RESUMEN
The dual roles of H2S as an endogenously synthesized respiratory substrate and as a toxin raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in the mitochondrial H2S oxidation pathway, using CoQ as an electron acceptor, and connects to the electron transport chain at the level of complex III. We have discovered that at high H2S concentrations, which are known to inhibit complex IV, a new redox cycle is established between SQOR and complex II, operating in reverse. Under these conditions, the purine nucleotide cycle and the malate aspartate shuttle furnish fumarate, which supports complex II reversal and leads to succinate accumulation. Complex II knockdown in colonocytes decreases the efficiency of H2S clearance while targeted knockout of complex II in intestinal epithelial cells significantly decreases the levels of thiosulfate, a biomarker of H2S oxidation, to approximately one-third of the values seen in serum and urine samples from control mice. These data establish the physiological relevance of this newly discovered redox circuitry between SQOR and complex II for prioritizing H2S oxidation and reveal the quantitatively significant contribution of intestinal epithelial cells to systemic H2S metabolism.
Asunto(s)
Sulfuro de Hidrógeno , Quinona Reductasas , Animales , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Sulfuro de Hidrógeno/metabolismo , Ratones , Oxidación-Reducción , Quinona Reductasas/genética , Quinona Reductasas/metabolismoRESUMEN
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.
Asunto(s)
Glucólisis , Sulfuro de Hidrógeno/metabolismo , Mitocondrias/metabolismo , NAD/metabolismo , Transducción de Señal , Células HCT116 , Células HT29 , HumanosRESUMEN
Hydrogen sulfide (H2 S) is an environmental toxin and a heritage of ancient microbial metabolism that has stimulated new interest following its discovery as a neuromodulator. While many physiological responses have been attributed to low H2 S levels, higher levels inhibit complex IV in the electron transport chain. To prevent respiratory poisoning, a dedicated set of enzymes that make up the mitochondrial sulfide oxidation pathway exists to clear H2 S. The committed step in this pathway is catalyzed by sulfide quinone oxidoreductase (SQOR), which couples sulfide oxidation to coenzyme Q10 reduction in the electron transport chain. The SQOR reaction prevents H2 S accumulation and generates highly reactive persulfide species as products; these can be further oxidized or can modify cysteine residues in proteins by persulfidation. Here, we review the kinetic and structural characteristics of human SQOR, and how its unconventional redox cofactor configuration and substrate promiscuity lead to sulfide clearance and potentially expand the signaling potential of H2 S. This dual role of SQOR makes it a promising target for H2 S-based therapeutics.
Asunto(s)
Sulfuro de Hidrógeno/metabolismo , Quinona Reductasas/metabolismo , Dominio Catalítico , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Sulfuro de Hidrógeno/química , Mitocondrias/metabolismo , Oxidación-Reducción , Fosforilación Oxidativa , Quinona Reductasas/química , Quinona Reductasas/clasificación , Especificidad por Sustrato , Ubiquinona/análogos & derivados , Ubiquinona/químicaRESUMEN
Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in sulfide clearance, coupling H2S oxidation to coenzyme Q reduction. Recent structures of human SQOR revealed a sulfur atom bridging the SQOR active site cysteines in a trisulfide configuration. Here, we assessed the importance of this cofactor using kinetic, crystallographic, and computational modeling approaches. Cyanolysis of SQOR proceeds via formation of an intense charge transfer complex that subsequently decays to eliminate thiocyanate. We captured a disulfanyl-methanimido thioate intermediate in the SQOR crystal structure, revealing how cyanolysis leads to reversible loss of SQOR activity that is restored in the presence of sulfide. Computational modeling and MD simulations revealed an â¼105-fold rate enhancement for nucleophilic addition of sulfide into the trisulfide versus a disulfide cofactor. The cysteine trisulfide in SQOR is thus critical for activity and provides a significant catalytic advantage over a cysteine disulfide.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Sulfuros/metabolismo , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Sulfuros/químicaRESUMEN
Hydrogen sulfide, a signaling molecule formed mainly from cysteine, is catabolized by sulfide:quinone oxidoreductase (gene SQOR). Toxic hydrogen sulfide exposure inhibits complex IV. We describe children of two families with pathogenic variants in SQOR. Exome sequencing identified variants; SQOR enzyme activity was measured spectrophotometrically, protein levels evaluated by western blotting, and mitochondrial function was assayed. In family A, following a brief illness, a 4-year-old girl presented comatose with lactic acidosis and multiorgan failure. After stabilization, she remained comatose, hypotonic, had neurostorming episodes, elevated lactate, and Leigh-like lesions on brain imaging. She died shortly after. Her 8-year-old sister presented with a rapidly fatal episode of coma with lactic acidosis, and lesions in the basal ganglia and left cortex. Muscle and liver tissue had isolated decreased complex IV activity, but normal complex IV protein levels and complex formation. Both patients were homozygous for c.637G > A, which we identified as a founder mutation in the Lehrerleut Hutterite with a carrier frequency of 1 in 13. The resulting p.Glu213Lys change disrupts hydrogen bonding with neighboring residues, resulting in severely reduced SQOR protein and enzyme activity, whereas sulfide generating enzyme levels were unchanged. In family B, a boy had episodes of encephalopathy and basal ganglia lesions. He was homozygous for c.446delT and had severely reduced fibroblast SQOR enzyme activity and protein levels. SQOR dysfunction can result in hydrogen sulfide accumulation, which, consistent with its known toxicity, inhibits complex IV resulting in energy failure. In conclusion, SQOR deficiency represents a new, potentially treatable, cause of Leigh disease.
Asunto(s)
Sulfuro de Hidrógeno/metabolismo , Enfermedad de Leigh/enzimología , Mitocondrias/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Quinona Reductasas/fisiología , Acidosis Láctica/patología , Encefalopatías/patología , Preescolar , Complejo IV de Transporte de Electrones/metabolismo , Familia , Femenino , Homocigoto , Humanos , Sulfuro de Hidrógeno/química , Cinética , Enfermedad de Leigh/metabolismo , Imagen por Resonancia Magnética , Masculino , Oxidación-Reducción , Quinona Reductasas/químicaRESUMEN
Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal α helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH2) and that FMNH2 can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH2 using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH2 The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET.
Asunto(s)
Mononucleótido de Flavina/metabolismo , Hidroquinonas/metabolismo , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/metabolismo , Ubiquinona/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , FMN Reductasa/genética , FMN Reductasa/metabolismo , Humanos , Hipoglucemiantes/farmacología , Cinética , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pioglitazona , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiazoles/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Óxido Nítrico/metabolismo , Humanos , Unión Proteica , Transducción de SeñalRESUMEN
Buildup of hydrogen sulfide (H2S), which functions as a signaling molecule but is toxic at high concentrations, is averted by its efficient oxidation by the mitochondrial sulfide oxidation pathway. The first step in this pathway is catalyzed by a flavoprotein, sulfide quinone oxidoreductase (SQR), which converts H2S to a persulfide and transfers electrons to coenzyme Q via a flavin cofactor. All previous studies on human SQR have used detergent-solubilized protein. Here, we embedded human SQR in nanodiscs (ndSQR) and studied highly homogenous preparations by steady-state and rapid-kinetics techniques. ndSQR exhibited higher catalytic rates in its membranous environment than in its solubilized state. Stopped-flow spectroscopic data revealed that transfer of the sulfane sulfur from an SQR-bound cysteine persulfide intermediate to a small-molecule acceptor is the rate-limiting step. The physiological acceptor of sulfane sulfur from SQR has been the subject of controversy; we report that the kinetic analysis of ndSQR is consistent with glutathione rather than sulfite being the predominant acceptor at physiologically relevant concentrations of the respective metabolites. The identity of the acceptor has an important bearing on how the sulfide oxidation pathway is organized. Our data are more consistent with the reaction sequence for sulfide oxidation being: H2S â glutathione persulfide â sulfite â sulfate, than with a more convoluted route that would result if sulfite were the primary acceptor of sulfane sulfur. In summary, nanodisc-incorporated human SQR exhibits enhanced catalytic performance, and pre-steady-state kinetics characterization of the complete SQR catalytic cycle indicates that GSH serves as the physiologically relevant sulfur acceptor.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Sulfuro de Hidrógeno/metabolismo , Modelos Moleculares , Nanopartículas/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Biocatálisis , Cisteína/química , Transporte de Electrón , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/genética , Glutatión/metabolismo , Humanos , Cinética , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatidilcolinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Ubiquinona/metabolismoRESUMEN
The human mitochondrial outer membrane protein mitoNEET is a novel target of the type II diabetes drug pioglitazone. The C-terminal cytosolic domain of mitoNEET hosts a redox-active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine residues and one histidine residue. Here we report that human mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells. In vitro studies show that purified mitoNEET [2Fe-2S] clusters can be partially reduced by monothiols such as reduced glutathione, L-cysteine or N-acetyl-L-cysteine and fully reduced by dithiothreitol or the E. coli thioredoxin/thioredoxin reductase system under anaerobic conditions. Importantly, thiol-reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by hydrogen peroxide without disruption of the clusters in vitro and in E. coli cells, indicating that mitoNEET may act as a sensor of oxidative signals to regulate mitochondrial functions via its [2Fe-2S] clusters. Furthermore, the binding of the type II diabetes drug pioglitazone in mitoNEET effectively inhibits the thiol-mediated reduction of [2Fe-2S] clusters, suggesting that pioglitazone may modulate the function of mitoNEET by blocking the thiol-mediated reduction of [2Fe-2S] clusters in the protein.
Asunto(s)
Peróxido de Hidrógeno/química , Hipoglucemiantes/química , Proteínas Hierro-Azufre/química , Proteínas Mitocondriales/química , Oxidantes/química , Tiazolidinedionas/química , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Hipoglucemiantes/farmacología , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Pioglitazona , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazolidinedionas/farmacologíaRESUMEN
Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Proteínas Portadoras/genética , Análisis Mutacional de ADN , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas Hierro-Azufre/genética , Mutagénesis Sitio-Dirigida , Unión ProteicaRESUMEN
YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/enzimología , Hierro/metabolismo , Zinc/metabolismo , ADN-Topoisomerasas de Tipo I/química , Escherichia coli/metabolismo , Hierro/química , Zinc/químicaRESUMEN
Human mitochondrial protein mitoNEET is a novel target of type II diabetes drug pioglitazone, and contains a redox active [2Fe-2S] cluster that is hosted by a unique ligand arrangement of three cysteine and one histidine residues. Here we report that zinc ion can compete for the [2Fe-2S] cluster binding site in human mitoNEET and potentially modulate the physiological function of mitoNEET. When recombinant mitoNEET is expressed in Escherichia coli cells grown in M9 minimal media, purified mitoNEET contains very little or no iron-sulfur clusters. Addition of exogenous iron or zinc ion in the media produces mitoNEET bound with a [2Fe-2S] cluster or zinc, respectively. Mutations of the amino acid residues that hosting the [2Fe-2S] cluster in mitoNEET diminish the zinc binding activity, indicating that zinc ion and the [2Fe-2S] cluster may share the same binding site in mitoNEET. Finally, excess zinc ion effectively inhibits the [2Fe-2S] cluster assembly in mitoNEET in E. coli cells, suggesting that zinc ion may impede the function of mitoNEET by blocking the [2Fe-2S] cluster assembly in the protein.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/metabolismo , Zinc/metabolismo , Unión Competitiva , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Iones/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/química , Terapia Molecular Dirigida , Pioglitazona , Tiazolidinedionas/farmacologíaRESUMEN
Escherichia coli topoisomerase I (TopA) cleaves and rejoins one strand of double-stranded DNA to relax the negatively supercoiled DNA. Structurally, TopA contains an N-terminal catalytic fragment and a C-terminal zinc-binding region that is required for relaxation of the negatively supercoiled DNA. Here we report that E. coli TopA is an iron and zinc binding protein. The UV-Vis absorption measurements and metal content analyses reveal that TopA purified from E. coli cells grown in the rich LB medium contains both iron and zinc. However, TopA purified from E. coli cells grown in the M9 minimal medium has negligible amounts of zinc or iron and no topoisomerase activity. Nevertheless, supplement of exogenous zinc or iron in E. coli cells grown in the M9 minimal medium produces the zinc- or iron-bound TopA, respectively. Whereas the zinc-bound TopA is fully active to relax the negatively supercoiled DNA, the iron-bound TopA has little or no enzyme activity. Furthermore, excess iron in the M9 minimal medium is able to compete with the zinc binding in TopA in E. coli cells and attenuate the topoisomerase activity, suggesting that E. coli TopA may be modulated by iron and zinc binding in vivo.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/enzimología , Hierro/metabolismo , Zinc/metabolismo , Sitios de Unión , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , Escherichia coli/citología , Espectrofotometría UltravioletaRESUMEN
IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 µM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Aconitato Hidratasa/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Unión a Hierro/genética , Oxígeno , EspectrofotometríaRESUMEN
The enzymes involved in H2S homeostasis regulate its production from sulfur-containing amino acids and its oxidation to thiosulfate and sulfate. Two gatekeepers in this homeostatic circuit are cystathionine beta-synthase, which commits homocysteine to cysteine, and sulfide quinone oxidoreductase, which commits H2S to oxidation via a mitochondrial pathway. Inborn errors at either locus affect sulfur metabolism, increasing homocysteine-derived H2S synthesis in the case of CBS deficiency and reducing complex IV activity in the case of SQOR deficiency. In this review, we focus on structural perspectives on the reaction mechanisms and regulation of these two enzymes, which are key to understanding H2S homeostasis in health and its dysregulation and potential targeting in disease.
Asunto(s)
Sulfuro de Hidrógeno , Cistationina betasintasa/metabolismo , Cisteína , Homeostasis , Oxidación-ReducciónRESUMEN
MitoNEET is a mitochondrial outer membrane protein that hosts a redox active [2Fe-2S] cluster in the C-terminal cytosolic domain. Increasing evidence has shown that mitoNEET has an essential role in regulating energy metabolism in human cells. Previously, we reported that the [2Fe-2S] clusters in mitoNEET can be reduced by the reduced flavin mononucleotide (FMNH2) and oxidized by oxygen or ubiquinone-2, suggesting that mitoNEET may act as a novel redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone. Here, we explore the FMN binding site in mitoNEET by using FMN analogs and find that lumiflavin, like FMN, at nanomolar concentrations can mediate the redox transition of the mitoNEET [2Fe-2S] clusters in the presence of flavin reductase and NADH (100 µM) under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that both FMN and lumiflavin can dramatically change the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters and form a covalently bound complex with mitoNEET under blue light exposure, suggesting that FMN/lumiflavin has specific interactions with the [2Fe-2S] clusters in mitoNEET. In contrast, lumichrome, another FMN analog, fails to mediate the redox transition of the mitoNEET [2Fe-2S] clusters and has no effect on the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters under blue light exposure. Instead, lumichrome can effectively inhibit the FMNH2-mediated reduction of the mitoNEET [2Fe-2S] clusters, indicating that lumichrome may act as a potential inhibitor to block the electron transfer activity of mitoNEET.
Asunto(s)
Mononucleótido de Flavina , Proteínas Hierro-Azufre , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Mononucleótido de Flavina/metabolismo , Humanos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-ReducciónRESUMEN
Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.
Asunto(s)
Butiratos/química , Coenzima A/metabolismo , Quinona Reductasas/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Disulfuros/química , Glutatión/análogos & derivados , Glutatión/química , Humanos , Sulfuro de Hidrógeno/química , Cinética , Mitocondrias/enzimología , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de Proteína , Quinona Reductasas/química , Especificidad por Sustrato , Sulfuros/química , Sulfuros/metabolismoRESUMEN
The mitochondrial sulfide oxidation pathway prevents the toxic accumulation of hydrogen sulfide (H2S), a signaling molecule that is maintained at low steady-state concentrations. Sulfide quinone oxidoreductase (SQR), an inner mitochondrial membrane-anchored protein, catalyzes the first and committing step in this pathway, oxidizing H2S to persulfide. The catalytic cycle comprises sulfide addition to the active site cysteine disulfide in SQR followed by sulfur transfer to a small molecule acceptor, while a pair of electrons moves from sulfide, to FAD, to coenzyme Q. While its ability to oxidize H2S is well characterized, SQR exhibits a remarkable degree of substrate promiscuity in vitro that could undermine its canonical enzyme activity. To assess how its promiscuity might be contained in vivo, we have used spectroscopic and kinetic analyses to characterize the reactivity of alternate substrates with SQR embedded in nanodiscs ( ndSQR) versus detergent-solubilized enzyme ( sSQR). We find that the membrane environment of ndSQR suppresses the unwanted addition of GSH but enhances sulfite addition, which might become significant under pathological conditions characterized by elevated sulfite levels. We demonstrate that methanethiol, a toxic sulfur compound produced in significant quantities by colonic and oral microbiota, can add to the SQR cysteine disulfide and also serve as a sulfur acceptor, potentially interfering with sulfide oxidation when its concentrations are elevated. These studies demonstrate that the membrane environment and substrate availability combine to minimize promiscuous reactions that would otherwise disrupt sulfide homeostasis.
Asunto(s)
Sulfuro de Hidrógeno/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Biocatálisis , Glutatión/química , Humanos , Cinética , Nanoestructuras/química , Oxidación-Reducción , Fosfatidilcolinas/química , Especificidad por Sustrato , Compuestos de Sulfhidrilo/química , Sulfitos/químicaRESUMEN
MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, 1 molecule of flavin nucleotide is sufficient to reduce about 100 molecules of the mitoNEET [2Fe-2S] clusters in 4min under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane α-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10Å, which could facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético , Proteínas Hierro-Azufre/metabolismo , Proteínas Mitocondriales/metabolismo , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/patología , Dinitrocresoles/química , Dinitrocresoles/metabolismo , Transporte de Electrón , Humanos , Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Mitocondriales/química , Simulación del Acoplamiento Molecular , NAD/metabolismo , Nucleótidos/química , Nucleótidos/metabolismo , Oxidorreductasas/metabolismo , Pioglitazona , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Tiazolidinedionas/uso terapéuticoRESUMEN
The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe-2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe-2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe-2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe-2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe-2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe-2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.