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

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.

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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

Journal of Biological Chemistry ◽

10.1074/jbc.m113.460360 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14200-14211 ◽

Cited By ~ 20

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.

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

Biochemical Journal ◽

10.1042/bj20101507 ◽

2010 ◽

Vol 432 (3) ◽

pp. 429-436 ◽

Cited By ~ 26

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.

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

Biochemistry ◽

10.1021/bi901393t ◽

2009 ◽

Vol 48 (45) ◽

pp. 10782-10792 ◽

Cited By ~ 19

Author(s):

Michael R. Reyda ◽

Corey J. Fugate ◽

Joseph T. Jarrett

Keyword(s):

Cluster Assembly ◽

Biotin Synthase ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur

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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

Journal of Biological Chemistry ◽

10.1074/jbc.m112.416602 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16680-16689 ◽

Cited By ~ 29

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.

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A novel role of the mitochondrial iron-sulfur cluster assembly protein ISCU-1/ISCU in longevity and stress response

GeroScience ◽

10.1007/s11357-021-00327-z ◽

2021 ◽

Author(s):

Yi Sheng ◽

Guang Yang ◽

Kaitlyn Casey ◽

Shayla Curry ◽

Mason Oliver ◽

...

Keyword(s):

Stress Response ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Mitochondrial Iron

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Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms22041598 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1598

Author(s):

Amber L. Hendricks ◽

Christine Wachnowsky ◽

Brian Fries ◽

Insiya Fidai ◽

James A. Cowan

Keyword(s):

Cluster Assembly ◽

Paramagnetic Resonance ◽

Iron Sulfur Cluster ◽

Sulfur Transfer ◽

Disease States ◽

Isolation And Characterization ◽

Iron Sulfur ◽

Natural Iron ◽

Lipoyl Synthase

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

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Multiple Sense and Antisense Promoters Contribute to the Regulated Expression of the isc-suf Operon for Iron-Sulfur Cluster Assembly in Rhodobacter

Microorganisms ◽

10.3390/microorganisms7120671 ◽

2019 ◽

Vol 7 (12) ◽

pp. 671 ◽

Cited By ~ 1

Author(s):

Xin Nie ◽

Bernhard Remes ◽

Gabriele Klug

Keyword(s):

Rhodobacter Sphaeroides ◽

Photosynthetic Electron Transport ◽

Regulatory Proteins ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Iron Sulfur Clusters ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Photosynthetic Complexes ◽

The One

A multitude of biological functions relies on iron-sulfur clusters. The formation of photosynthetic complexes goes along with an additional demand for iron-sulfur clusters for bacteriochlorophyll synthesis and photosynthetic electron transport. However, photooxidative stress leads to the destruction of iron-sulfur clusters, and the released iron promotes the formation of further reactive oxygen species. A balanced regulation of iron-sulfur cluster synthesis is required to guarantee the supply of this cofactor, on the one hand, but also to limit stress, on the other hand. The phototrophic alpha-proteobacterium Rhodobacter sphaeroides harbors a large operon for iron-sulfur cluster assembly comprising the iscRS and suf genes. IscR (iron-sulfur cluster regulator) is an iron-dependent regulator of isc-suf genes and other genes with a role in iron metabolism. We applied reporter gene fusions to identify promoters of the isc-suf operon and studied their activity alone or in combination under different conditions. Gel-retardation assays showed the binding of regulatory proteins to individual promoters. Our results demonstrated that several promoters in a sense and antisense direction influenced isc-suf expression and the binding of the IscR, Irr, and OxyR regulatory proteins to individual promoters. These findings demonstrated a complex regulatory network of several promoters and regulatory proteins that helped to adjust iron-sulfur cluster assembly to changing conditions in Rhodobacter sphaeroides.

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