal Women

To identify novel susceptibility variants for osteoporosis in Korean postmenopausal women, we performed a genome-wide association analysis of 1180 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 405 individuals with osteoporosis and 722 normal controls of the Korean Association Resource cohort. A logistic regression analysis revealed 72 nsSNPs that showed a significant association with osteoporosis (p<0.05). The top 10 nsSNPs showing the lowest p-values (p = 5.2×10^-4–8.5×10^-3) were further studied to investigate their effects at the protein level. Based on the results of an in silico prediction of the protein’s functional effect based on amino acid alterations and a sequence conservation evaluation of the amino acid residues at the positions of the nsSNPs among orthologues, we selected one nsSNP in the SQRDL gene (rs1044032, SQRDL I264T) as a meaningful genetic variant associated with postmenopausal osteoporosis. To assess whether the SQRDL I264T variant played a functional role in the pathogenesis of osteoporosis, we examined the in vitro effect of the nsSNP on bone remodeling. Overexpression of the SQRDL I264T variant in the preosteoblast MC3T3-E1 cells significantly increased alkaline phosphatase activity, mineralization, and the mRNA expression of osteoblastogenesis markers, Runx2, Sp7, and Bglap genes, whereas the SQRDL wild type had no effect or a negative effect on osteoblast differentiation. Overexpression of the SQRDL I264T variant did not affect osteoclast differentiation of the primary-cultured monocytes. The known effects of hydrogen sulfide (H₂S) on bone remodeling may explain the findings of the current study, which demonstrated the functional role of the H₂S-catalyzing enzyme SQRDL I264T variant in osteoblast differentiation. In conclusion, the results of the statistical and experimental analyses indicate that the SQRDL I264T nsSNP may be a significant susceptibility variant for osteoporosis in Korean postmenopausal women that is involved in osteoblast differentiation.     

Taxonomic distribution,structure/function relationship and metabolic context of the two families of sulfide dehydrogenases: SQR and FCSD

Hydrogen sulfide (H₂S) is a versatile molecule with different functions in living organisms: it can work as a metabolite of sulfur and energetic metabolism or as a signaling molecule in higher Eukaryotes. H₂S is also highly toxic since it is able to inhibit heme cooper oxygen reductases, preventing oxidative phosphorylation. Due to the fact that it can both inhibit and feed the respiratory chain, the immediate role of H₂S on energy metabolism crucially relies on its bioavailability, meaning that studying the central players involved in the H₂S homeostasis is key for understanding sulfide metabolism.Two different enzymes with sulfide oxidation activity (sulfide dehydrogenases) are known: flavocytochrome c sulfide dehydrogenase (FCSD), a sulfide:cytochrome c oxidoreductase; and sulfide:quinone oxidoreductase (SQR).In this work we performed a thorough bioinformatic study of SQRs and FCSDs and integrated all published data. We systematized several properties of these proteins: (i) nature of flavin binding, (ii) capping loops and (iii) presence of key amino acid residues. We also propose an update to the SQR classification system and discuss the role of these proteins in sulfur metabolism.     

Complexities of complex II: Sulfide metabolism in vivo

High levels of H₂S produced by gut microbiota can block oxygen utilization by inhibiting mitochondrial complex IV. Kumar et al. have shown how cells respond to this inhibition by using the mitochondrial sulfide oxidation pathway and reverse electron transport. The reverse activity of mitochondrial complex II (succinate-quinone oxidoreductase, i.e., fumarate reduction) generates oxidized coenzyme Q, which is then reduced by the mitochondrial sulfide quinone oxidoreductase to oxidize H₂S. This newly identified redox circuitry points to the importance of complex II reversal in mitochondria during periods of hypoxia and cellular stress.     

The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H₂O₂ in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H₂O₂ levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H₂O₂ is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).     

Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

Sulfide oxidation in gram-negative bacteria by expression of the sulfide-quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone.

The oxidation of sulfide was studied in recombinant bacteria expressing the sulfide-quinone reductase gene (sqr) from Rhodobacter capsulatus. Sulfide was oxidized by the Escherichia coli strain W3110 harboring the sqr construct (pKKSQ) under anaerobic conditions and nitrate was utilized as a terminal electron acceptor. Following the oxidation, elemental sulfur and nitrite were produced as the final reaction products. This activity was retained in the membrane preparation and was sensitive towards antimycin A, stigmatellin, and azide. As a consequence of the ubiquinone deficiency, this activity was markedly decreased. In additon, by recovery of ubiquinone, the oxidation was also restored to rates similar to those of the wild-type strain. These results indicate that sulfide oxidation in this strain occurs via the quinone pool in vivo, and that this sulfide-quinone reductase (SQR) in particular utilizes ubiquinone as a more appropriate electron acceptor than menaquinone or demetylmenaquinone. To our knowledge, this is the first study to show a direct interaction between SQR and ubiquinone in cells. When expressed in Pseudomonas putida and Rhizobium meliloti, the SQR conferred on these organisms the ability to oxidize sulfide as well as E. coli in vivo.     

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Background

Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer''s disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage.

Methodology and Principal Findings

H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors.

Innovation

This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences.

Significance

H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.     

Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense

Hydrogen peroxide (H₂O₂) is a key signaling molecule that also induces apoptosis. Thus, cells must rapidly sense and tightly control H₂O₂ levels. Well-characterized cellular responses to exogenous H₂O₂ involve oxidation of specific cytosolic protein-based thiols but sensing of H₂O₂ generated by mitochondrial respiration is less well described. Here we provide substantial biochemical evidence that the heme enzyme Ccp1 (cytochrome c peroxidase), which is targeted to the intermembrane space, functions primarily as a mitochondrial H₂O₂ sensing and signaling protein in Saccharomyces cerevisiae. Key evidence for a sensing role for Ccp1 is the significantly higher H₂O₂ accumulation in ccp1-null cells(ccp1Δ) vs ccp1^W191F cells producing the catalytically inactive Ccp1^W191F variant. In fact, intracellular H₂O₂ levels (ccp1Δ>wildtype >ccp1^W191F) correlate inversely with the activity of the mitochondrial (and peroxisomal) heme catalase, Cta1 (ccp1Δ<wildtype <ccp1^W191F). Mitochondrial Sod2 activity also varies in the three strains (ccp1Δ>wildtype >ccp1^W191F) and ccp1Δ cells exhibit low superoxide levels. Notably, Ccp1^W191F is a more persistent H₂O₂ signaling protein than wild-type Ccp1, and this enhanced mitochondrial H₂O₂ signaling decreases the mitochondrial fitness of ccp1^W191F cells. However, these cells are fully protected from a bolus (0.4 mM) of exogenous H₂O₂ added after 12 h of growth, whereas the viability of ccp1Δ cells drops below 20%, which additionally associates Ccp1 with Yap1-dependent H₂O₂ signaling. Combined, our results strongly implicate Ccp1, independent of its peroxidase activity, in mitochondrial H₂O₂ sensing and signaling to maintain reactive oxygen species homeostasis.     

Anaerobic Expression of Escherichia coli Succinate Dehydrogenase: Functional Replacement of Fumarate Reductase in the Respiratory Chain during Anaerobic Growth

Succinate-ubiquinone oxidoreductase (SQR) from Escherichia coli is expressed maximally during aerobic growth, when it catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid cycle and reduces ubiquinone in the membrane. The enzyme is similar in structure and function to fumarate reductase (menaquinol-fumarate oxidoreductase [QFR]), which participates in anaerobic respiration by E. coli. Fumarate reductase, which is proficient in succinate oxidation, is able to functionally replace SQR in aerobic respiration when conditions are used to allow the expression of the frdABCD operon aerobically. SQR has not previously been shown to be capable of supporting anaerobic growth of E. coli because expression of the enzyme complex is largely repressed by anaerobic conditions. In order to obtain expression of SQR anaerobically, plasmids which utilize the P_FRD promoter of the frdABCD operon fused to the sdhCDAB genes to drive expression were constructed. It was found that, under anaerobic growth conditions where fumarate is utilized as the terminal electron acceptor, SQR would function to support anaerobic growth of E. coli. The levels of amplification of SQR and QFR were similar under anaerobic growth conditions. The catalytic properties of SQR isolated from anaerobically grown cells were measured and found to be identical to those of enzyme produced aerobically. The anaerobic expression of SQR gave a greater yield of enzyme complex than was found in the membrane from aerobically grown cells under the conditions tested. In addition, it was found that anaerobic expression of SQR could saturate the capacity of the membrane for incorporation of enzyme complex. As has been seen with the amplified QFR complex, E. coli accommodates the excess SQR produced by increasing the amount of membrane. The excess membrane was found in tubular structures that could be seen in thin-section electron micrographs.     

Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H₂S) and thiosulfate (an intermediate in the oxidation of H₂S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H₂S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.     

Sickle Cell Hemoglobin in the Ferryl State Promotes βCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10)

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H₂O₂) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H₂O₂ molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H₂O₂ at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized βCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.     

Microsomal Prostaglandin E Synthase Type 2 (mPGES2) Is a Glutathione-dependent Heme Protein,and Dithiothreitol Dissociates the Bound Heme to Produce Active Prostaglandin E2 Synthase in Vitro

An x-ray study indicated that microsomal prostaglandin E synthase type 2 (mPGES2) is a heme-bound protein and catalyzes prostaglandin (PG) H₂ degradation, but not PGE₂ formation (Yamada, T., and Takusagawa, F. (2007) Biochemistry 46, 8414–8424). In response to the x-ray study, Watanabe et al. claimed that mPGES2 is a heme-free protein and that both the heme-free and heme-bound proteins have PGE₂ synthesis activity in the presence of dithiothreitol (Watanabe, K., Ito, S., and Yamamoto, S. (2008) Biochem. Biophys. Res. Commun. 367, 782–786). To resolve the contradictory results, the heme-binding scheme of mPGES2 was further characterized in vivo and in vitro by absorption and fluorescence spectroscopies. A substantial amount of heme-bound mPGES2 was detected in cell extracts. The heme content in mPGES2 was increased along with an increase in Fe³⁺ in the culture medium. Heme-free mPGES2 was converted to the heme-bound form by mixing it with pig liver extract, indicating that mPGES2 is capable of forming a complex with heme in mammalian cells. Heme binds to mPGES2 only in the presence of glutathione. The newly determined heme dissociation constant (2.9 nm) supports strongly that mPGES2 is a heme-bound protein in vivo. The bound heme was not dissociated by oxidation by H₂O₂ or reduction by glutathione or 2-mercaptoethanol. However, reduction by dithiothreitol (an artificial reducing compound) induced the bound heme to dissociate from mPGES2 and released heme-free mPGES2, which exhibited PGE₂ synthesis activity in vitro. Imidazole bound to mPGES2 by stacking on the bound heme and inhibited heme oxidation by H₂O₂ and reduction by dithiothreitol.