RESUMEN
It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.
Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Proteínas Hierro-Azufre , Hierro , Proteínas Represoras , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Hierro/metabolismoRESUMEN
Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E. coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E. coli Fur also binds a [2Fe-2S] cluster in wildtype E. coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box in vitro, and are inactive to complement the function of Fur in vivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E. coli cells.
Asunto(s)
Escherichia coli , Proteínas Hierro-Azufre , Hierro , Escherichia coli/genética , Escherichia coli/metabolismo , Homeostasis , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , MutaciónRESUMEN
Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.
Asunto(s)
Escherichia coli , Proteínas Hierro-Azufre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismoRESUMEN
Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). Since its discovery, Fur has been assumed to bind ferrous iron and regulate expression of target genes. However, the iron-bound Fur has never been isolated from any bacteria. In previous studies, we have shown that Escherichia coli Fur and Haemophilus influenzae Fur bind a [2Fe-2S] cluster via the conserved Cys-93 and Cys-96 when expressed in the E. coli mutant cells in which intracellular free iron content is elevated. Here we report that Fur homologs from Vibrio cholerae and Helicobacter pylori which contain Cys-93 and Cys-96 can also bind a [2Fe-2S] cluster. On the other hand, Fur homolog from Magnetospirillum gryphiswaldense MSR-1 which has no cysteine residues fails to bind any [2Fe-2S] clusters. Interestingly, different Fur proteins with the conserved Cys-93 and Cys-96 have distinct binding activities for the [2Fe-2S] cluster, with H. influenzae Fur having the highest, followed by E. coli Fur, V. cholera Fur, and H. pylori Fur. Binding of the [2Fe-2S] cluster in the Fur proteins is significantly decreased when expressed in wild-type E. coli cells, indicating that binding of the [2Fe-2S] clusters in Fur proteins is regulated by the levels of intracellular free iron content. Finally, unlike the [2Fe-2S] clusters in E. coli ferredoxin, the [2Fe-2S] clusters in the Fur proteins are not stable and quickly release ferrous iron when the clusters are reduced, suggesting that Fur may undergo reversible binding of the [2Fe-2S] cluster in response to intracellular free iron content in bacteria.
Asunto(s)
Helicobacter pylori , Proteínas Hierro-Azufre , Vibrio cholerae , Cisteína/química , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Vibrio cholerae/metabolismoRESUMEN
The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular "free" iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the "iron-bound" Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron-sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is â¼31% in the E. coli iscA/sufA mutant cells and is decreased to â¼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Hierro/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Cisteína/química , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Haemophilus influenzae/metabolismo , Quelantes del Hierro/química , Ligandos , Mutagénesis Sitio-Dirigida , Unión Proteica , Alineación de Secuencia , Espectroscopía de MossbauerRESUMEN
While zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases in Escherichia coli Here, we report that the intracellular zinc overload in E. coli cells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene cluster iscSUA-hscBA-fdx-iscX in E. coli cells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of the E. coli mutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin in E. coli cells.IMPORTANCE Zinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload in Escherichia coli cells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Proteínas Hierro-Azufre/genética , Zinc/toxicidad , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Proteínas Hierro-Azufre/metabolismoRESUMEN
Human mitochondrial matrix protein Miner2 hosts two [2Fe-2S] clusters via two CDGSH (Cys-Asp-Gly-Ser-His) motifs. Unlike other iron-sulfur clusters in proteins, the reduced CDGSH-type [2Fe-2S] clusters in Miner2 are able to bind nitric oxide (NO) and form stable NO-bound [2Fe-2S] clusters without disruption of the clusters. Here we report that the NO-bound Miner2 [2Fe-2S] clusters can quickly release NO upon the visible light excitation. The UV-visible and Electron Paramagnetic Resonance (EPR) measurements show that the NO-bound Miner2 [2Fe-2S] clusters are converted to the reduced Miner2 [2Fe-2S] clusters upon the light excitation under anaerobic conditions, suggesting that NO binding in the reduced Miner2 [2Fe-2S] clusters is reversible. Additional studies reveal that binding of NO effectively inhibits the redox transition of the Miner2 [2Fe-2S] clusters, indicating that NO may modulate the physiological activity of Miner2 in mitochondria by directly binding to the CDGSH-type [2Fe-2S] clusters in the protein.
Asunto(s)
Proteínas Hierro-Azufre/efectos de la radiación , Hierro/química , Proteínas Mitocondriales/efectos de la radiación , Óxido Nítrico/metabolismo , Azufre/química , Escherichia coli/genética , Humanos , Proteínas Hierro-Azufre/química , Luz , Proteínas Mitocondriales/química , Oxidación-ReducciónRESUMEN
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
While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells.IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they are under aerobic conditions. Under anaerobic conditions, E. coli cells accumulate excess intracellular copper, which specifically targets iron-sulfur proteins by blocking iron-sulfur cluster biogenesis. Since iron-sulfur proteins are involved in diverse and vital physiological processes, inhibition of iron-sulfur cluster biogenesis by copper disrupts multiple cellular functions and ultimately inhibits cell growth. The results from this study illustrate a new interplay between intracellular copper toxicity and iron-sulfur cluster biogenesis in bacterial cells under anaerobic conditions.
Asunto(s)
Cobre/metabolismo , Escherichia coli/metabolismo , Hierro/metabolismo , Azufre/metabolismo , Anaerobiosis , Cobre/toxicidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Oxígeno/metabolismoRESUMEN
In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from L-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2'-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5'-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess L-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.
Asunto(s)
Liasas de Carbono-Azufre/genética , Proteínas Portadoras/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Alanina/química , Liasas de Carbono-Azufre/metabolismo , Proteínas Portadoras/metabolismo , Cisteína/química , Proteínas de Escherichia coli/metabolismo , Hierro/química , Proteínas Hierro-Azufre/metabolismo , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Fosfato de Piridoxal/química , Proteínas Recombinantes/metabolismo , Sulfuros/químicaRESUMEN
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
MitoNEET is the first iron-sulfur protein found in mitochondrial outer membrane. Abnormal expression of mitoNEET in cells has been linked to several types of cancer, type II diabetes, and neurodegenerative diseases. Structurally, mitoNEET is anchored to mitochondrial outer membrane via its N-terminal single transmembrane alpha helix. The C-terminal cytosolic domain of mitoNEET binds a [2Fe-2S] cluster via three cysteine and one histidine residues. It has been shown that mitoNEET has a crucial role in energy metabolism, iron homeostasis, and free radical production in cells. However, the exact function of mitoNEET remains elusive. Previously, we reported that the C-terminal soluble domain of mitoNEET has a specific binding site for flavin mononucleotide (FMN) and can transfer electrons from FMNH2 to oxygen or ubiquinone-2 via its [2Fe-2S] cluster. Here we have constructed a hybrid protein using the N-terminal transmembrane domain of Escherichia coli YneM and the C-terminal soluble domain of human mitoNEET and assembled the hybrid protein YneM-mitoNEET into phospholipid nanodiscs. The results show that the [2Fe-S] clusters in the nanodisc-bound YneM-mitoNEET can be rapidly reduced by FMNH2 which is reduced by flavin reductase using NADH as the electron donor. Addition of lumichrome, a FMN analog, effectively inhibits the FMNH2-mediated reduction of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET. The reduced [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET are quickly oxidized by oxygen under aerobic conditions or by ubiquinone-10 in the nanodiscs under anaerobic conditions. Because NADH oxidation is required for cellular glycolytic activity, we propose that the mitochondrial outer membrane protein mitoNEET may promote glycolysis by transferring electrons from FMNH2 to oxygen or ubiquinone-10 in mitochondria.
Asunto(s)
Diabetes Mellitus Tipo 2 , Proteínas Hierro-Azufre , Diabetes Mellitus Tipo 2/metabolismo , Electrones , Escherichia coli/genética , Escherichia coli/metabolismo , Mononucleótido de Flavina/metabolismo , Humanos , Proteínas Hierro-Azufre/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , NAD/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Ubiquinona/metabolismoRESUMEN
MitoNEET is a mitochondrial outer membrane protein that regulates energy metabolism, iron homeostasis, and production of reactive oxygen species in cells. Aberrant expression of mitoNEET in tissues has been linked to type II diabetes, neurodegenerative diseases, and several types of cancer. Structurally, the N-terminal domain of mitoNEET has a single transmembrane alpha helix that anchors the protein to mitochondrial outer membrane. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine and one histidine residues. Here we report that the reduced [2Fe-2S] cluster in the C-terminal cytosolic domain of mitoNEET (mitoNEET45-108) is able to bind nitric oxide (NO) without disruption of the cluster. Importantly, binding of NO at the reduced [2Fe-2S] cluster effectively inhibits the redox transition of the cluster in mitoNEET45-108. While the NO-bound [2Fe-2S] cluster in mitoNEET45-108 is stable, light excitation releases NO from the NO-bound [2Fe-2S] cluster and restores the redox transition activity of the cluster in mitoNEET45-108. The results suggest that NO may regulate the electron transfer activity of mitoNEET in mitochondrial outer membrane via reversible binding to its reduced [2Fe-2S] cluster.
RESUMEN
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
A human homologue of the iron-sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV-visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron centre and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2x1019 M-1, and that the iron-bound hIscA1 is able to provide the iron for the iron-sulfur cluster assembly in a proposed scaffold protein, IscU of E. coli, in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring the cell growth of E. coli in the M9 minimal medium under aerobic conditions. The results suggest that hIscA1, like E. coli IscA, is an iron-binding protein that may act as an iron chaperone for biogenesis of iron-sulfur clusters.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Hierro/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Humanos , Proteínas Hierro-Azufre/química , CinéticaRESUMEN
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.