scholarly journals IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

2013 ◽  
Vol 288 (23) ◽  
pp. 16680-16689 ◽  
Author(s):  
Mineaki Seki ◽  
Yukiko Takeda ◽  
Kazuhiro Iwai ◽  
Kiyoji Tanaka
Keyword(s):  
Cluster Assembly ◽  
Core Complex ◽  
Core Component ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
The Core ◽  
Iron Sulfur ◽  
External Component

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.

scholarly journals Iron–sulfur cluster biosynthesis

2008 ◽  
Vol 36 (6) ◽  
pp. 1112-1119 ◽  
Author(s):  
Sibali Bandyopadhyay ◽  
Kala Chandramouli ◽  
Michael K. Johnson
Keyword(s):  
Scaffold Proteins ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Eukaryotic Organisms ◽  
Almost All ◽  
Cluster Biosynthesis

Iron–sulfur (Fe–S) clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of Fe–S cluster biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of Fe–S cluster assembly machinery have been established in bacteria, termed the NIF, ISC and SUF systems, and, in each case, the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of pre-formed clusters to apo proteins. A molecular level understanding of the complex processes of Fe–S cluster assembly and transfer is now beginning to emerge from the combination of in vivo and in vitro approaches. The present review highlights recent developments in understanding the mechanism of Fe–S cluster assembly and transfer involving the ubiquitous U-type scaffold proteins and the potential roles of accessory proteins such as Nfu proteins and monothiol glutaredoxins in the assembly, storage or transfer of Fe–S clusters.

scholarly journals Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

2013 ◽  
Vol 288 (20) ◽  
pp. 14200-14211 ◽  
Author(s):  
Sylvain Boutigny ◽  
Avneesh Saini ◽  
Edward E. K. Baidoo ◽  
Natasha Yeung ◽  
Jay D. Keasling ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Carrier Protein ◽  
Cluster Assembly ◽  
Carrier Proteins ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur ◽  
Initial Cluster ◽  
Initial Assembly

The biosynthesis of iron sulfur (FeS) clusters, their trafficking from initial assembly on scaffold proteins via carrier proteins to final incorporation into FeS apoproteins, is a highly coordinated process enabled by multiprotein systems encoded in iscRSUAhscBAfdx and sufABCDSE operons in Escherichia coli. Although these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors such as monothiol glutaredoxin, GrxD, and the FeS carrier protein NfuA are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins and in the final stages of FeS protein assembly by transferring clusters to client FeS apoproteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA, and the methylthiolase MiaB, a radical S-adenosyl-l-methionine-dependent enzyme involved in the maturation of a subset of tRNAs. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo. We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins could play complementary roles in maturation and repair of MiaB.

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.

scholarly journals A Complex between Biotin Synthase and the Iron−Sulfur Cluster Assembly Chaperone HscA That Enhances in Vivo Cluster Assembly

Biochemistry ◽  
2009 ◽  
Vol 48 (45) ◽  
pp. 10782-10792 ◽  
Author(s):  
Michael R. Reyda ◽  
Corey J. Fugate ◽  
Joseph T. Jarrett
Keyword(s):  
Cluster Assembly ◽  
Biotin Synthase ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

Biogenesis and Transfer of Iron-Sulfur Clusters from Acidithiobacillus ferrooxidans

2013 ◽  
Vol 825 ◽  
pp. 198-201 ◽  
Author(s):  
Jian She Liu ◽  
Lin Qian ◽  
Chun Li Zheng
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Binding Protein ◽  
Cluster Assembly ◽  
Cysteine Desulfurase ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins

Iron-sulfur (Fe-S) proteins are ubiquitous and participate in multiple essential functions of life. However, little is currently known about the mechanisms of iron-sulfur biosynthesis and transfer in acidophilic microorganisms. In this study, the IscS, IscU and IscA proteins from Acidithiobacillus ferrooxidans were successfully expressed in Escherichia coli and purified by affinity chromatography. The IscS was a cysteine desulfurase which catalyzes desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly. Purified IscU did not have an iron-sulfur cluster but could act as a scaffold protein to assemble the [2Fe-2S] cluster in vitro. The IscA was a [4Fe-4S] cluster binding protein, but it also acted as an iron binding protein. Further studies indicated that the iron sulfur clusters could be transferred from pre-assembled scaffold proteins to apo-form iron sulfur proteins, the reconstituted iron sulfur proteins could restore their physiological activities.

scholarly journals In Vivo Demonstration of FNR Dimers in Response to Lower O2 Availability

2007 ◽  
Vol 189 (7) ◽  
pp. 2930-2932 ◽  
Author(s):  
Adrian J. Jervis ◽  
Jeffrey Green
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Current Model ◽  
In Vitro Studies ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Two Hybrid

ABSTRACT Escherichia coli FNR is an O2-sensing transcription factor. In vitro studies indicate that anaerobic iron-sulfur cluster acquisition promotes FNR dimerization. Here, two-hybrid assays show that iron-sulfur cluster-dependent FNR dimers are formed in vivo in response to lower O2 availability, consistent with the current model of FNR activation.

Research on isc Operon in Acidithiobacillus ferrooxidans ATCC 23270

2007 ◽  
Vol 20-21 ◽  
pp. 509-512 ◽  
Author(s):  
Jian She Liu ◽  
Yan Fei Zhang ◽  
Mei Mei Geng ◽  
Jia Zeng ◽  
Guan Zhou Qiu
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Purification And Characterization ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins

The highly conserved operon iron–sulfur cluster (iscSUA) is essential for the general biogenesis and transfer of iron–sulfur proteins in bacteria. In this study, expression, purification and characterization of the proteins of the isc operon (iscSUA) of Acidithiobacillus ferrooxidans ATCC 23270 was studied. Assembly and transfer of [Fe4S4] in vitro during the isc proteins and other iron sulfur proteins was studied in order to detect the pathway and mechanism of [Fe4S4] assembly and transfer in vivo. The [Fe4S4] cluster was successfully assembled in iron-sulfur proteins in vitro in the presence of Fe2+ and sulfide, and it was successfully transferred from IscA or IscU to iron- sulfur proteins. Our results support and extend certain models of iron-sulfur clusters assembly and transfer.

scholarly journals Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

2008 ◽  
Vol 191 (5) ◽  
pp. 1490-1497 ◽  
Author(s):  
Jeffrey M. Boyd ◽  
Randy M. Drevland ◽  
Diana M. Downs ◽  
David E. Graham
Keyword(s):  
Genetic System ◽  
Growth Defect ◽  
Cluster Assembly ◽  
Carrier Proteins ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Amino Terminal ◽  
Iron Sulfur ◽  
Major Cluster

ABSTRACT Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.

scholarly journals The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions

2020 ◽  
Vol 295 (33) ◽  
pp. 11891-11901 ◽  
Author(s):  
Brigitta Németh ◽  
Henrik Land ◽  
Ann Magnuson ◽  
Anders Hofer ◽  
Gustav Berggren
Keyword(s):  
Gas Production ◽  
Cluster Assembly ◽  
Paramagnetic Resonance ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Spectroscopy Studies ◽  
Electron Paramagnetic

[FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H2 gas production. H2 formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase in in vitro assays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly, i.e. in binding and delivering the precatalyst.

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.

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