scholarly journals NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

2015 ◽  
Vol 197 (13) ◽  
pp. 2239-2247 ◽  
Author(s):  
Chao Gao ◽  
Yujiao Wang ◽  
Yingxin Zhang ◽  
Min Lv ◽  
Peipei Dou ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Gene Cluster ◽  
Pseudomonas Stutzeri ◽  
Lactate Metabolism ◽  
Lactate Oxidation ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Lactate Utilization

ABSTRACTNAD-independentl-lactate dehydrogenases (l-iLDHs) play important roles inl-lactate utilization of different organisms. All of the previously reportedl-iLDHs were flavoproteins that catalyze the oxidation ofl-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA,lldB, andlldC) encoding a novel type ofl-iLDH was identified inPseudomonas stutzeriA1501. When the gene cluster was expressed inEscherichia coli, distinctivel-iLDH activity was detected. The expressedl-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis of the purifiedl-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded bylldA,lldB, andlldC, respectively). Purifiedl-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containingl-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for thel-lactate oxidation. LldABC has narrow substrate specificity, and onlyl-lactate anddl-2-hydrobutyrate were rapidly oxidized. Mg2+could activatel-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for thel-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for thel-lactate metabolism ofP. stutzeriA1501. LldABC is the first purified and characterizedl-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor.IMPORTANCEProviding new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independentl-lactate dehydrogenase (l-iLDH) encoded by the gene clusterlldABCis indispensable for thel-lactate metabolism inPseudomonas stutzeriA1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containingl-iLDH in other microbes, LldABC inP. stutzeriA1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

scholarly journals AVibrio choleraeBolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Aurore Fleurie ◽  
Abdelrahim Zoued ◽  
Laura Alvarez ◽  
Kelly M. Hines ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Morphology ◽  
Cell Shape ◽  
Cell Envelope ◽  
Intestinal Colonization ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Morphological Defects

ABSTRACTBolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins inVibrio cholerae, the cholera pathogen. LikeEscherichia coli,V. choleraeencodes two BolA proteins, BolA and IbaG. However, in marked contrast toE. coli, wherebolAis linked to cell shape andibaGis not, inV. cholerae,bolAmutants lack morphological defects, whereasibaGproved critical for the generation and/or maintenance of the pathogen’s morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. choleraecultures were not observed in stationary-phase cultures. TheV. choleraeΔibaGmutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. choleraehad reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant’s morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis ofibaG’s genetic interactions suggested thatibaGis involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest thatV. choleraeIbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCEBolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogenVibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of theV. choleraecell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governsV. choleraecell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

scholarly journals Zinc Toxicity and Iron-Sulfur Cluster Biogenesis inEscherichia coli

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Jianghui Li ◽  
Xiaojun Ren ◽  
Bingqian Fan ◽  
Zhaoyang Huang ◽  
Wu Wang ◽  
...  
Keyword(s):  
Binding Activity ◽  
Zinc Toxicity ◽  
Cluster Assembly ◽  
Intracellular Zinc ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Assembly Proteins

ABSTRACTWhile 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 inEscherichia coli. Here, we report that the intracellular zinc overload inE. colicells 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 clusteriscSUA-hscBA-fdx-iscXinE. colicells, 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 theE. colimutant 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 inE. colicells.IMPORTANCEZinc 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 inEscherichia colicells 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.

scholarly journals Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters

2014 ◽  
Vol 82 (4) ◽  
pp. 1390-1401 ◽  
Author(s):  
Jyoti Velayudhan ◽  
Joyce E. Karlinsey ◽  
Elaine R. Frawley ◽  
Lynne A. Becker ◽  
Margaret Nartea ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Salmonella Enterica ◽  
Active Sites ◽  
Nitrosative Stress ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Serovar Typhimurium

ABSTRACTLabile [4Fe-4S]2+clusters found at the active sites of many dehydratases are susceptible to damage by univalent oxidants that convert the clusters to an inactive [3Fe-4S]1+form. Bacteria repair damaged clusters in a process that does not requirede novoprotein synthesis or the Isc and Suf cluster assembly pathways. The current study investigates the participation of the bacterial frataxin ortholog CyaY and the YggX protein, which are proposed to play roles in iron trafficking and iron-sulfur cluster repair. Previous reports found that individual mutations incyaYoryggXwere not associated with phenotypic changes inEscherichia coliandSalmonella entericaserovar Typhimurium, suggesting that CyaY and YggX might have functionally redundant roles. However, we have found that individual mutations incyaYoryggXconfer enhanced susceptibility to hydrogen peroxide inSalmonella entericaserovar Typhimurium. In addition, inactivation of thestm3944open reading frame, which is located immediately upstream ofcyaYand which encodes a putative inner membrane protein, dramatically enhances the hydrogen peroxide sensitivity of acyaYmutant. Overexpression of STM3944 reduces the elevated intracellular free iron levels observed in anS. Typhimuriumfurmutant and also reduces the total cellular iron content under conditions of iron overload, suggesting that thestm3944-encoded protein may mediate iron efflux. Mutations incyaYandyggXhave different effects on the activities of the iron-sulfur cluster-containing aconitase, serine deaminase, and NADH dehydrogenase I enzymes ofS. Typhimurium under basal conditions or following recovery from oxidative stress. In addition,cyaYandyggXmutations have additive effects on 6-phosphogluconate dehydratase-dependent growth during nitrosative stress, and acyaYmutation reducesSalmonellavirulence in mice. Collectively, these results indicate that CyaY and YggX play distinct supporting roles in iron-sulfur cluster biosynthesis and the repair of labile clusters damaged by univalent oxidants.Salmonellaexperiences oxidative and nitrosative stress within host phagocytes, and CyaY-dependent maintenance of labile iron-sulfur clusters appears to be important forSalmonellavirulence.

scholarly journals Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Guoqiang Tan ◽  
Jing Yang ◽  
Tang Li ◽  
Jin Zhao ◽  
Shujuan Sun ◽  
...  
Keyword(s):  
Copper Toxicity ◽  
Anaerobic Conditions ◽  
Iron Sulfur Cluster ◽  
Aerobic Conditions ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins ◽  
Intracellular Copper

ABSTRACT 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.

scholarly journals Evolution of the Cytosolic Iron-Sulfur Cluster Assembly Machinery in Blastocystis Species and Other Microbial Eukaryotes

2013 ◽  
Vol 13 (1) ◽  
pp. 143-153 ◽  
Author(s):  
Anastasios D. Tsaousis ◽  
Eleni Gentekaki ◽  
Laura Eme ◽  
Daniel Gaston ◽  
Andrew J. Roger
Keyword(s):  
Subcellular Fractionation ◽  
Comparative Genomic ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Evolutionary Diversification ◽  
Iron Sulfur ◽  
History Of ◽  
Phylogenomic Analyses

ABSTRACT The cytosolic iron/sulfur cluster assembly (CIA) machinery is responsible for the assembly of cytosolic and nuclear iron/sulfur clusters, cofactors that are vital for all living cells. This machinery is uniquely found in eukaryotes and consists of at least eight proteins in opisthokont lineages, such as animals and fungi. We sought to identify and characterize homologues of the CIA system proteins in the anaerobic stramenopile parasite Blastocystis sp. strain NandII. We identified transcripts encoding six of the components—Cia1, Cia2, MMS19, Nbp35, Nar1, and a putative Tah18—and showed using immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation that the last three of them localized to the cytoplasm of the cell. We then used comparative genomic and phylogenetic approaches to investigate the evolutionary history of these proteins. While most Blastocystis homologues branch with their eukaryotic counterparts, the putative Blastocystis Tah18 seems to have a separate evolutionary origin and therefore possibly a different function. Furthermore, our phylogenomic analyses revealed that all eight CIA components described in opisthokonts originated before the diversification of extant eukaryotic lineages and were likely already present in the last eukaryotic common ancestor (LECA). The Nbp35, Nar1 Cia1, and Cia2 proteins have been conserved during the subsequent evolutionary diversification of eukaryotes and are present in virtually all extant lineages, whereas the other CIA proteins have patchy phylogenetic distributions. Cia2 appears to be homologous to SufT, a component of the prokaryotic sulfur utilization factors (SUF) system, making this the first reported evolutionary link between the CIA and any other Fe/S biogenesis pathway. All of our results suggest that the CIA machinery is an ubiquitous biosynthetic pathway in eukaryotes, but its apparent plasticity in composition raises questions regarding how it functions in nonmodel organisms and how it interfaces with various iron/sulfur cluster systems (i.e., the iron/sulfur cluster, nitrogen fixation, and/or SUF system) found in eukaryotic cells.

scholarly journals Key Enzymes for Anaerobic Lactate Metabolism in Geobacter sulfurreducens

2020 ◽  
Vol 87 (2) ◽  
Author(s):  
Toshiyuki Ueki
Keyword(s):  
Lactate Dehydrogenase ◽  
Environmental Biotechnology ◽  
Anaerobic Metabolism ◽  
Geobacter Sulfurreducens ◽  
Full Potential ◽  
Lactate Metabolism ◽  
Content Type ◽  
Key Enzymes ◽  
Lactate Utilization ◽  
Succinyl Coa Synthetase

ABSTRACT Growth of Geobacter sulfurreducens PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the sucCD genes, encoding a succinyl-coenzyme A (CoA) synthetase. To further investigate the function of the succinyl-CoA synthetase, the sucCD genes were deleted from G. sulfurreducens. The mutant showed defective growth on lactate but not on acetate. Introduction of the sucCD genes into the mutant restored the full potential to grow on lactate. These results verify the importance of the succinyl-CoA synthetase in growth on lactate. Genome analysis of Geobacter species identified candidate genes, GSU1623, GSU1624, and GSU1620, for lactate dehydrogenase. Deletion mutants of the identified genes for d-lactate dehydrogenase (ΔGSU1623 ΔGSU1624 mutant) or l-lactate dehydrogenase (ΔGSU1620 mutant) could not grow on d-lactate or l-lactate but could grow on acetate and l- or d-lactate, respectively. Introduction of the respective genes into the mutants allowed growth on the corresponding lactate stereoisomer. These results suggest that the identified genes were essential for d- or l-lactate utilization. The lacZ reporter assay demonstrated that the putative promoter regions were more active during growth on lactate than during growth on acetate, indicating that the genes for the lactate dehydrogenases were expressed more during growth on lactate than during growth on acetate. The gene deletion phenotypes and the expression profiles indicate that there are metabolic switches between lactate and acetate. This study advances the understanding of anaerobic lactate utilization in G. sulfurreducens. IMPORTANCE Lactate is a microbial fermentation product as well as a source of carbon and electrons for microorganisms in the environment. Furthermore, lactate is a common amendment for stimulation of microbial growth in environmental biotechnology applications. However, anaerobic metabolism of lactate has been poorly studied for environmentally relevant microorganisms. Geobacter species are found in various environments and environmental biotechnology applications. By employing genomic and genetic approaches, succinyl-CoA synthetase and lactate dehydrogenase were identified as key enzymes in anaerobic metabolism of lactate in Geobacter sulfurreducens, a representative Geobacter species. Differential gene expression during growth on lactate and acetate was observed, demonstrating that G. sulfurreducens could metabolically switch to adapt to available substrates in the environment. The findings provide new insights into basic physiology in lactate metabolism as well as cellular responses to growth conditions in the environment and can be informative for the application of lactate in environmental biotechnology.

scholarly journals Cysteine Desulfurase IscS2 Plays a Role in Oxygen Resistance in Clostridium difficile

2018 ◽  
Vol 86 (8) ◽  
Author(s):  
Nicole Giordano ◽  
Jessica L. Hastie ◽  
Ashley D. Smith ◽  
Elissa D. Foss ◽  
Daniela F. Gutierrez-Munoz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Clostridium Difficile ◽  
The United States ◽  
Growth Defect ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Low Levels

ABSTRACT Clostridium difficile is an anaerobic, spore-forming bacterium capable of colonizing the gastrointestinal tract of humans following disruption of the normal microbiota, typically from antibiotic therapy for an unrelated infection. With approximately 500,000 confirmed infections leading to 29,000 deaths per year in the United States, C. difficile infection (CDI) is an urgent public health threat. We previously determined that C. difficile survives in up to 3% oxygen. Low levels of oxygen are present in the intestinal tract, with the higher concentrations being associated with the epithelial cell surface. Additionally, antibiotic treatment, the greatest risk factor for CDI, increases the intestinal oxygen concentration. Therefore, we hypothesized that the C. difficile genome encodes mechanisms for survival during oxidative stress. Previous data have shown that cysteine desulfurases involved in iron-sulfur cluster assembly are involved in protecting bacteria from oxidative stress. In this study, deletion of a putative cysteine desulfurase (Cd630_12790/IscS2) involved in the iron-sulfur cluster (Isc) system caused a severe growth defect in the presence of 2% oxygen. Additionally, this mutant delayed colonization in a conventional mouse model of CDI and failed to colonize in a germfree model, which has higher intestinal oxygen levels. These data imply an undefined role for this cysteine desulfurase in protecting C. difficile from low levels of oxygen in the gut.

Disruption of the glycolate dehydrogenase gene in the high-CO2-requiring mutant HCR89 of Chlamydomonas reinhardtii

2005 ◽  
Vol 83 (7) ◽  
pp. 820-833 ◽  
Author(s):  
Yoshiko Nakamura ◽  
Saradadevi Kanakagiri ◽  
Kyujung Van ◽  
Wei He ◽  
Martin H Spalding
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Dehydrogenase Activity ◽  
Insertional Mutagenesis ◽  
Higher Plants ◽  
Glycolate Oxidase ◽  
Lactate Oxidation ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Glycolate Dehydrogenase

One of the most notable contrasts between the photorespiratory pathway of higher plants and that of many of the green algae including Chlamydomonas reinhardtii lies in the enzymes that serve for oxidation of glycolate to glyoxylate. The gene disrupted by insertional mutagenesis in a high-CO2-requiring mutant, HCR89, of C. reinhardtii was determined to encode glycolate dehydrogenase (EC 1.1.99.14), which serves as the counterpart of glycolate oxidase (EC 1.1.3.15) in classical higher plant photorespiration. Neither glycolate nor D-lactate oxidation from the membrane fraction of HCR89 was detected. Excretion of over-accumulated glycolate into media due to the absence of glycolate dehydrogenase activity was observed for HCR89 under both high- and low-CO2 conditions. Chlamydomonas glycolate dehydrogenase, CrGDH, with a molecular mass of 118 851 Da, comprises a relatively hydrophobic N-terminal region, a FAD-containing domain homologous to the D subunit of the glycolate oxidase complex from Escherischia coli, and an iron–sulfur cluster containing domain homologous to the C subunit of anaerobic glycerol-3-phosphate dehydrogenase complex from Escherichia coli. The second Cys residue in the second iron–sulfur cluster motif of CrGDH is replaced by Asp, as CxxDxxCxxxCP, indicating the second iron–sulfur cluster coordinates most likely 3Fe–4S instead of 4Fe–4S. The membrane association of the glycolate dehydrogenase activity agrees with three predicted transmembrane regions on the iron–sulfur domain.Key words: algae, Chlamydomonas, CO2, glycolate, lactate, mitochondria, photorespiration, photosynthesis.

scholarly journals Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Xiaojun Ren ◽  
Feng Liang ◽  
Zhengfen He ◽  
Bingqian Fan ◽  
Zhirong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cold Stress ◽  
Stress Conditions ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

ABSTRACT Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli. Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system. IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

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