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.

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 In vivo evidence for the iron-binding activity of an iron–sulfur cluster assembly protein IscA in Escherichia coli

2010 ◽  
Vol 432 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Wu Wang ◽  
Hao Huang ◽  
Guoqiang Tan ◽  
Fan Si ◽  
Min Liu ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Binding Activity ◽  
Anaerobic Growth ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

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.

Complementary roles of SufA and IscA in the biogenesis of iron–sulfur clusters in Escherichia coli

2007 ◽  
Vol 409 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Jianxin Lu ◽  
Juanjuan Yang ◽  
Guoqiang Tan ◽  
Huangen Ding
Keyword(s):  
Cell Growth ◽  
Double Mutant ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Iron Binding Protein

Biogenesis of iron–sulfur clusters requires a concerted delivery of iron and sulfur to target proteins. It is now clear that sulfur in iron–sulfur clusters is derived from L-cysteine via cysteine desulfurases. However, the specific iron donor for the iron–sulfur cluster assembly still remains elusive. Previous studies showed that IscA, a member of the iron–sulfur cluster assembly machinery in Escherichia coli, is a novel iron-binding protein, and that the iron-bound IscA can provide iron for the iron–sulfur cluster assembly in a proposed scaffold IscU in vitro. However, genetic studies have indicated that IscA is not essential for the cell growth of E. coli. In the present paper, we report that SufA, an IscA paralogue in E. coli, may represent the redundant activity of IscA. Although deletion of IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA in E. coli results in a severe growth phenotype in minimal medium under aerobic growth conditions. Cell growth is restored when either IscA or SufA is re-introduced into the iscA−/sufA− double mutant, demonstrating further that either IscA or SufA is sufficient for their functions in vivo. Purified SufA, like IscA, is an iron-binding protein that can provide iron for the iron–sulfur cluster assembly in IscU in the presence of a thioredoxin reductase system which emulates the intracellular redox potential. Site-directed mutagenesis studies show that the SufA/IscA variants that lose the specific iron-binding activity fail to restore the cell growth of the iscA−/sufA− double mutant. The results suggest that SufA and IscA may constitute the redundant cellular activities to recruit intracellular iron and deliver iron for the iron–sulfur cluster assembly in E. coli.

scholarly journals Iron-binding activity of human iron–sulfur cluster assembly protein hIscA1

2010 ◽  
Vol 428 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Jianxin Lu ◽  
Jacob P. Bitoun ◽  
Guoqiang Tan ◽  
Wu Wang ◽  
Wenguang Min ◽  
...  
Keyword(s):  
Cell Growth ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Visible Absorption ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Mononuclear Iron ◽  
Iron Sulfur

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. 2×1019 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.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

Crystal Structure of IscA, an Iron-sulfur Cluster Assembly Protein from Escherichia coli

2004 ◽  
Vol 338 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Jill R. Cupp-Vickery ◽  
Jonathan J. Silberg ◽  
Dennis T. Ta ◽  
Larry E. Vickery
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

scholarly journals The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e21992 ◽  
Author(s):  
Clara Iannuzzi ◽  
Salvatore Adinolfi ◽  
Barry D. Howes ◽  
Ricardo Garcia-Serres ◽  
Martin Clémancey ◽  
...  
Keyword(s):  
Scaffold Protein ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

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 Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Elliot I. Corless ◽  
Erin L. Mettert ◽  
Patricia J. Kiley ◽  
Edwin Antony
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Protein A ◽  
Fold Increase ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
E Coli ◽  
Protein Levels ◽  
Iron Sulfur

ABSTRACT Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered “SufFeScient” derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

Sign in / Sign up

Export Citation Format

Share Document