The mitochondrial Hsp70 chaperone Ssq1 facilitates Fe/S cluster transfer from Isu1 to Grx5 by complex formation.

PubMed

Uzarska, Marta A; Dutkiewicz, Rafal; Freibert, Sven-Andreas; Lill, Roland; Mühlenhoff, Ulrich

2013-06-01

The mitochondrial Hsp70 chaperone Ssq1 plays a dedicated role in the maturation of iron-sulfur (Fe/S) proteins, an essential process of mitochondria. Similar to its bacterial orthologue HscA, Ssq1 binds to the scaffold protein Isu1, thereby facilitating dissociation of the newly synthesized Fe/S cluster on Isu1 and its transfer to target apoproteins. Here we use in vivo and in vitro approaches to show that Ssq1 also interacts with the monothiol glutaredoxin 5 (Grx5) at a binding site different from that of Isu1. Grx5 binding does not stimulate the ATPase activity of Ssq1 and is most pronounced for the ADP-bound form of Ssq1, which interacts with Isu1 most tightly. The vicinity of Isu1 and Grx5 on the Hsp70 chaperone facilitates rapid Fe/S cluster transfer from Isu1 to Grx5. Grx5 and its bound Fe/S cluster are required for maturation of all cellular Fe/S proteins, regardless of the type of bound Fe/S cofactor and subcellular localization. Hence Grx5 functions as a late-acting component of the core Fe/S cluster (ISC) assembly machinery linking the Fe/S cluster synthesis reaction on Isu1 with late assembly steps involving Fe/S cluster targeting to dedicated apoproteins.

Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

PubMed Central

Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

2016-01-01

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20

PubMed Central

Maio, N.; Rouault, T. A.

2017-01-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045

Fe-S cluster biogenesis in Gram-positive bacteria: SufU is a zinc-dependent sulfur transfer protein.

PubMed

Selbach, Bruna P; Chung, Alexander H; Scott, Aubrey D; George, Simon J; Cramer, Stephen P; Dos Santos, Patricia C

2014-01-14

The biosynthesis of Fe-S clusters in Bacillus subtilis and other Gram-positive bacteria is catalyzed by the SufCDSUB system. The first step in this pathway involves the sulfur mobilization from the free amino acid cysteine to a sulfur acceptor protein SufU via a PLP-dependent cysteine desulfurase SufS. In this reaction scheme, the formation of an enzyme S-covalent intermediate is followed by the binding of SufU. This event leads to the second half of the reaction where a deprotonated thiol of SufU promotes the nucleophilic attack onto the persulfide intermediate of SufS. Kinetic analysis combined with spectroscopic methods identified that the presence of a zinc atom tightly bound to SufU (Ka = 10(17) M(-1)) is crucial for its structural and catalytic competency. Fe-S cluster assembly experiments showed that despite the high degree of sequence and structural similarity to the ortholog enzyme IscU, the B. subtilis SufU does not act as a standard Fe-S cluster scaffold protein. The involvement of SufU as a dedicated agent of sulfur transfer, rather than as an assembly scaffold, in the biogenesis of Fe-S clusters in Gram-positive microbes indicates distinct strategies used by bacterial systems to assemble Fe-S clusters.

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation*

PubMed Central

Netz, Daili J. A.; Pierik, Antonio J.; Stümpfig, Martin; Bill, Eckhard; Sharma, Anil K.; Pallesen, Leif J.; Walden, William E.; Lill, Roland

2012-01-01

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo. PMID:22362766

Roles of Fe-S proteins: from cofactor synthesis to iron homeostasis to protein synthesis.

PubMed

Pain, Debkumar; Dancis, Andrew

2016-06-01

Fe-S cluster assembly is an essential process for all cells. Impairment of Fe-S cluster assembly creates diseases in diverse and surprising ways. In one scenario, the loss of function of lipoic acid synthase, an enzyme with Fe-S cluster cofactor in mitochondria, impairs activity of various lipoamide-dependent enzymes with drastic consequences for metabolism. In a second scenario, the heme biosynthetic pathway in red cell precursors is specifically targeted, and iron homeostasis is perturbed, but lipoic acid synthesis is unaffected. In a third scenario, tRNA modifications arising from action of the cysteine desulfurase and/or Fe-S cluster proteins are lost, which may lead to impaired protein synthesis. These defects can then result in cancer, neurologic dysfunction or type 2 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

PubMed

Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

2016-02-15

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Human mitochondrial MIA40 (CHCHD4) is a component of the Fe-S cluster export machinery.

PubMed

Murari, Anjaneyulu; Thiriveedi, Venkata Ramana; Mohammad, Fareed; Vengaldas, Viswamithra; Gorla, Madhavi; Tammineni, Prasad; Krishnamoorthy, Thanuja; Sepuri, Naresh Babu V

2015-10-15

Mitochondria play an essential role in synthesis and export of iron-sulfur (Fe-S) clusters to other sections of a cell. Although the mechanism of Fe-S cluster synthesis is well elucidated, information on the identity of the proteins involved in the export pathway is limited. The present study identifies hMIA40 (human mitochondrial intermembrane space import and assembly protein 40), also known as CHCHD4 (coiled-coil-helix-coiled-coil-helix domain-containing 4), as a component of the mitochondrial Fe-S cluster export machinery. hMIA40 is an iron-binding protein with the ability to bind iron in vivo and in vitro. hMIA40 harbours CPC (Cys-Pro-Cys) motif-dependent Fe-S clusters that are sensitive to oxidation. Depletion of hMIA40 results in accumulation of iron in mitochondria concomitant with decreases in the activity and stability of Fe-S-containing cytosolic enzymes. Intriguingly, overexpression of either the mitochondrial export component or cytosolic the Fe-S cluster assembly component does not have any effect on the phenotype of hMIA40-depleted cells. Taken together, our results demonstrate an indispensable role for hMIA40 for the export of Fe-S clusters from mitochondria. © 2015 Authors; published by Portland Press Limited.

Exome sequencing identifies NFS1 deficiency in a novel Fe-S cluster disease, infantile mitochondrial complex II/III deficiency.

PubMed

Farhan, Sali M K; Wang, Jian; Robinson, John F; Lahiry, Piya; Siu, Victoria M; Prasad, Chitra; Kronick, Jonathan B; Ramsay, David A; Rupar, C Anthony; Hegele, Robert A

2014-01-01

Iron-sulfur (Fe-S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe-S proteins, to assist in various key biochemical pathways. Mutations in Fe-S proteins often disrupt Fe-S cluster assembly leading to a spectrum of severe disorders such as Friedreich's ataxia or iron-sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe-S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

Structural and Functional Analyses of the Proteins Involved in the Iron-Sulfur Cluster Biosynthesis

NASA Astrophysics Data System (ADS)

Wada, Kei

The iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups that are required to maintain such fundamental life processes as respiratory chain, photosynthesis and the regulation of gene expression. Assembly of intracellular Fe-S cluster requires the sophisticated biosynthetic systems called ISC and SUF machineries. To shed light on the molecular mechanism of Fe-S cluster assembly mediated by SUF machinery, several structures of the SUF components and their sub-complex were determined. The structural findings together with biochemical characterization of the core-complex (SufB-SufC-SufD complex) have led me to propose a working model for the cluster biosynthesis in the SUF machinery.

Fe-S cluster assembly in the supergroup Excavata.

PubMed

Peña-Diaz, Priscila; Lukeš, Julius

2018-04-05

The majority of established model organisms belong to the supergroup Opisthokonta, which includes yeasts and animals. While enlightening, this focus has neglected protists,  organisms that represent the bulk of eukaryotic diversity and are often regarded as primitive eukaryotes. One of these is the "supergroup" Excavata, which comprises unicellular flagellates of diverse lifestyles and contains species of medical importance, such as Trichomonas, Giardia, Naegleria, Trypanosoma and Leishmania. Excavata exhibits a continuum in mitochondrial forms, ranging from classical aerobic, cristae-bearing mitochondria to mitochondria-related organelles, such as hydrogenosomes and mitosomes, to the extreme case of a complete absence of the organelle. All forms of mitochondria house a machinery for the assembly of Fe-S clusters, ancient cofactors required in various biochemical activities needed to sustain every extant cell. In this review, we survey what is known about the Fe-S cluster assembly in the supergroup Excavata. We aim to bring attention to the diversity found in this group, reflected in gene losses and gains that have shaped the Fe-S cluster biogenesis pathways.

Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis†

PubMed Central

Bridwell-Rabb, Jennifer; Iannuzzi, Clara; Pastore, Annalisa; Barondeau, David P.

2012-01-01

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich’s ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homolog CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule. PMID:22352884

Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis.

PubMed

Bridwell-Rabb, Jennifer; Iannuzzi, Clara; Pastore, Annalisa; Barondeau, David P

2012-03-27

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Response of Fe-S cluster assembly machinery of Escherichia coli to mechanical stress in a model of amino-acid crystal fermentation.

PubMed

Okutani, Satoshi; Iwai, Takayoshi; Iwatani, Shintaro; Matsuno, Kiyoshi; Takahashi, Yasuhiro; Hase, Toshiharu

2015-09-01

During amino-acid crystal fermentation, mechanical stress on bacterial cells caused by crystal collision often impacts negatively on bacterial growth and amino-acid production. When Escherichia coli cells were cultivated under mechanical stress of polyvinyl chloride particles as a model of the crystal fermentation, activities of iron-sulfur (Fe-S) cluster-containing enzymes were apparently decreased. Based on an assumption that function of Fe-S cluster assembly machinery would be elevated to recover the enzyme activities in such stressed cells, we analyzed levels of various components of Fe-S cluster assembly machinery by western blotting. It was found that the expression of HscA, a chaperon component of the machinery, was up-regulated and that shorter forms of HscA with the N-terminal region truncated were accumulated, suggesting an important role of HscA against the mechanical stress. An overexpression of HscA gene in E. coli cells gave a positive effect on rescue of the stress-induced decrease of the activity of Fe-S cluster-containing enzyme. These results may provide a new strategy to alleviate the mechanical stress during the amino-acid crystal fermentation. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Proteomic analysis of protein-protein interactions within the Cysteine Sulfinate Desulfinase Fe-S cluster biogenesis system.

PubMed

Bolstad, Heather M; Botelho, Danielle J; Wood, Matthew J

2010-10-01

Fe-S cluster biogenesis is of interest to many fields, including bioenergetics and gene regulation. The CSD system is one of three Fe-S cluster biogenesis systems in E. coli and is comprised of the cysteine desulfurase CsdA, the sulfur acceptor protein CsdE, and the E1-like protein CsdL. The biological role, biochemical mechanism, and protein targets of the system remain uncharacterized. Here we present that the active site CsdE C61 has a lowered pK(a) value of 6.5, which is nearly identical to that of C51 in the homologous SufE protein and which is likely critical for its function. We observed that CsdE forms disulfide bonds with multiple proteins and identified the proteins that copurify with CsdE. The identification of Fe-S proteins and both putative and established Fe-S cluster assembly (ErpA, glutaredoxin-3, glutaredoxin-4) and sulfur trafficking (CsdL, YchN) proteins supports the two-pathway model, in which the CSD system is hypothesized to synthesize both Fe-S clusters and other sulfur-containing cofactors. We suggest that the identified Fe-S cluster assembly proteins may be the scaffold and/or shuttle proteins for the CSD system. By comparison with previous analysis of SufE, we demonstrate that there is some overlap in the CsdE and SufE interactomes.

Separate Fe-S Scaffold And Carrier Functions For SufB2C2 And SufA During In Vitro Maturation Of [2Fe-2S] Fdx

PubMed Central

Chahal, Harsimranjit K.; Outten, F. Wayne

2012-01-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of Fe-S clusters proceeds via complex pathways involving multiple protein complexes. In the Suf Fe-S cluster biogenesis system, SufB may be a scaffold for nascent Fe-S cluster assembly whereas SufA is proposed to act as either a scaffold or an Fe-S cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB2C2 and SufBC2D and the specific functions of these complexes in Fe-S cluster assembly are not clear. Here we compare the ability of the SufB2C2 and SufBC2D complexes as well as SufA to promote in vitro maturation of the [2Fe-2S] ferredoxin (Fdx). We found that SufB2C2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed Fe-S cluster to Fdx. Furthermore, cluster transfer from [4Fe-4S] SufB2C2 or SufBC2D to Fdx will proceed through a SufA intermediate to Fdx is SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe-4S] SufB2C2 to Fdx and from SufBC2D to [2Fe-2S] SufA or Fdx. These studies indicate that SufB2C2 can serve as a terminal scaffold to load the SufA Fe-S cluster carrier for in vitro maturation of [2Fe-2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction. PMID:23018275

Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin

2010-02-12

Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold,more » in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.« less

[Nitrogen oxide is involved in the regulation of the Fe-S cluster assembly in proteins and the formation of biofilms by Escherichia coli cells].

PubMed

Vasil'eva, S V; Streltsova, D A; Starostina, I A; Sanina, N A

2013-01-01

The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants deltaiscA and deltasufA, and the double paralog mutant deltaiscA/sufA with deletions in the alternative pathways of Fe2+ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe2+ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CE Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.

The Diabetes Drug Target MitoNEET Governs a Novel Trafficking Pathway to Rebuild an Fe-S Cluster into Cytosolic Aconitase/Iron Regulatory Protein 1*

PubMed Central

Ferecatu, Ioana; Gonçalves, Sergio; Golinelli-Cohen, Marie-Pierre; Clémancey, Martin; Martelli, Alain; Riquier, Sylvie; Guittet, Eric; Latour, Jean-Marc; Puccio, Hélène; Drapier, Jean-Claude; Lescop, Ewen; Bouton, Cécile

2014-01-01

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis. PMID:25012650

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

PubMed

Ferecatu, Ioana; Gonçalves, Sergio; Golinelli-Cohen, Marie-Pierre; Clémancey, Martin; Martelli, Alain; Riquier, Sylvie; Guittet, Eric; Latour, Jean-Marc; Puccio, Hélène; Drapier, Jean-Claude; Lescop, Ewen; Bouton, Cécile

2014-10-10

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

PubMed Central

Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

2014-01-01

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes. PMID:25271645

Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

PubMed

Anwar, Shadab; Dikhit, Manas Ranjan; Singh, Krishn Pratap; Kar, Rajiv Kumar; Zaidi, Amir; Sahoo, Ganesh Chandra; Roy, Awadh Kishore; Nozaki, Tomoyoshi; Das, Pradeep; Ali, Vahab

2014-01-01

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity.

PubMed

Mena, Natalia P; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C; Núñez, Marco T

2011-06-03

Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease. Copyright © 2011 Elsevier Inc. All rights reserved.

Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

PubMed Central

Maio, Nunziata; Rouault, Tracey. A.

2014-01-01

Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

His86 from the N-terminus of frataxin coordinates iron and is required for Fe-S cluster synthesis.

PubMed

Gentry, Leslie E; Thacker, Matthew A; Doughty, Reece; Timkovich, Russell; Busenlehner, Laura S

2013-09-03

Human frataxin has a vital role in the biosynthesis of iron-sulfur (Fe-S) clusters in mitochondria, and its deficiency causes the neurodegenerative disease Friedreich's ataxia. Proposed functions for frataxin in the Fe-S pathway include iron donation to the Fe-S cluster machinery and regulation of cysteine desulfurase activity to control the rate of Fe-S production, although further molecular detail is required to distinguish these two possibilities. It is well established that frataxin can coordinate iron using glutamate and aspartate side chains on the protein surface; however, in this work we identify a new iron coordinating residue in the N-terminus of human frataxin using complementary spectroscopic and structural approaches. Further, we demonstrate that His86 in this N-terminal region is required for high affinity iron coordination and iron assembly of Fe-S clusters by ISCU as part of the Fe-S cluster biosynthetic complex. If a binding site that includes His86 is important for Fe-S cluster synthesis as part of its chaperone function, this raises the possibility that either iron binding at the acidic surface of frataxin may be spurious or that it is required for protein-protein interactions with the Fe-S biosynthetic quaternary complex. Our data suggest that iron coordination to frataxin may be significant to the Fe-S cluster biosynthesis pathway in mitochondria.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

PubMed

Cory, Seth A; Van Vranken, Jonathan G; Brignole, Edward J; Patra, Shachin; Winge, Dennis R; Drennan, Catherine L; Rutter, Jared; Barondeau, David P

2017-07-03

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.

Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

PubMed

Galeano, B K; Ranatunga, W; Gakh, O; Smith, D Y; Thompson, J R; Isaya, G

2017-06-21

Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

Glutaredoxin S15 Is Involved in Fe-S Cluster Transfer in Mitochondria Influencing Lipoic Acid-Dependent Enzymes, Plant Growth, and Arsenic Tolerance in Arabidopsis1[OPEN

PubMed Central

2016-01-01

Glutaredoxins (Grxs) are small proteins that function as oxidoreductases with roles in deglutathionylation of proteins, reduction of antioxidants, and assembly of iron-sulfur (Fe-S) cluster-containing enzymes. Which of the 33 Grxs in Arabidopsis (Arabidopsis thaliana) perform roles in Fe-S assembly in mitochondria is unknown. We have examined in detail the function of the monothiol GrxS15 in plants. Our results show its exclusive mitochondrial localization, and we are concluding it is the major or only Grx in this subcellular location. Recombinant GrxS15 has a very low deglutathionylation and dehydroascorbate reductase activity, but it binds a Fe-S cluster. Partially removing GrxS15 from mitochondria slowed whole plant growth and respiration. Native GrxS15 is shown to be especially important for lipoic acid-dependent enzymes in mitochondria, highlighting a putative role in the transfer of Fe-S clusters in this process. The enhanced effect of the toxin arsenic on the growth of GrxS15 knockdown plants compared to wild type highlights the role of mitochondrial glutaredoxin Fe-S-binding in whole plant growth and toxin tolerance. PMID:26672074

Iron loading site on the Fe-S cluster assembly scaffold protein is distinct from the active site.

PubMed

Rodrigues, Andria V; Kandegedara, Ashoka; Rotondo, John A; Dancis, Andrew; Stemmler, Timothy L

2015-06-01

Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal titration calorimetry and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential scanning calorimetry was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray absorption spectroscopy was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

Interactions of iron-bound frataxin with ISCU and ferredoxin on the cysteine desulfurase complex leading to Fe-S cluster assembly.

PubMed

Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

2018-06-01

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the components of the biological machine that carries out the assembly of iron‑sulfur clusters in human mitochondria. Our results show that FXN tightly binds a single Fe 2+ but not Fe 3+ . While FXN (with or without bound Fe 2+ ) does not bind the scaffold protein ISCU directly, the two proteins interact mutually when each is bound to the cysteine desulfurase complex ([NFS1] 2 :[ISD11] 2 :[Acp] 2 ), abbreviated as (NIA) 2 , where "N" represents the cysteine desulfurase (NFS1), "I" represents the accessory protein (ISD11), and "A" represents acyl carrier protein (Acp). FXN binds (NIA) 2 weakly in the absence of ISCU but more strongly in its presence. Fe 2+ -FXN binds to the (NIA) 2 -ISCU 2 complex without release of iron. However, upon the addition of both l-cysteine and a reductant (either reduced FDX2 or DTT), Fe 2+ is released from FXN as consistent with Fe 2+ -FXN being the proximal source of iron for Fe-S cluster assembly. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Malfunctioning of the iron-sulfur cluster assembly machinery in Saccharomyces cerevisiae produces oxidative stress via an iron-dependent mechanism, causing dysfunction in respiratory complexes.

PubMed

Gomez, Mauricio; Pérez-Gallardo, Rocío V; Sánchez, Luis A; Díaz-Pérez, Alma L; Cortés-Rojo, Christian; Meza Carmen, Victor; Saavedra-Molina, Alfredo; Lara-Romero, Javier; Jiménez-Sandoval, Sergio; Rodríguez, Francisco; Rodríguez-Zavala, José S; Campos-García, Jesús

2014-01-01

Biogenesis and recycling of iron-sulfur (Fe-S) clusters play important roles in the iron homeostasis mechanisms involved in mitochondrial function. In Saccharomyces cerevisiae, the Fe-S clusters are assembled into apoproteins by the iron-sulfur cluster machinery (ISC). The aim of the present study was to determine the effects of ISC gene deletion and consequent iron release under oxidative stress conditions on mitochondrial functionality in S. cerevisiae. Reactive oxygen species (ROS) generation, caused by H2O2, menadione, or ethanol, was associated with a loss of iron homeostasis and exacerbated by ISC system dysfunction. ISC mutants showed increased free Fe2+ content, exacerbated by ROS-inducers, causing an increase in ROS, which was decreased by the addition of an iron chelator. Our study suggests that the increment in free Fe2+ associated with ROS generation may have originated from mitochondria, probably Fe-S cluster proteins, under both normal and oxidative stress conditions, suggesting that Fe-S cluster anabolism is affected. Raman spectroscopy analysis and immunoblotting indicated that in mitochondria from SSQ1 and ISA1 mutants, the content of [Fe-S] centers was decreased, as was formation of Rieske protein-dependent supercomplex III2IV2, but this was not observed in the iron-deficient ATX1 and MRS4 mutants. In addition, the activity of complexes II and IV from the electron transport chain (ETC) was impaired or totally abolished in SSQ1 and ISA1 mutants. These results confirm that the ISC system plays important roles in iron homeostasis, ROS stress, and in assembly of supercomplexes III2IV2 and III2IV1, thus affecting the functionality of the respiratory chain.

Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

PubMed

Ciesielski, Szymon J; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A

2016-04-01

Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands. © 2016 Ciesielski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mena, Natalia P.; Millennium Institute of Cell Dynamics and Biotechnology, Santiago; Bulteau, Anne Laure

2011-06-03

Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters aremore » involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex I and iron accumulation are hallmarks of idiopathic Parkinson's disease, the findings reported here may have relevance for understanding the pathophysiology of this disease.« less

Role of Nfu1 and Bol3 in iron-sulfur cluster transfer to mitochondrial clients

PubMed Central

Melber, Andrew; Na, Un; Vashisht, Ajay; Weiler, Benjamin D; Lill, Roland; Wohlschlegel, James A; Winge, Dennis R

2016-01-01

Iron-sulfur (Fe-S) clusters are essential for many cellular processes, ranging from aerobic respiration, metabolite biosynthesis, ribosome assembly and DNA repair. Mutations in NFU1 and BOLA3 have been linked to genetic diseases with defects in mitochondrial Fe-S centers. Through genetic studies in yeast, we demonstrate that Nfu1 functions in a late step of [4Fe-4S] cluster biogenesis that is of heightened importance during oxidative metabolism. Proteomic studies revealed Nfu1 physical interacts with components of the ISA [4Fe-4S] assembly complex and client proteins that need [4Fe-4S] clusters to function. Additional studies focused on the mitochondrial BolA proteins, Bol1 and Bol3 (yeast homolog to human BOLA3), revealing that Bol1 functions earlier in Fe-S biogenesis with the monothiol glutaredoxin, Grx5, and Bol3 functions late with Nfu1. Given these observations, we propose that Nfu1, assisted by Bol3, functions to facilitate Fe-S transfer from the biosynthetic apparatus to the client proteins preventing oxidative damage to [4Fe-4S] clusters. DOI: http://dx.doi.org/10.7554/eLife.15991.001 PMID:27532773

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

PubMed Central

Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K.; Smith, Douglas Y.; Söderberg, Christopher A. G.; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

2016-01-01

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. PMID:26941001

Glutaredoxin S15 Is Involved in Fe-S Cluster Transfer in Mitochondria Influencing Lipoic Acid-Dependent Enzymes, Plant Growth, and Arsenic Tolerance in Arabidopsis.

PubMed

Ströher, Elke; Grassl, Julia; Carrie, Chris; Fenske, Ricarda; Whelan, James; Millar, A Harvey

2016-03-01

Glutaredoxins (Grxs) are small proteins that function as oxidoreductases with roles in deglutathionylation of proteins, reduction of antioxidants, and assembly of iron-sulfur (Fe-S) cluster-containing enzymes. Which of the 33 Grxs in Arabidopsis (Arabidopsis thaliana) perform roles in Fe-S assembly in mitochondria is unknown. We have examined in detail the function of the monothiol GrxS15 in plants. Our results show its exclusive mitochondrial localization, and we are concluding it is the major or only Grx in this subcellular location. Recombinant GrxS15 has a very low deglutathionylation and dehydroascorbate reductase activity, but it binds a Fe-S cluster. Partially removing GrxS15 from mitochondria slowed whole plant growth and respiration. Native GrxS15 is shown to be especially important for lipoic acid-dependent enzymes in mitochondria, highlighting a putative role in the transfer of Fe-S clusters in this process. The enhanced effect of the toxin arsenic on the growth of GrxS15 knockdown plants compared to wild type highlights the role of mitochondrial glutaredoxin Fe-S-binding in whole plant growth and toxin tolerance. © 2016 American Society of Plant Biologists. All Rights Reserved.

Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria.

PubMed

Outten, F Wayne

2015-06-01

Fe-S clusters play critical roles in cellular function throughout all three kingdoms of life. Consequently, Fe-S cluster biogenesis systems are present in most organisms. The Suf (sulfur formation) system is the most ancient of the three characterized Fe-S cluster biogenesis pathways, which also include the Isc and Nif systems. Much of the first work on the Suf system took place in Gram-negative Proteobacteria used as model organisms. These early studies led to a wealth of biochemical, genetic, and physiological information on Suf function. From those studies we have learned that SufB functions as an Fe-S scaffold in conjunction with SufC (and in some cases SufD). SufS and SufE together mobilize sulfur for cluster assembly and SufA traffics the complete Fe-S cluster from SufB to target apo-proteins. However, recent progress on the Suf system in other organisms has opened up new avenues of research and new hypotheses about Suf function. This review focuses primarily on the most recent discoveries about the Suf pathway and where those new models may lead the field. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Human frataxin is an allosteric switch that activates the Fe-S cluster biosynthetic complex.

PubMed

Tsai, Chi-Lin; Barondeau, David P

2010-11-02

Cellular depletion of the human protein frataxin is correlated with the neurodegenerative disease Friedreich's ataxia and results in the inactivation of Fe-S cluster proteins. Most researchers agree that frataxin functions in the biogenesis of Fe-S clusters, but its precise role in this process is unclear. Here we provide in vitro evidence that human frataxin binds to a Nfs1, Isd11, and Isu2 complex to generate the four-component core machinery for Fe-S cluster biosynthesis. Frataxin binding dramatically changes the K(M) for cysteine from 0.59 to 0.011 mM and the catalytic efficiency (k(cat)/K(M)) of the cysteine desulfurase from 25 to 7900 M⁻¹s⁻¹. Oxidizing conditions diminish the levels of both complex formation and frataxin-based activation, whereas ferrous iron further stimulates cysteine desulfurase activity. Together, these results indicate human frataxin functions with Fe(2+) as an allosteric activator that triggers sulfur delivery and Fe-S cluster assembly. We propose a model in which cellular frataxin levels regulate human Fe-S cluster biosynthesis that has implications for mitochondrial dysfunction, oxidative stress response, and both neurodegenerative and cardiovascular disease.

Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria

PubMed Central

Outten, F. Wayne

2014-01-01

Fe-S clusters play critical roles in cellular function throughout all three kingdoms of life. Consequently, Fe-S cluster biogenesis systems are present in most organisms. The Suf (sulfur formation) system is the most ancient of the three characterized Fe-S cluster biogenesis pathways, which also include the Isc and Nif systems. Much of the first work on the Suf system took place in Gram-negative Proteobacteria used as model organisms. These early studies led to a wealth of biochemical, genetic, and physiological information on Suf function. From those studies we have learned that SufB functions as an Fe-S scaffold in conjunction with SufC (and in some cases SufD). SufS and SufE together mobilize sulfur for cluster assembly and SufA traffics the complete Fe-S cluster from SufB to target apo-proteins. However, recent progress on the Suf system in other organisms has opened up new avenues of research and new hypotheses about Suf function. This review focuses primarily on the most recent discoveries about the Suf pathway and where those new models may lead the field. PMID:25447545

Fe-S Cluster Hsp70 Chaperones: The ATPase Cycle and Protein Interactions.

PubMed

Dutkiewicz, Rafal; Nowak, Malgorzata; Craig, Elizabeth A; Marszalek, Jaroslaw

2017-01-01

Hsp70 chaperones and their obligatory J-protein cochaperones function together in many cellular processes. Via cycles of binding to short stretches of exposed amino acids on substrate proteins, Hsp70/J-protein chaperones not only facilitate protein folding but also drive intracellular protein transport, biogenesis of cellular structures, and disassembly of protein complexes. The biogenesis of iron-sulfur (Fe-S) clusters is one of the critical cellular processes that require Hsp70/J-protein action. Fe-S clusters are ubiquitous cofactors critical for activity of proteins performing diverse functions in, for example, metabolism, RNA/DNA transactions, and environmental sensing. This biogenesis process can be divided into two sequential steps: first, the assembly of an Fe-S cluster on a conserved scaffold protein, and second, the transfer of the cluster from the scaffold to a recipient protein. The second step involves Hsp70/J-protein chaperones. Via binding to the scaffold, chaperones enable cluster transfer to recipient proteins. In eukaryotic cells mitochondria have a key role in Fe-S cluster biogenesis. In this review, we focus on methods that enabled us to dissect protein interactions critical for the function of Hsp70/J-protein chaperones in the mitochondrial process of Fe-S cluster biogenesis in the yeast Saccharomyces cerevisiae. © 2017 Elsevier Inc. All rights reserved.

Mammalian Fe-S cluster biogenesis and its implication in disease.

PubMed

Beilschmidt, Lena K; Puccio, Hélène M

2014-05-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are ubiquitous and essential. Due to their chemical versatility, Fe-S clusters are implicated in a wide range of protein functions including mitochondrial respiration and DNA repair. Composed of iron and sulfur, they are sensible to oxygen and their biogenesis requires a highly conserved protein machinery that facilitates assembly of the cluster as well as its insertion into apoproteins. Mitochondria are the central cellular compartment for Fe-S cluster biogenesis in eukaryotic cells and the importance of proper function of this biogenesis for life is highlighted by a constantly increasing number of human genetic diseases that are associated with dysfunction of this Fe-S cluster biogenesis pathway. Although these disorders are rare and appear dissimilar, common aspects are found among them. This review will give an overview on what is known on mammalian Fe-S cluster biogenesis today, by putting it into the context of what is known from studies from lower model organisms, and focuses on the associated diseases, by drawing attention to the respective mutations. Finally, it outlines the importance of adequate cellular and murine models to uncover not only each protein function, but to resolve their role and requirement throughout the mammalian organism. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Friedreich's Ataxia Variants I154F and W155R Diminish Frataxin-Based Activation of the Iron-Sulfur Cluster Assembly Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Chi-Lin; Bridwell-Rabb, Jennifer; Barondeau, David P

2011-11-07

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ~15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defectivemore » in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.« less

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

PubMed Central

Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

2015-01-01

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

A Complex Between Biotin Synthase and The Iron-Sulfur Cluster Assembly Chaperone HscA That Enhances In Vivo Cluster Assembly†

PubMed Central

Reyda, Michael R.; Fugate, Corey J.; Jarrett, Joseph T.

2009-01-01

Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 carbons of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S]2+ cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. In order for BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S]2+ cluster in BioB must be reassembled, a process that could be carried out in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70-class molecular chaperone, whose yeast homolog has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in S. cerevesiae. In the present work we show that in E. coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S]2+ cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner and a high affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB:HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein:protein interaction. PMID:19821612

NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi.

PubMed

Nývltová, Eva; Šuták, Robert; Harant, Karel; Šedinová, Miroslava; Hrdy, Ivan; Paces, Jan; Vlček, Čestmír; Tachezy, Jan

2013-04-30

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.

NIF-type iron-sulfur cluster assembly system is duplicated and distributed in the mitochondria and cytosol of Mastigamoeba balamuthi

PubMed Central

Nývltová, Eva; Šuták, Robert; Harant, Karel; Šedinová, Miroslava; Hrdý, Ivan; Pačes, Jan; Vlček, Čestmír; Tachezy, Jan

2013-01-01

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an ε-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments. PMID:23589868

Biogenesis and functions of mammalian iron-sulfur proteins in the regulation of iron homeostasis and pivotal metabolic pathways.

PubMed

Rouault, Tracey A; Maio, Nunziata

2017-08-04

Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin.

PubMed

Webert, Holger; Freibert, Sven-Andreas; Gallo, Angelo; Heidenreich, Torsten; Linne, Uwe; Amlacher, Stefan; Hurt, Ed; Mühlenhoff, Ulrich; Banci, Lucia; Lill, Roland

2014-10-31

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

PubMed

Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-05-06

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fe-S cluster biogenesis in isolated mammalian mitochondria: coordinated use of persulfide sulfur and iron and requirements for GTP, NADH, and ATP.

PubMed

Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K; Dancis, Andrew; Pain, Debkumar

2015-01-02

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [(35)S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-(35)S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the (35)S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

PubMed

Hu, Xueyun; Kato, Yukako; Sumida, Akihiro; Tanaka, Ayumi; Tanaka, Ryouichi

2017-04-01

Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC 2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC 2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC 2 D is the major, and possibly sole scaffold in plastids. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Staphylococcus aureus SufT: an essential iron-sulphur cluster assembly factor in cells experiencing a high-demand for lipoic acid.

PubMed

Mashruwala, Ameya A; Roberts, Christina A; Bhatt, Shiven; May, Kerrie L; Carroll, Ronan K; Shaw, Lindsey N; Boyd, Jeffrey M

2016-12-01

Staphylococcus aureus SufT is composed solely of the domain of unknown function 59 (DUF59) and has a role in the maturation of iron-sulphur (Fe-S) proteins. We report that SufT is essential for S. aureus when growth is heavily reliant upon lipoamide-utilizing enzymes, but dispensable when this reliance is decreased. LipA requires Fe-S clusters for lipoic acid (LA) synthesis and a ΔsufT strain had phenotypes suggestive of decreased LA production and decreased activities of lipoamide-requiring enzymes. Fermentative growth, a null clpC allele, or decreased flux through the TCA cycle diminished the demand for LA and rendered SufT non-essential. Abundance of the Fe-S cluster carrier Nfu was increased in a ΔclpC strain and a null clpC allele was unable to suppress the LA requirement of a ΔsufT Δnfu strain. Over-expression of nfu suppressed the LA requirement of the ΔsufT strain. We propose a model wherein SufT, and by extension the DUF59, is essential for the maturation of holo-LipA in S. aureus cells experiencing a high demand for lipoamide-dependent enzymes. The findings presented suggest that the demand for products of Fe-S enzymes is a factor governing the usage of one Fe-S cluster assembly factor over another in the maturation of apo-proteins. © 2016 John Wiley & Sons Ltd.

Evolution of Fe/S cluster biogenesis in the anaerobic parasite Blastocystis

PubMed Central

Tsaousis, Anastasios D.; Ollagnier de Choudens, Sandrine; Gentekaki, Eleni; Long, Shaojun; Gaston, Daniel; Stechmann, Alexandra; Vinella, Daniel; Py, Béatrice; Fontecave, Marc; Barras, Frédéric; Lukeš, Julius; Roger, Andrew J.

2012-01-01

Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei, its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite’s cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress. PMID:22699510

Turning Saccharomyces cerevisiae into a Frataxin-Independent Organism

PubMed Central

Yoon, Heeyong; Knight, Simon A. B.; Pandey, Alok; Pain, Jayashree; Turkarslan, Serdar; Pain, Debkumar; Dancis, Andrew

2015-01-01

Frataxin (Yfh1 in yeast) is a conserved protein and deficiency leads to the neurodegenerative disease Friedreich’s ataxia. Frataxin is a critical protein for Fe-S cluster assembly in mitochondria, interacting with other components of the Fe-S cluster machinery, including cysteine desulfurase Nfs1, Isd11 and the Isu1 scaffold protein. Yeast Isu1 with the methionine to isoleucine substitution (M141I), in which the E. coli amino acid is inserted at this position, corrected most of the phenotypes that result from lack of Yfh1 in yeast. This suppressor Isu1 behaved as a genetic dominant. Furthermore frataxin-bypass activity required a completely functional Nfs1 and correlated with the presence of efficient scaffold function. A screen of random Isu1 mutations for frataxin-bypass activity identified only M141 substitutions, including Ile, Cys, Leu, or Val. In each case, mitochondrial Nfs1 persulfide formation was enhanced, and mitochondrial Fe-S cluster assembly was improved in the absence of frataxin. Direct targeting of the entire E. coli IscU to ∆yfh1 mitochondria also ameliorated the mutant phenotypes. In contrast, expression of IscU with the reverse substitution i.e. IscU with Ile to Met change led to worsening of the ∆yfh1 phenotypes, including severely compromised growth, increased sensitivity to oxygen, deficiency in Fe-S clusters and heme, and impaired iron homeostasis. A bioinformatic survey of eukaryotic Isu1/prokaryotic IscU database entries sorted on the amino acid utilized at the M141 position identified unique groupings, with virtually all of the eukaryotic scaffolds using Met, and the preponderance of prokaryotic scaffolds using other amino acids. The frataxin-bypassing amino acids Cys, Ile, Leu, or Val, were found predominantly in prokaryotes. This amino acid position 141 is unique in Isu1, and the frataxin-bypass effect likely mimics a conserved and ancient feature of the prokaryotic Fe-S cluster assembly machinery. PMID:25996596

Biogenesis of cytosolic ribosomes requires the essential iron–sulphur protein Rli1p and mitochondria

PubMed Central

Kispal, Gyula; Sipos, Katalin; Lange, Heike; Fekete, Zsuzsanna; Bedekovics, Tibor; Janáky, Tamás; Bassler, Jochen; Aguilar Netz, Daili J; Balk, Janneke; Rotte, Carmen; Lill, Roland

2005-01-01

Mitochondria perform a central function in the biogenesis of cellular iron–sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p. PMID:15660134

Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems.

PubMed

Dos Santos, Patricia C; Johnson, Deborah C; Ragle, Brook E; Unciuleac, Mihaela-Carmen; Dean, Dennis R

2007-04-01

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.

Fe-S cluster coordination of the chromokinesin KIF4A alters its sub-cellular localization during mitosis.

PubMed

Ben-Shimon, Lilach; Paul, Viktoria D; David-Kadoch, Galit; Volpe, Marina; Stümpfig, Martin; Bill, Eckhard; Mühlenhoff, Ulrich; Lill, Roland; Ben-Aroya, Shay

2018-05-30

Fe-S clusters act as co-factors of proteins with diverse functions, e.g. in DNA repair. Down-regulation of the cytosolic iron-sulfur protein assembly (CIA) machinery promotes genomic instability by the inactivation of multiple DNA repair pathways. Furthermore, CIA deficiencies are associated with so far unexplained mitotic defects. Here, we show that CIA2B and MMS19, constituents of the CIA targeting complex involved in facilitating Fe-S cluster insertion into cytosolic and nuclear target proteins, co-localize with components of the mitotic machinery. Down-regulation of CIA2B and MMS19 impairs the mitotic cycle. We identify the chromokinesin KIF4A as a mitotic component involved in these effects. KIF4A binds a Fe-S cluster in vitro through its conserved cysteine-rich domain. We demonstrate in vivo that this domain is required for the mitosis-related KIF4A localization and for the mitotic defects associated with KIF4A knockout. KIF4A is the first identified mitotic component carrying such a post-translational modification. These findings suggest that the lack of Fe-S clusters in KIF4A upon down-regulation of the CIA targeting complex contributes to the mitotic defects. © 2018. Published by The Company of Biologists Ltd.

Cancer-Related NEET Proteins Transfer 2Fe-2S Clusters to Anamorsin, a Protein Required for Cytosolic Iron-Sulfur Cluster Biogenesis.

PubMed

Lipper, Colin H; Paddock, Mark L; Onuchic, José N; Mittler, Ron; Nechushtai, Rachel; Jennings, Patricia A

2015-01-01

Iron-sulfur cluster biogenesis is executed by distinct protein assembly systems. Mammals have two systems, the mitochondrial Fe-S cluster assembly system (ISC) and the cytosolic assembly system (CIA), that are connected by an unknown mechanism. The human members of the NEET family of 2Fe-2S proteins, nutrient-deprivation autophagy factor-1 (NAF-1) and mitoNEET (mNT), are located at the interface between the mitochondria and the cytosol. These proteins have been implicated in cancer cell proliferation, and they can transfer their 2Fe-2S clusters to a standard apo-acceptor protein. Here we report the first physiological 2Fe-2S cluster acceptor for both NEET proteins as human Anamorsin (also known as cytokine induced apoptosis inhibitor-1; CIAPIN-1). Anamorsin is an electron transfer protein containing two iron-sulfur cluster-binding sites that is required for cytosolic Fe-S cluster assembly. We show, using UV-Vis spectroscopy, that both NAF-1 and mNT can transfer their 2Fe-2S clusters to apo-Anamorsin with second order rate constants similar to those of other known human 2Fe-2S transfer proteins. A direct protein-protein interaction of the NEET proteins with apo-Anamorsin was detected using biolayer interferometry. Furthermore, electrospray mass spectrometry of holo-Anamorsin prepared by cluster transfer shows that it receives both of its 2Fe-2S clusters from the NEETs. We propose that mNT and NAF-1 can provide parallel routes connecting the mitochondrial ISC system and the CIA. 2Fe-2S clusters assembled in the mitochondria are received by NEET proteins and when needed transferred to Anamorsin, activating the CIA.

Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins†

PubMed Central

Gao, Huanyao; Subramanian, Sowmya; Couturier, Jérémy; Naik, Sunil; Kim, Sung-Kun; Leustek, Thomas; Knaff, David B.; Wu, Hui-Chen; Vignols, Florence; Huynh, Boi Hanh; Rouhier, Nicolas; Johnson, Michael K.

2013-01-01

Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism, resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S]2+ and [4Fe-4S]2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for maturation of chloroplastic Fe-S proteins via intact, rapid and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16, but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S]2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1) and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2, but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters. PMID:24032747

Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

PubMed Central

Fung, Danny Ka Chun; Lau, Wai Yin; Chan, Wing Tat

2013-01-01

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli. PMID:23893112

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.

PubMed

Urbina, H D; Silberg, J J; Hoff, K G; Vickery, L E

2001-11-30

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

Levels of control exerted by the Isc iron-sulfur cluster system on biosynthesis of the formate hydrogenlyase complex.

PubMed

Pinske, Constanze; Jaroschinsky, Monique; Sawers, R Gary

2013-06-01

The membrane-associated formate hydrogenlyase (FHL) complex of bacteria like Escherichia coli is responsible for the disproportionation of formic acid into the gaseous products carbon dioxide and dihydrogen. It comprises minimally seven proteins including FdhF and HycE, the catalytic subunits of formate dehydrogenase H and hydrogenase 3, respectively. Four proteins of the FHL complex have iron-sulphur cluster ([Fe-S]) cofactors. Biosynthesis of [Fe-S] is principally catalysed by the Isc or Suf systems and each comprises proteins for assembly and for delivery of [Fe-S]. This study demonstrates that the Isc system is essential for biosynthesis of an active FHL complex. In the absence of the IscU assembly protein no hydrogen production or activity of FHL subcomponents was detected. A deletion of the iscU gene also resulted in reduced intracellular formate levels partially due to impaired synthesis of pyruvate formate-lyase, which is dependent on the [Fe-S]-containing regulator FNR. This caused reduced expression of the formate-inducible fdhF gene. The A-type carrier (ATC) proteins IscA and ErpA probably deliver [Fe-S] to specific apoprotein components of the FHL complex because mutants lacking either protein exhibited strongly reduced hydrogen production. Neither ATC protein could compensate for the lack of the other, suggesting that they had independent roles in [Fe-S] delivery to complex components. Together, the data indicate that the Isc system modulates FHL complex biosynthesis directly by provision of [Fe-S] as well as indirectly by influencing gene expression through the delivery of [Fe-S] to key regulators and enzymes that ultimately control the generation and oxidation of formate.

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis[C][W][OA

PubMed Central

Luo, Dexian; Bernard, Delphine G.; Balk, Janneke; Hai, Huang; Cui, Xiaofeng

2012-01-01

Eukaryotic organisms have evolved a set of strategies to safeguard genome integrity, but the underlying mechanisms remain poorly understood. Here, we report that ASYMMETRIC LEAVES1/2 ENHANCER7 (AE7), an Arabidopsis thaliana gene encoding a protein in the evolutionarily conserved Domain of Unknown Function 59 family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal, whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly accumulated DNA damage that activates the DNA damage response to arrest the cell cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are highly conserved in eukaryotes and are involved in the biogenesis of cytosolic and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic [4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1. Additionally, mutations in the gene encoding the mitochondrial ATP binding cassette transporter ATM3/ABCB25, which is required for the activity of cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity similar to that of ae7-1. These results indicate that AE7 is a central member of the CIA pathway, linking plant mitochondria to nuclear genome integrity through assembly of Fe-S proteins. PMID:23104832

The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion

PubMed Central

Paul, Viktoria Désirée; Mühlenhoff, Ulrich; Stümpfig, Martin; Seebacher, Jan; Kugler, Karl G; Renicke, Christian; Taxis, Christof; Gavin, Anne-Claude; Pierik, Antonio J; Lill, Roland

2015-01-01

Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery. DOI: http://dx.doi.org/10.7554/eLife.08231.001 PMID:26182403

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.

PubMed

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-09-30

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN 42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN 42-210 ] 24 ·[NFS1] 24 ·[ISD11] 24 ·[ISCU] 24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN 42-210 ] 24 ·[ISCU] 24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN 42-210 trimer at each of its eight vertices. Binding of 12 [NFS1] 2 ·[ISD11] 2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN 42-210 to ISCU. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery*

PubMed Central

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y.; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

2016-01-01

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42–210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42–210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42–210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42–210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42–210 to ISCU. PMID:27519411

The Role of SufS Is Restricted to Fe-S Cluster Biosynthesis in Escherichia coli.

PubMed

Bühning, Martin; Valleriani, Angelo; Leimkühler, Silke

2017-04-11

In Escherichia coli, two different systems that are important for the coordinate formation of Fe-S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe-S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S-containing proteins, including the MoaA protein, which is involved in the conversion of 5'GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.

Deficiency of Arabidopsis thaliana frataxin alters activity of mitochondrial Fe-S proteins and induces oxidative stress.

PubMed

Busi, Maria V; Maliandi, María V; Valdez, Hugo; Clemente, Marina; Zabaleta, Eduardo J; Araya, Alejandro; Gomez-Casati, Diego F

2006-12-01

Frataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO(2) assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.

Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation

PubMed Central

Ferecatu, Ioana; Canal, Frédéric; Fabbri, Lucilla; Mazure, Nathalie M.; Bouton, Cécile

2018-01-01

Biogenesis of iron-sulfur clusters (ISC) is essential to almost all forms of life and involves complex protein machineries. This process is initiated within the mitochondrial matrix by the ISC assembly machinery. Cohort and case report studies have linked mutations in ISC assembly machinery to severe mitochondrial diseases. The voltage-dependent anion channel (VDAC) located within the mitochondrial outer membrane regulates both cell metabolism and apoptosis. Recently, the C-terminal truncation of the VDAC1 isoform, termed VDAC1-ΔC, has been observed in chemoresistant late-stage tumor cells grown under hypoxic conditions with activation of the hypoxia-response nuclear factor HIF-1α. These cells harbored atypical enlarged mitochondria. Here, we show for the first time that depletion of several proteins of the mitochondrial ISC machinery in normoxia leads to a similar enlarged mitochondria phenotype associated with accumulation of VDAC1-ΔC. This truncated form of VDAC1 accumulates in the absence of HIF-1α and HIF-2α activations and confers cell resistance to drug-induced apoptosis. Furthermore, we show that when hypoxia and siRNA knock-down of the ISC machinery core components are coupled, the cell phenotype is further accentuated, with greater accumulation of VDAC1-ΔC. Interestingly, we show that hypoxia promotes the downregulation of several proteins (ISCU, NFS1, FXN) involved in the early steps of mitochondrial Fe-S cluster biogenesis. Finally, we have identified the mitochondria-associated membrane (MAM) localized Fe-S protein CISD2 as a link between ISC machinery downregulation and accumulation of anti-apoptotic VDAC1-ΔC. Our results are the first to associate dysfunction in Fe-S cluster biogenesis with cleavage of VDAC1, a form which has previously been shown to promote tumor resistance to chemotherapy, and raise new perspectives for targets in cancer therapy. PMID:29596470

[Fe-S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite.

PubMed

Charan, Manish; Choudhary, Hadi Hasan; Singh, Nidhi; Sadik, Mohammad; Siddiqi, Mohammad Imran; Mishra, Satish; Habib, Saman

2017-08-01

The relict plastid (apicoplast) of the malaria parasite is the site for important biochemical pathways and is essential for parasite survival. The sulfur mobilization (SUF) pathway of iron-sulfur [Fe-S] cluster assembly in the apicoplast of Plasmodium spp. is of interest due to its absence in the human host suggesting the possibility of antimalarial intervention through apicoplast [Fe-S] biogenesis. We report biochemical characterization of components of the Plasmodium falciparum apicoplast SUF pathway after the first step of SUF. In vitro interaction experiments and in vivo cross-linking showed that apicoplast-encoded PfSufB and apicoplast-targeted PfSufC and PfSufD formed a complex. The PfSufB-C 2 -D complex could function as a scaffold to assemble [4Fe-4S] clusters in vitro and activity of the PfSufC ATPase was enhanced by PfSufD. Two carrier proteins, the NifU-like protein PfNfu and the A-type carrier PfSufA are homodimers, the former mediating transfer of [4Fe-4S] from the scaffold to a model [4Fe-4S] target protein with higher efficiency. Conditional knockout of SufS, the enzyme catalyzing the first step of SUF, by selective excision in the mosquito stages of Plasmodium berghei severely impaired development of sporozoites in oocysts establishing essentiality of the SUF machinery in the vector. Our results delineate steps of the complete apicoplast SUF pathway and demonstrate its critical role in the parasite life cycle. © 2017 Federation of European Biochemical Societies.

The mitochondrial proteins AtHscB and AtIsu1 involved in Fe-S cluster assembly interact with the Hsp70-type chaperon AtHscA2 and modulate its catalytic activity.

PubMed

Leaden, Laura; Busi, Maria V; Gomez-Casati, Diego F

2014-11-01

Arabidopsis plants contain two genes coding for mitochondrial Hsp70-type chaperon-like proteins, AtHscA1 (At4g37910) and AtHscA2 (At5g09590). Both genes are homologs of the Ssq1 gene involved in Fe-S cluster assembly in yeast. Protein-protein interaction studies showed that AtHscA2 interacts with AtIsu1 and AtHscB, two Arabidopsis homologs of the Isu1 protein and the Jac1 yeast co-chaperone. Moreover, this interaction could modulate the activity of AtHscA2. In the presence of a 1:5:5 molar ratio of AtHscA2:AtIsu1:AtHscB we observed an increase in the V(max) and a decrease in the S(0.5) for ATP of AtHscA2. Furthermore, an increase of about 28-fold in the catalytic efficiency of AtHscA2 was also observed. Results suggest that AtHscA2 in cooperation with AtIsu1 and AtHscB play an important role in the regulation of the Fe-S assembly pathway in plant mitochondria. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Impaired mitochondrial Fe-S cluster biogenesis activates the DNA damage response through different signaling mediators.

PubMed

Pijuan, Jordi; María, Carlos; Herrero, Enrique; Bellí, Gemma

2015-12-15

Fe-S cluster biogenesis machinery is required for multiple DNA metabolism processes. In this work, we show that, in Saccharomyces cerevisiae, defects at different stages of the mitochondrial Fe-S cluster assembly machinery (ISC) result in increased spontaneous mutation rate and hyper-recombination, accompanied by an increment in Rad52-associated DNA repair foci and a higher phosphorylated state of γH2A histone, altogether supporting the presence of constitutive DNA lesions. Furthermore, ISC assembly machinery deficiency elicits a DNA damage response that upregulates ribonucleotide reductase activity by promoting the reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and Rnr4 enzyme subunits. Depending on the impaired stage of the ISC machinery, different signaling pathway mediators contribute to such a response, converging on Dun1. Thus, cells lacking the glutaredoxin Grx5, which are compromised at the core ISC system, show Mec1- and Rad53-independent Dun1 activation, whereas both Mec1 and Chk1 are required when the non-core ISC member Iba57 is absent. Grx5-null cells exhibit a strong dependence on the error-free post-replication repair and the homologous recombination pathways, demonstrating that a DNA damage response needs to be activated upon ISC impairment to preserve cell viability. © 2015. Published by The Company of Biologists Ltd.

The iron-sulfur cluster assembly machineries in plants: current knowledge and open questions

PubMed Central

Couturier, Jérémy; Touraine, Brigitte; Briat, Jean-François; Gaymard, Frédéric; Rouhier, Nicolas

2013-01-01

Many metabolic pathways and cellular processes occurring in most sub-cellular compartments depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated protein machineries. Recent advances have been made in the knowledge of the functions of individual components through a combination of genetic, biochemical and structural approaches, primarily in prokaryotes and non-plant eukaryotes. Whereas most of the components of these machineries are conserved between kingdoms, their complexity is likely increased in plants owing to the presence of additional assembly proteins and to the existence of expanded families for several assembly proteins. This review focuses on the new actors discovered in the past few years, such as glutaredoxin, BOLA and NEET proteins as well as MIP18, MMS19, TAH18, DRE2 for the cytosolic machinery, which are integrated into a model for the plant Fe-S cluster biogenesis systems. It also discusses a few issues currently subjected to an intense debate such as the role of the mitochondrial frataxin and of glutaredoxins, the functional separation between scaffold, carrier and iron-delivery proteins and the crosstalk existing between different organelles. PMID:23898337

In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2.

PubMed

Hoff, Kevin G; Culler, Stephanie J; Nguyen, Peter Q; McGuire, Ryan M; Silberg, Jonathan J; Smolke, Christina D

2009-12-24

A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins. Copyright 2009 Elsevier Ltd. All rights reserved.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

PubMed

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

PubMed Central

Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

2018-01-01

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle. PMID:29491838

Divergence of Erv1-Associated Mitochondrial Import and Export Pathways in Trypanosomes and Anaerobic Protists

PubMed Central

Basu, Somsuvro; Leonard, Joanne C.; Desai, Nishal; Mavridou, Despoina A. I.; Tang, Kong Ho; Goddard, Alan D.

2013-01-01

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O2, unexpectedly find O2 and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O2 by TbERV1 is not dependent upon a simple O2 channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes. PMID:23264646

Simultaneous depletion of Atm and Mdl rebalances cytosolic Fe-S cluster assembly but not heme import into the mitochondrion of Trypanosoma brucei.

PubMed

Horáková, Eva; Changmai, Piya; Paris, Zdeněk; Salmon, Didier; Lukeš, Julius

2015-11-01

ABC transporter mitochondrial 1 (Atm1) and multidrug resistance-like 1 (Mdl) are mitochondrial ABC transporters. Although Atm1 was recently suggested to transport different forms of glutathione from the mitochondrion, which are used for iron-sulfur (Fe-S) cluster maturation in the cytosol, the function of Mdl remains elusive. In Trypanosoma brucei, we identified one homolog of each of these genes, TbAtm and TbMdl, which were downregulated either separately or simultaneously using RNA interference. Individual depletion of TbAtm and TbMdl led to limited growth defects. In cells downregulated for TbAtm, the enzymatic activities of the Fe-S cluster proteins aconitase and fumarase significantly decreased in the cytosol but not in the mitochondrion. Downregulation of TbMdl did not cause any change in activities of the Fe-S proteins. Unexpectedly, the simultaneous downregulation of TbAtm and TbMdl did not result in any growth defect, nor were the Fe-S cluster protein activities altered in either the cytosolic or mitochondrial compartments. Additionally, TbAtm and TbMdl were able to partially restore the growth of the Saccharomyces cerevisiae Δatm1 and Δmdl2 null mutants, respectively. Because T. brucei completely lost the heme b biosynthesis pathway, this cofactor has to be obtained from the host. Based on our results, TbMdl is a candidate for mitochondrial import of heme b, which was markedly decreased in both TbMdl and TbAtm + TbMdl knockdowns. Moreover, the levels of heme a were strongly decreased in the same knockdowns, suggesting that TbMdl plays a key role in heme a biosynthesis, thus affecting the overall heme homeostasis in T. brucei. © 2015 FEBS.

2008 GRC Iron Sulfur Enzymes-Conference to be held June 8-13, 2008

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cramer, Stephen; Gray, Nancy Ryan

2009-01-01

Iron-sulfur proteins are among the most common and ancient enzymes and electron-transfer agents in nature. They play key roles in photosynthesis, respiration, and the metabolism of small molecules such as H2, CO, and N2. The Iron Sulfur Enzyme Gordon Research Conference evolved from an earlier GRC on Nitrogen Fixation that began in 1994. The scope of the current meeting has broadened to include all enzymes or metalloproteins in which Fe-S bonds play a key role. This year's meeting will focus on the biosynthesis of Fe-S clusters, as well as the structure and mechanism of key Fe-S enzymes such as hydrogenase,more » nitrogenase and its homologues, radical SAM enzymes, and aconitase-related enzymes. Recent progress on the role of Fe-S enzymes in health, disease, DNA/RNA-processing, and alternative bio-energy systems will also be highlighted. This conference will assemble a broad, diverse, and international group of biologists and chemists who are investigating fundamental issues related to Fe-S enzymes, on atomic, molecular, organism, and environmental scales. The topics to be addressed will include: Biosynthesis & Genomics of Fe-S Enzymes; Fundamental Fe-S Chemistry; Hydrogen and Fe-S Enzymes; Nitrogenase & Homologous Fe-S Enzymes; Fe-S Enzymes in Health & Disease; Radical SAM and Aconitase-Related Fe-S Enzymes; Fe-S Enzymes and Synthetic Analogues in BioEnergy; and Fe-S Enzymes in Geochemistry and the Origin of Life.« less

Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.

PubMed

Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth Anne

2012-06-26

Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical iron-requiring processes under changing environmental conditions.

Fe-S Clusters Emerging as Targets of Therapeutic Drugs

PubMed Central

2017-01-01

Fe-S centers exhibit strong electronic plasticity, which is of importance for insuring fine redox tuning of protein biological properties. In accordance, Fe-S clusters are also highly sensitive to oxidation and can be very easily altered in vivo by different drugs, either directly or indirectly due to catabolic by-products, such as nitric oxide species (NOS) or reactive oxygen species (ROS). In case of metal ions, Fe-S cluster alteration might be the result of metal liganding to the coordinating sulfur atoms, as suggested for copper. Several drugs presented through this review are either capable of direct interaction with Fe-S clusters or of secondary Fe-S clusters alteration following ROS or NOS production. Reactions leading to Fe-S cluster disruption are also reported. Due to the recent interest and progress in Fe-S biology, it is very likely that an increasing number of drugs already used in clinics will emerge as molecules interfering with Fe-S centers in the near future. Targeting Fe-S centers could also become a promising strategy for drug development. PMID:29445445

How Is Fe-S Cluster Formation Regulated?

PubMed

Mettert, Erin L; Kiley, Patricia J

2015-01-01

Iron-sulfur (Fe-S) clusters are fundamental to numerous biological processes in most organisms, but these protein cofactors can be prone to damage by various oxidants (e.g., O2, reactive oxygen species, and reactive nitrogen species) and toxic levels of certain metals (e.g., cobalt and copper). Furthermore, their synthesis can also be directly influenced by the level of available iron in the environment. Consequently, the cellular need for Fe-S cluster biogenesis varies with fluctuating growth conditions. To accommodate changes in Fe-S demand, microorganisms employ diverse regulatory strategies to tailor Fe-S cluster biogenesis according to their surroundings. Here, we review the mechanisms that regulate Fe-S cluster formation in bacteria, primarily focusing on control of the Isc and Suf Fe-S cluster biogenesis systems in the model bacterium Escherichia coli.

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein (ISCU) Mutation Leads to Development of Mitochondrial Myopathy*

PubMed Central

Saha, Prasenjit Prasad; Kumar, S. K. Praveen; Srivastava, Shubhi; Sinha, Devanjan; Pareek, Gautam; D'Silva, Patrick

2014-01-01

Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044G→C), compound heterozygous patients with severe myopathy have been identified to carry the c.149G→A missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms. PMID:24573684

Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System.

PubMed

Ozer, Hatice K; Dlouhy, Adrienne C; Thornton, Jeremy D; Hu, Jingjing; Liu, Yilin; Barycki, Joseph J; Balk, Janneke; Outten, Caryn E

2015-11-13

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Impact of mutations within the [Fe-S] cluster or the lipoic acid biosynthesis pathways on mitochondrial protein expression profiles in fibroblasts from patients.

PubMed

Lebigot, E; Gaignard, P; Dorboz, I; Slama, A; Rio, M; de Lonlay, P; Héron, B; Sabourdy, F; Boespflug-Tanguy, O; Cardoso, A; Habarou, F; Ottolenghi, C; Thérond, P; Bouton, C; Golinelli-Cohen, M P; Boutron, A

2017-11-01

Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron‑sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy.

PubMed

Albetel, Angela-Nadia; Outten, Caryn E

2018-01-01

Monothiol glutaredoxins (Grxs) with a conserved Cys-Gly-Phe-Ser (CGFS) active site are iron-sulfur (Fe-S) cluster-binding proteins that interact with a variety of partner proteins and perform crucial roles in iron metabolism including Fe-S cluster transfer, Fe-S cluster repair, and iron signaling. Various analytical and spectroscopic methods are currently being used to monitor and characterize glutaredoxin Fe-S cluster-dependent interactions at the molecular level. The electronic, magnetic, and vibrational properties of the protein-bound Fe-S cluster provide a convenient handle to probe the structure, function, and coordination chemistry of Grx complexes. However, some limitations arise from sample preparation requirements, complexity of individual techniques, or the necessity for combining multiple methods in order to achieve a complete investigation. In this chapter, we focus on the use of UV-visible circular dichroism spectroscopy as a fast and simple initial approach for investigating glutaredoxin Fe-S cluster-dependent interactions. © 2018 Elsevier Inc. All rights reserved.

Structure and functional dynamics of the mitochondrial Fe/S cluster synthesis complex.

PubMed

Boniecki, Michal T; Freibert, Sven A; Mühlenhoff, Ulrich; Lill, Roland; Cygler, Miroslaw

2017-11-03

Iron-sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status.

Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway.

PubMed

Boyd, Eric S; Thomas, Khaleh M; Dai, Yuyuan; Boyd, Jeff M; Outten, F Wayne

2014-09-23

Iron-sulfur (Fe-S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe-S clusters and the fundamental requirement for Fe-S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth's atmosphere. Intriguingly, Fe-S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe-S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe-S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe-S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB-SufC scaffold complex. This analysis provides a new framework for the study of Fe-S cluster biogenesis pathways and Fe-S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen.

A-Type Carrier Protein ErpA Is Essential for Formation of an Active Formate-Nitrate Respiratory Pathway in Escherichia coli K-12

PubMed Central

Pinske, Constanze

2012-01-01

A-type carrier (ATC) proteins of the Isc (iron-sulfur cluster) and Suf (sulfur mobilization) iron-sulfur ([Fe-S]) cluster biogenesis pathways are proposed to traffic preformed [Fe-S] clusters to apoprotein targets. In this study, we analyzed the roles of the ATC proteins ErpA, IscA, and SufA in the maturation of the nitrate-inducible, multisubunit anaerobic respiratory enzymes formate dehydrogenase N (Fdh-N) and nitrate reductase (Nar). Mutants lacking SufA had enhanced activities of both enzymes. While both Fdh-N and Nar activities were strongly reduced in an iscA mutant, both enzymes were inactive in an erpA mutant and in a mutant unable to synthesize the [Fe-S] cluster scaffold protein IscU. It could be shown for both Fdh-N and Nar that loss of enzyme activity correlated with absence of the [Fe-S] cluster-containing small subunit. Moreover, a slowly migrating form of the catalytic subunit FdnG of Fdh-N was observed, consistent with impeded twin arginine translocation (TAT)-dependent transport. The highly related Fdh-O enzyme was also inactive in the erpA mutant. Although the Nar enzyme has its catalytic subunit NarG localized in the cytoplasm, it also exhibited aberrant migration in an erpA iscA mutant, suggesting that these modular enzymes lack catalytic integrity due to impaired cofactor biosynthesis. Cross-complementation experiments demonstrated that multicopy IscA could partially compensate for lack of ErpA with respect to Fdh-N activity but not Nar activity. These findings suggest that ErpA and IscA have overlapping roles in assembly of these anaerobic respiratory enzymes but demonstrate that ErpA is essential for the production of active enzymes. PMID:22081393

Tumor-initiating cells of breast and prostate origin show alterations in the expression of genes related to iron metabolism

PubMed Central

Tomkova, Veronika; Korenkova, Vlasta; Langerova, Lucie; Simonova, Ekaterina; Zjablovskaja, Polina; Alberich-Jorda, Meritxell; Neuzil, Jiri; Truksa, Jaroslav

2017-01-01

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy. PMID:28031527

The intermembrane space protein Erv1 of Trypanosoma brucei is essential for mitochondrial Fe-S cluster assembly and operates alone.

PubMed

Haindrich, Alexander C; Boudová, Michala; Vancová, Marie; Diaz, Priscila Peña; Horáková, Eva; Lukeš, Julius

2017-06-01

Sulfhydryl oxidase Erv1 is a ubiquitous and conserved protein of the mitochondrial intermembrane space that plays a role in the transport of small sulfur-containing proteins. In higher eukaryotes, Erv1 interacts with the mitochondrial import protein Mia40. However, Trypanosoma brucei lacks an obvious Mia40 homologue in its genome. Here we show by tandem affinity purification and mass spectrometry that in this excavate protist, Erv1 functions without a Mia40 homologue and most likely any other interaction partner. Down-regulation of TbErv1 caused a reduction of the mitochondrial membrane potential already within 24h to less than 50% when compared with control cells. The depletion of TbErv1 was accompanied by accumulation of trCOIV precursor, with a concomitant reduction of aconitase activity both in the cytosol and mitochondrion. Overall, TbErv1 seems to have a role in the mitochondrial translocation and Fe-S cluster assembly in the organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation.

PubMed

Tong, Wing-Hang; Maio, Nunziata; Zhang, De-Liang; Palmieri, Erika M; Ollivierre, Hayden; Ghosh, Manik C; McVicar, Daniel W; Rouault, Tracey A

2018-05-22

Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)-activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)-mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders.

Metal Sulfide Cluster Complexes and their Biogeochemical Importance in the Environment

NASA Astrophysics Data System (ADS)

Luther, George W.; Rickard, David T.

2005-10-01

Aqueous clusters of FeS, ZnS and CuS constitute a major fraction of the dissolved metal load in anoxic oceanic, sedimentary, freshwater and deep ocean vent environments. Their ubiquity explains how metals are transported in anoxic environmental systems. Thermodynamic and kinetic considerations show that they have high stability in oxic aqueous environments, and are also a significant fraction of the total metal load in oxic river waters. Molecular modeling indicates that the clusters are very similar to the basic structural elements of the first condensed phase forming from aqueous solutions in the Fe-S, Zn-S and Cu-S systems. The structure of the first condensed phase is determined by the structure of the cluster in solution. This provides an alternative explanation of Ostwald's Rule, where the most soluble, metastable phases form before the stable phases. For example, in the case of FeS, we showed that the first condensed phase is nanoparticulate, metastable mackinawite with a particle size of 2 nm consisting of about 150 FeS subunits, representing the end of a continuum between aqueous FeS clusters and condensed material. These metal sulfide clusters and nanoparticles are significant in biogeochemistry. Metal sulfide clusters reduce sulfide and metal toxicity and help drive ecology. FeS cluster formation drives vent ecology and AgS cluster formation detoxifies Ag in Daphnia magna neonates. We also note a new reaction between FeS and DNA and discuss the potential role of FeS clusters in denaturing DNA.

Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET.

PubMed

Mons, Cécile; Ferecatu, Ioana; Riquier, Sylvie; Lescop, Ewen; Bouton, Cécile; Golinelli-Cohen, Marie-Pierre

2017-01-01

MitoNEET is the first identified Fe-S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe-2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe-S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe-S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe-S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe-S cluster in an Fe-S repair process. This chapter describes methods to study in vitro mitoNEET Fe-S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods. © 2017 Elsevier Inc. All rights reserved.

The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation.

PubMed

Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

2016-05-19

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

PubMed Central

Wang, Xiaokang; Li, Qi; Yuan, Wei; Cao, Zhendong; Qi, Bei; Kumar, Suresh; Li, Yan; Qian, Weiqiang

2016-01-01

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis. PMID:27193999

TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation

PubMed Central

Maio, Nunziata; Palmieri, Erika M.; Ollivierre, Hayden; Ghosh, Manik C.

2018-01-01

Given the essential roles of iron-sulfur (Fe-S) cofactors in mediating electron transfer in the mitochondrial respiratory chain and supporting heme biosynthesis, mitochondrial dysfunction is a common feature in a growing list of human Fe-S cluster biogenesis disorders, including Friedreich ataxia and GLRX5-related sideroblastic anemia. Here, our studies showed that restriction of Fe-S cluster biogenesis not only compromised mitochondrial oxidative metabolism but also resulted in decreased overall histone acetylation and increased H3K9me3 levels in the nucleus and increased acetylation of α-tubulin in the cytosol by decreasing the lipoylation of the pyruvate dehydrogenase complex, decreasing levels of succinate dehydrogenase and the histone acetyltransferase ELP3, and increasing levels of the tubulin acetyltransferase MEC17. Previous studies have shown that the metabolic shift in Toll-like receptor (TLR)–activated myeloid cells involves rapid activation of glycolysis and subsequent mitochondrial respiratory failure due to nitric oxide (NO)–mediated damage to Fe-S proteins. Our studies indicated that TLR activation also actively suppresses many components of the Fe-S cluster biogenesis machinery, which exacerbates NO-mediated damage to Fe-S proteins by interfering with cluster recovery. These results reveal new regulatory pathways and novel roles of the Fe-S cluster biogenesis machinery in modifying the epigenome and acetylome and provide new insights into the etiology of Fe-S cluster biogenesis disorders. PMID:29784770

Monomeric Yeast Frataxin is an Iron-Binding Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook,J.; Bencze, K.; Jankovic, A.

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly)more » share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Monomeric Yeast Frataxin is an Iron Binding Protein†

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, J.; Bencze, K; Jankovic, A

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) sharemore » requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.« less

Sulfur mobilization for Fe-S cluster assembly by the essential SUF pathway in the Plasmodium falciparum apicoplast and its inhibition.

PubMed

Charan, Manish; Singh, Nidhi; Kumar, Bijay; Srivastava, Kumkum; Siddiqi, Mohammad Imran; Habib, Saman

2014-06-01

The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis.

PubMed

Garcia-Serres, Ricardo; Clémancey, Martin; Latour, Jean-Marc; Blondin, Geneviève

2018-01-19

Fe/S cluster biogenesis involves a complex machinery comprising several mitochondrial and cytosolic proteins. Fe/S cluster biosynthesis is closely intertwined with iron trafficking in the cell. Defects in Fe/S cluster elaboration result in severe diseases such as Friedreich ataxia. Deciphering this machinery is a challenge for the scientific community. Because iron is a key player, 57 Fe-Mössbauer spectroscopy is especially appropriate for the characterization of Fe species and monitoring the iron distribution. This minireview intends to illustrate how Mössbauer spectroscopy contributes to unravel steps in Fe/S cluster biogenesis. Studies were performed on isolated proteins that may be present in multiple protein complexes. Since a few decades, Mössbauer spectroscopy was also performed on whole cells or on isolated compartments such as mitochondria and vacuoles, affording an overview of the iron trafficking. This minireview aims at presenting selected applications of 57 Fe-Mössbauer spectroscopy to Fe/S cluster biogenesis.

Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus

PubMed Central

Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Mike, Laura A.; Skaar, Eric P.; Boyd, Jeffrey M.

2015-01-01

Summary Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activity of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus. PMID:26135358

Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus.

PubMed

Rosario-Cruz, Zuelay; Chahal, Harsimranjit K; Mike, Laura A; Skaar, Eric P; Boyd, Jeffrey M

2015-10-01

Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus. © 2015 John Wiley & Sons Ltd.

Monothiol glutaredoxins and A-type proteins: partners in Fe-S cluster trafficking.

PubMed

Mapolelo, Daphne T; Zhang, Bo; Randeniya, Sajini; Albetel, Angela-Nadia; Li, Haoran; Couturier, Jérémy; Outten, Caryn E; Rouhier, Nicolas; Johnson, Michael K

2013-03-07

Monothiol glutaredoxins (Grxs) are proposed to function in Fe-S cluster storage and delivery, based on their ability to exist as apo monomeric forms and dimeric forms containing a subunit-bridging [Fe(2)S(2)](2+) cluster, and to accept [Fe(2)S(2)](2+) clusters from primary scaffold proteins. In addition yeast cytosolic monothiol Grxs interact with Fra2 (Fe repressor of activation-2), to form a heterodimeric complex with a bound [Fe(2)S(2)](2+) cluster that plays a key role in iron sensing and regulation of iron homeostasis. In this work, we report on in vitro UV-visible CD studies of cluster transfer between homodimeric monothiol Grxs and members of the ubiquitous A-type class of Fe-S cluster carrier proteins ((Nif)IscA and SufA). The results reveal rapid, unidirectional, intact and quantitative cluster transfer from the [Fe(2)S(2)](2+) cluster-bound forms of A. thaliana GrxS14, S. cerevisiae Grx3, and A. vinelandii Grx-nif homodimers to A. vinelandii(Nif)IscA and from A. thaliana GrxS14 to A. thaliana SufA1. Coupled with in vivo evidence for interaction between monothiol Grxs and A-type Fe-S cluster carrier proteins, the results indicate that these two classes of proteins work together in cellular Fe-S cluster trafficking. However, cluster transfer is reversed in the presence of Fra2, since the [Fe(2)S(2)](2+) cluster-bound heterodimeric Grx3-Fra2 complex can be formed by intact [Fe(2)S(2)](2+) cluster transfer from (Nif)IscA. The significance of these results for Fe-S cluster biogenesis or repair and the cellular regulation of the Fe-S cluster status are discussed.

Biogenesis of iron-sulfur clusters in mammalian cells: new insights and relevance to human disease

PubMed Central

Rouault, Tracey A.

2012-01-01

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors composed of iron and inorganic sulfur. They are required for the function of proteins involved in a wide range of activities, including electron transport in respiratory chain complexes, regulatory sensing, photosynthesis and DNA repair. The proteins involved in the biogenesis of Fe-S clusters are evolutionarily conserved from bacteria to humans, and many insights into the process of Fe-S cluster biogenesis have come from studies of model organisms, including bacteria, fungi and plants. It is now clear that several rare and seemingly dissimilar human diseases are attributable to defects in the basic process of Fe-S cluster biogenesis. Although these diseases –which include Friedreich’s ataxia (FRDA), ISCU myopathy, a rare form of sideroblastic anemia, an encephalomyopathy caused by dysfunction of respiratory chain complex I and multiple mitochondrial dysfunctions syndrome – affect different tissues, a feature common to many of them is that mitochondrial iron overload develops as a secondary consequence of a defect in Fe-S cluster biogenesis. This Commentary outlines the basic steps of Fe-S cluster biogenesis as they have been defined in model organisms. In addition, it draws attention to refinements of the process that might be specific to the subcellular compartmentalization of Fe-S cluster biogenesis proteins in some eukaryotes, including mammals. Finally, it outlines several important unresolved questions in the field that, once addressed, should offer important clues into how mitochondrial iron homeostasis is regulated, and how dysfunction in Fe-S cluster biogenesis can contribute to disease. PMID:22382365

A Sinorhizobium meliloti RpoH-Regulated Gene Is Involved in Iron-Sulfur Protein Metabolism and Effective Plant Symbiosis under Intrinsic Iron Limitation.

PubMed

Sasaki, Shohei; Minamisawa, Kiwamu; Mitsui, Hisayuki

2016-09-01

In Sinorhizobium meliloti, RpoH-type sigma factors have a global impact on gene expression during heat shock and play an essential role in symbiosis with leguminous plants. Using mutational analysis of a set of genes showing highly RpoH-dependent expression during heat shock, we identified a gene indispensable for effective symbiosis. This gene, designated sufT, was located downstream of the sufBCDS homologs that specify the iron-sulfur (Fe/S) cluster assembly pathway. The identified transcription start site was preceded by an RpoH-dependent promoter consensus sequence. SufT was related to a conserved protein family of unknown molecular function, of which some members are involved in Fe/S cluster metabolism in diverse organisms. A sufT mutation decreased bacterial growth in both rich and minimal media, tolerance to stresses such as iron starvation, and activities of some Fe/S cluster-dependent enzymes. These results support the involvement of SufT in SUF (sulfur mobilization) system-mediated Fe/S protein metabolism. Furthermore, we isolated spontaneous pseudorevertants of the sufT mutant with partially recovered growth; each of them had a mutation in rirA This gene encodes a global iron regulator whose loss increases the intracellular iron content. Deletion of rirA in the original sufT mutant improved growth and restored Fe/S enzyme activities and effective symbiosis. These results suggest that enhanced iron availability compensates for the lack of SufT in the maintenance of Fe/S proteins. Although RpoH-type sigma factors of the RNA polymerase are present in diverse proteobacteria, their role as global regulators of protein homeostasis has been studied mainly in the enteric gammaproteobacterium Escherichia coli In the soil alphaproteobacterium Sinorhizobium meliloti, the rpoH mutations have a strong impact on symbiosis with leguminous plants. We found that sufT is a unique member of the S. meliloti RpoH regulon; sufT contributes to Fe/S protein metabolism and effective symbiosis under intrinsic iron limitation exerted by RirA, a global iron regulator. Our study provides insights into the RpoH regulon function in diverse proteobacteria adapted to particular ecological niches and into the mechanism of conserved Fe/S protein biogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Analysis of NFU-1 metallocofactor binding-site substitutions-impacts on iron-sulfur cluster coordination and protein structure and function.

PubMed

Wesley, Nathaniel A; Wachnowsky, Christine; Fidai, Insiya; Cowan, J A

2017-11-01

Iron-sulfur (Fe/S) clusters are ancient prosthetic groups found in numerous metalloproteins and are conserved across all kingdoms of life due to their diverse, yet essential functional roles. Genetic mutations to a specific subset of mitochondrial Fe/S cluster delivery proteins are broadly categorized as disease-related under multiple mitochondrial dysfunction syndrome (MMDS), with symptoms indicative of a general failure of the metabolic system. Multiple mitochondrial dysfunction syndrome 1 (MMDS1) arises as a result of the missense mutation in NFU1, an Fe/S cluster scaffold protein, which substitutes a glycine near the Fe/S cluster-binding pocket to a cysteine (p.Gly208Cys). This substitution has been shown to promote protein dimerization such that cluster delivery to NFU1 is blocked, preventing downstream cluster trafficking. However, the possibility of this additional cysteine, located adjacent to the cluster-binding site, serving as an Fe/S cluster ligand has not yet been explored. To fully understand the consequences of this Gly208Cys replacement, complementary substitutions at the Fe/S cluster-binding pocket for native and Gly208Cys NFU1 were made, along with six other variants. Herein, we report the results of an investigation on the effect of these substitutions on both cluster coordination and NFU1 structure and function. The data suggest that the G208C substitution does not contribute to cluster binding. Rather, replacement of the glycine at position 208 changes the oligomerization state as a result of global structural alterations that result in the downstream effects manifest as MMDS1, but does not perturb the coordination chemistry of the Fe-S cluster. © 2017 Federation of European Biochemical Societies.

Genetic approaches of the Fe-S cluster biogenesis process in bacteria: Historical account, methodological aspects and future challenges.

PubMed

Py, Béatrice; Barras, Frédéric

2015-06-01

Since their discovery in the 50's, Fe-S cluster proteins have attracted much attention from chemists, biophysicists and biochemists. However, in the 80's they were joined by geneticists who helped to realize that in vivo maturation of Fe-S cluster bound proteins required assistance of a large number of factors defining complex multi-step pathways. The question of how clusters are formed and distributed in vivo has since been the focus of much effort. Here we review how genetics in discovering genes and investigating processes as they unfold in vivo has provoked seminal advances toward our understanding of Fe-S cluster biogenesis. The power and limitations of genetic approaches are discussed. As a final comment, we argue how the marriage of classic strategies and new high-throughput technologies should allow genetics of Fe-S cluster biology to be even more insightful in the future. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of a Nfs1p-bound persulfide intermediate in Fe-S cluster synthesis by intact mitochondria.

PubMed

Pandey, Alok; Yoon, Heeyong; Lyver, Elise R; Dancis, Andrew; Pain, Debkumar

2012-09-01

Cysteine desulfurases generate a covalent persulfide intermediate from cysteine, and this activated form of sulfur is essential for the synthesis of iron-sulfur (Fe-S) clusters. In yeast mitochondria, there is a complete machinery for Fe-S cluster synthesis, including a cysteine desulfurase, Nfs1p. Here we show that following supplementation of isolated mitochondria with [(35)S]cysteine, a radiolabeled persulfide could be detected on Nfs1p. The persulfide persisted under conditions that did not permit Fe-S cluster formation, such as nucleotide and/or iron depletion of mitochondria. By contrast, under permissive conditions, the radiolabeled Nfs1p persulfide was greatly reduced and radiolabeled aconitase was formed, indicating transfer of persulfide to downstream Fe-S cluster recipients. Nfs1p in mitochondria was found to be relatively more resistant to inactivation by N-ethylmaleimide (NEM) as compared with a prokaryotic cysteine desulfurase. Mitochondria treated with NEM (1 mM) formed the persulfide on Nfs1p but failed to generate Fe-S clusters on aconitase, likely due to inactivation of downstream recipient(s) of the Nfs1p persulfide. Thus the Nfs1p-bound persulfide as described here represents a precursor en route to Fe-S cluster synthesis in mitochondria. Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Properties of iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide cluster anions through photoelectron spectroscopy and density functional theory calculations.

PubMed

Yin, Shi; Bernstein, Elliot R

2016-10-21

A new magnetic-bottle time-of-flight photoelectron spectroscopy (PES) apparatus is constructed in our laboratory. The PES spectra of iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide [FeS m (SH) n - ; m, n = 0-3, 0 < (m + n) ≤ 3] cluster anions, obtained at 2.331 eV (532 nm) and 3.492 eV (355 nm) photon energies, are reported. The electronic structure and bonding properties of these clusters are additionally investigated at different levels of density functional theory. The most probable structures and ground state spin multiplicity for these cluster anions are tentatively assigned by comparing their theoretical first vertical detachment energies (VDEs) with their respective experiment values. The behavior of S and (SH) as ligands in these iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide cluster anions is investigated and compared. The experimental first VDEs for Fe(SH) 1-3 - cluster anions are lower than those found for their respective FeS 1-3 - cluster anions. The experimental first VDEs for FeS 1-3 - clusters are observed to increase for the first two S atoms bound to Fe - ; however, due to the formation of an S-S bond for the FeS 3 - cluster, its first VDE is found to be ∼0.41 eV lower than the first VDE for the FeS 2 - cluster. The first VDEs of Fe(SH) 1-3 - cluster anions are observed to increase with the increasing numbers of SH groups. The calculated partial charges of the Fe atom for ground state FeS 1-3 - and Fe(SH) 1-3 - clusters are apparently related to and correlated with their determined first VDEs. The higher first VDE is correlated with a higher, more positive partial charge for the Fe atom of these cluster anions. Iron sulfide/hydrosulfide mixed cluster anions are also explored in this work: the first VDE for FeS(SH) - is lower than that for FeS 2 - , but higher than that for Fe(SH) 2 - ; the first VDEs for FeS 2 (SH) - and FeS(SH) 2 - are close to that for FeS 3 - , but higher than that for Fe(SH) 3 - . The first VDEs of general iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide clusters [FeS m (SH) n - ; m, n = 0-3, 0 < (m + n) ≤ 3] are dependent on three properties of these anions: 1. the partial charge on the Fe atom, 2. disulfide bond formation (S-S) in the cluster, and 3. the number of hydrosulfide ligands in the cluster. The higher the partial charge on the Fe atom of these clusters, the larger the first VDE; however, cluster S-S bonding and more (SH) ligands in the cluster lower the cluster anion first VDE.

Fe-S Proteins that Regulate Gene Expression

PubMed Central

Mettert, Erin L.; Kiley, Patricia J.

2014-01-01

Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

Cysteine 295 indirectly affects Ni coordination of carbon monoxide dehydrogenase-II C-cluster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inoue, Takahiro; Takao, Kyosuke; Yoshida, Takashi

2013-11-08

Highlights: •CODH-II harbors a unique [Ni-Fe-S] cluster. •We substituted the ligand residues of Cys{sup 295} and His{sup 261}. •Dramatic decreases in Ni content upon substitutions were observed. •All substitutions did not affect Fe-S clusters assembly. •CO oxidation activity was decreased by the substitutions. -- Abstract: A unique [Ni–Fe–S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His{sup 261}, which coordinates one of the Fe atoms with Cys{sup 295}, is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys{sup 295}, we constructed CODH-II variants. Ala substitution formore » the Cys{sup 295} substitution resulted in the decrease of Ni content and didn’t result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe–S] clusters. This strongly suggests Cys{sup 295} indirectly and His{sup 261} together affect Ni-coordination in the C-cluster.« less

The iron-binding CyaY and IscX proteins assist the ISC-catalyzed Fe-S biogenesis in Escherichia coli.

PubMed

Roche, Béatrice; Huguenot, Allison; Barras, Frédéric; Py, Béatrice

2015-02-01

In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss of iron-sulfur (Fe-S) cluster protein activity, accumulation of mitochondrial iron and leads to the neurodegenerative disease Friedreich's ataxia. In contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not to cause any significant phenotype, questioning both its importance and its actual contribution to Fe-S cluster biogenesis. Because FXN is conserved between eukaryotes and prokaryotes, this surprising discrepancy prompted us to reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i) potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the maturation of Fe-S cluster-containing proteins and (iii) requires iron-rich conditions for its contribution to be significant. A genetic interaction was discovered between cyaY and iscX, the last gene of the isc operon. Deletion of both genes showed an additive effect on Fe-S cluster protein maturation, which led, among others, to increased resistance to aminoglycosides and increased sensitivity to lambda phage infection. Together, these in vivo results establish the importance of CyaY as a member of the ISC-mediated Fe-S cluster biogenesis pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona fide Fe-S cluster biogenesis factor. © 2014 John Wiley & Sons Ltd.

Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway.

PubMed

Ezraty, Benjamin; Vergnes, Alexandra; Banzhaf, Manuel; Duverger, Yohann; Huguenot, Allison; Brochado, Ana Rita; Su, Shu-Yi; Espinosa, Leon; Loiseau, Laurent; Py, Béatrice; Typas, Athanasios; Barras, Frédéric

2013-06-28

All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus

PubMed Central

Mashruwala, Ameya A.; Pang, Yun Y.; Rosario-Cruz, Zuelay; Chahal, Harsimranjit K.; Benson, Meredith A.; Anzaldi-Mike, Laura L.; Skaar, Eric P.; Torres, Victor J.; Nauseef, William M.; Boyd, Jeffrey M.

2015-01-01

Summary The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as a Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target. PMID:25388433

Tracing the `ninth sulfur' of the nitrogenase cofactor via a semi-synthetic approach

NASA Astrophysics Data System (ADS)

Tanifuji, Kazuki; Lee, Chi Chung; Sickerman, Nathaniel S.; Tatsumi, Kazuyuki; Ohki, Yasuhiro; Hu, Yilin; Ribbe, Markus W.

2018-05-01

The M-cluster is the [(homocitrate)MoFe7S9C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe4S4] clusters concomitant with the insertion of an interstitial carbon and a `ninth sulfur'. Combining synthetic [Fe4S4] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-l-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe8S8C] cluster intermediate, the L*-cluster, which is similar to the [Fe8S9C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.

Tracing the 'ninth sulfur' of the nitrogenase cofactor via a semi-synthetic approach.

PubMed

Tanifuji, Kazuki; Lee, Chi Chung; Sickerman, Nathaniel S; Tatsumi, Kazuyuki; Ohki, Yasuhiro; Hu, Yilin; Ribbe, Markus W

2018-05-01

The M-cluster is the [(homocitrate)MoFe 7 S 9 C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe 4 S 4 ] clusters concomitant with the insertion of an interstitial carbon and a 'ninth sulfur'. Combining synthetic [Fe 4 S 4 ] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-L-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe 8 S 8 C] cluster intermediate, the L*-cluster, which is similar to the [Fe 8 S 9 C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems.

Emerging critical roles of Fe-S clusters in DNA replication and repair

PubMed Central

Fuss, Jill O.; Tsai, Chi-Lin; Ishida, Justin P.; Tainer, John A.

2015-01-01

Fe-S clusters are partners in the origin of life that predate cells, acetyl-CoA metabolism, DNA, and the RNA world. The double helix solved the mystery of DNA replication by base pairing for accurate copying. Yet, for genome stability necessary to life, the double helix has equally important implications for damage repair. Here we examine striking advances that uncover Fe-S cluster roles both in copying the genetic sequence by DNA polymerases and in crucial repair processes for genome maintenance, as mutational defects cause cancer and degenerative disease. Moreover, we examine an exciting, controversial role for Fe-S clusters in a third element required for life – the long-range coordination and regulation of replication and repair events. By their ability to delocalize electrons over both Fe and S centers, Fe-S clusters have unbeatable features for protein conformational control and charge transfer via double-stranded DNA that may fundamentally transform our understanding of life, replication, and repair. PMID:25655665

Regulation of human Nfu activity in Fe-S cluster delivery-characterization of the interaction between Nfu and the HSPA9/Hsc20 chaperone complex.

PubMed

Wachnowsky, Christine; Liu, Yushi; Yoon, Taejin; Cowan, J A

2018-01-01

Iron-sulfur cluster biogenesis is a complex, but highly regulated process that involves de novo cluster formation from iron and sulfide ions on a scaffold protein, and subsequent delivery to final targets via a series of Fe-S cluster-binding carrier proteins. The process of cluster release from the scaffold/carrier for transfer to the target proteins may be mediated by a dedicated Fe-S cluster chaperone system. In human cells, the chaperones include heat shock protein HSPA9 and the J-type chaperone Hsc20. While the role of chaperones has been somewhat clarified in yeast and bacterial systems, many questions remain over their functional roles in cluster delivery and interactions with a variety of human Fe-S cluster proteins. One such protein, Nfu, has recently been recognized as a potential interaction partner of the chaperone complex. Herein, we examined the ability of human Nfu to function as a carrier by interacting with the human chaperone complex. Human Nfu is shown to bind to both chaperone proteins with binding affinities similar to those observed for IscU binding to the homologous HSPA9 and Hsc20, while Nfu can also stimulate the ATPase activity of HSPA9. Additionally, the chaperone complex was able to promote Nfu function by enhancing the second-order rate constants for Fe-S cluster transfer to target proteins and providing directionality in cluster transfer from Nfu by eliminating promiscuous transfer reactions. Together, these data support a hypothesis in which Nfu can serve as an alternative carrier protein for chaperone-mediated cluster release and delivery in Fe-S cluster biogenesis and trafficking. © 2017 Federation of European Biochemical Societies.

Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P

2012-08-01

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-Smore » cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k cat/K M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k cat/K M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.« less

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

PubMed

Nuñez, Nicole N; Majumdar, Chandrima; Lay, Kori T; David, Sheila S

2018-01-01

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S] 2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease. © 2018 Elsevier Inc. All rights reserved.

Thermodynamic performance testing of the orbiter flash evaporator system

NASA Technical Reports Server (NTRS)

Jaax, J. R.; Melgares, M. A.; Frahm, J. P.

1980-01-01

System level testing of the space shuttle orbiter's development flash evaporator system (FES) was performed in a thermal vacuum chamber capable of simulating ambient ascent, orbital, and entry temperature and pressure profiles. The test article included the evaporator assembly, high load and topping exhaust duct and nozzle assemblies, and feedwater supply assembly. Steady state and transient heat load, water pressure/temperature and ambient pressure/temperature profiles were imposed by especially designed supporting test hardware. Testing in 1978 verified evaporator and duct heater thermal design, determined FES performance boundaries, and assessed topping evaporator plume characteristics. Testing in 1979 combined the FES with the other systems in the orbiter active thermal control subsystem (ATCS). The FES met or exceeded all nominal and contingency performance requirements during operation with the integrated ATCS. During both tests stability problems were encountered during steady state operations which resulted in subsequent design changes to the water spray nozzle and valve plate assemblies.

Tangled web of interactions among proteins involved in iron–sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies☆

PubMed Central

Kim, Jin Hae; Bothe, Jameson R.; Alderson, T. Reid; Markley, John L.

2014-01-01

Proteins containing iron–sulfur (Fe–S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe–S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron sulfur cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins and small-angle X-ray scattering (SAXS), chemical crosslinking experiments, and functional assays, we have constructed working models for Fe–S cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe–S cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. PMID:25450980

Spectroscopic and functional characterization of iron-sulfur cluster-bound forms of Azotobacter vinelandii (Nif)IscA.

PubMed

Mapolelo, Daphne T; Zhang, Bo; Naik, Sunil G; Huynh, Boi Hanh; Johnson, Michael K

2012-10-16

The mechanism of [4Fe-4S] cluster assembly on A-type Fe-S cluster assembly proteins, in general, and the specific role of (Nif)IscA in the maturation of nitrogen fixation proteins are currently unknown. To address these questions, in vitro spectroscopic studies (UV-visible absorption/CD, resonance Raman and Mössbauer) have been used to investigate the mechanism of [4Fe-4S] cluster assembly on Azotobacter vinelandii(Nif)IscA, and the ability of (Nif)IscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that (Nif)IscA can rapidly and reversibly cycle between forms containing one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per homodimer via DTT-induced two-electron reductive coupling of two [2Fe-2S](2+) clusters and O(2)-induced [4Fe-4S](2+) oxidative cleavage. This unique type of cluster interconversion in response to cellular redox status and oxygen levels is likely to be important for the specific role of A-type proteins in the maturation of [4Fe-4S] cluster-containing proteins under aerobic growth or oxidative stress conditions. Only the [4Fe-4S](2+)-(Nif)IscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-(Nif)IscA was shown to accept clusters from [4Fe-4S] cluster-bound NifU via rapid intact cluster transfer, indicating a potential role as a cluster carrier for delivery of clusters assembled on NifU. Overall the results support the proposal that A-type proteins can function as carrier proteins for clusters assembled on U-type proteins and suggest that they are likely to supply [2Fe-2S] clusters rather than [4Fe-4S] for the maturation of [4Fe-4S] cluster-containing proteins under aerobic or oxidative stress growth conditions.

Spectroscopic and Functional Characterization of Iron-Sulfur Cluster-Bound Forms of Azotobacter vinelandii NifIscA†

PubMed Central

Mapolelo, Daphne T.; Zhang, Bo; Naik, Sunil G.; Huynh, Boi Hanh; Johnson, Michael K.

2012-01-01

The mechanism of [4Fe-4S] cluster assembly on A-type Fe-S cluster assembly proteins, in general, and the specific role of NifIscA in the maturation of nitrogen fixation proteins are currently unknown. To address these questions, in vitro spectroscopic studies (UV–visible absorption/CD, resonance Raman and Mössbauer) have been used to investigate the mechanism of [4Fe-4S] cluster assembly on Azotobacter vinelandii NifIscA, and the ability of NifIscA to accept clusters from NifU and to donate clusters to the apo form of the nitrogenase Fe-protein. The results show that NifIscA can rapidly and reversibly cycle between forms containing one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster per homodimer via DTT-induced two-electron reductive coupling of two [2Fe-2S]2+ clusters and O2-induced [4Fe-4S]2+ oxidative cleavage. This unique type of cluster interconversion in response to cellular redox status and oxygen levels is likely to be important for the specific role of A-type proteins in the maturation of [4Fe-4S] cluster-containing proteins under aerobic growth or oxidative stress conditions. Only the [4Fe-4S]2+-NifIscA was competent for rapid activation of apo-nitrogenase Fe protein under anaerobic conditions. Apo-NifIscA was shown to accept clusters from [4Fe-4S] cluster-bound NifU via rapid intact cluster transfer, indicating a potential role as a cluster carrier for delivery of clusters assembled on NifU. Overall the results support the proposal that A-type proteins can function as carrier proteins for clusters assembled on U-type proteins and suggest that they are likely to supply [2Fe-2S] clusters rather than [4Fe-4S] for the maturation of [4Fe-4S] cluster-containing proteins under aerobic or oxidative stress growth conditions. PMID:23003323

Methionine sulphoxide reductases protect iron-sulphur clusters from oxidative inactivation in yeast

PubMed Central

Sideri, Theodora C.; Willetts, Sylvia A.; Avery, Simon V.

2008-01-01

Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of microorganisms. Here we show that methionine redox-modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrΔ double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe-transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild type reproduced the Cu-resistance phenotype, and FeS cluster functions were found to be defective in the mxrΔ mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55Fe-labeling studies showed that FeS clusters are turned over more rapidly in the mxrΔ mutant than the wild type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes, in helping to protect the essential function of FeS clusters in aerobic settings. PMID:19202110

Resonant photoemission study of pyrite-type NiS2, CoS2 and FeS2

NASA Astrophysics Data System (ADS)

Fujimori, A.; Mamiya, K.; Mizokawa, T.; Miyadai, T.; Sekiguchi, T.; Takahashi, H.; Môri, N.; Suga, S.

1996-12-01

The electronic structure of pyrite-type NiS2, CoS2, and FeS2 has been studied by photoemission spectroscopy. From resonant photoemission studies and configuration-interaction cluster-model analysis of the spectra, NiS2 is found to be a charge-transfer-type insulator, the band gap of which is formed between the occupied S 3p and the empty Ni 3d states. Cluster-model calculations indicate that the short Fe-S distance favors the low-spin (S=0) ground state in FeS2 compared to the high-spin FeS. Resonant photoemission results indicate a sign of electron correlation in the nonmagnetic semiconductor FeS2.

Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes.

PubMed

Tsai, Chi-Lin; Tainer, John A

2018-01-01

[Fe-S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe-S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe-S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe-S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe-S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe-S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure-activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe-S] cluster respiratory enzymes. © 2018 Elsevier Inc. All rights reserved.

Properties of iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide cluster anions through photoelectron spectroscopy and density functional theory calculations

NASA Astrophysics Data System (ADS)

Yin, Shi; Bernstein, Elliot R.

2016-10-01

A new magnetic-bottle time-of-flight photoelectron spectroscopy (PES) apparatus is constructed in our laboratory. The PES spectra of iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide [FeSm(SH)n-; m, n = 0-3, 0 < (m + n) ≤ 3] cluster anions, obtained at 2.331 eV (532 nm) and 3.492 eV (355 nm) photon energies, are reported. The electronic structure and bonding properties of these clusters are additionally investigated at different levels of density functional theory. The most probable structures and ground state spin multiplicity for these cluster anions are tentatively assigned by comparing their theoretical first vertical detachment energies (VDEs) with their respective experiment values. The behavior of S and (SH) as ligands in these iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide cluster anions is investigated and compared. The experimental first VDEs for Fe(SH)1-3- cluster anions are lower than those found for their respective FeS1-3- cluster anions. The experimental first VDEs for FeS1-3- clusters are observed to increase for the first two S atoms bound to Fe-; however, due to the formation of an S-S bond for the FeS3- cluster, its first VDE is found to be ˜0.41 eV lower than the first VDE for the FeS2- cluster. The first VDEs of Fe(SH)1-3- cluster anions are observed to increase with the increasing numbers of SH groups. The calculated partial charges of the Fe atom for ground state FeS1-3- and Fe(SH)1-3- clusters are apparently related to and correlated with their determined first VDEs. The higher first VDE is correlated with a higher, more positive partial charge for the Fe atom of these cluster anions. Iron sulfide/hydrosulfide mixed cluster anions are also explored in this work: the first VDE for FeS(SH)- is lower than that for FeS2-, but higher than that for Fe(SH)2-; the first VDEs for FeS2(SH)- and FeS(SH)2- are close to that for FeS3-, but higher than that for Fe(SH)3-. The first VDEs of general iron sulfide, hydrosulfide, and mixed sulfide/hydrosulfide clusters [FeSm(SH)n-; m, n = 0-3, 0 < (m + n) ≤ 3] are dependent on three properties of these anions: 1. the partial charge on the Fe atom, 2. disulfide bond formation (S-S) in the cluster, and 3. the number of hydrosulfide ligands in the cluster. The higher the partial charge on the Fe atom of these clusters, the larger the first VDE; however, cluster S-S bonding and more (SH) ligands in the cluster lower the cluster anion first VDE.

Understanding the role of dynamics in the iron sulfur cluster molecular machine.

PubMed

di Maio, Danilo; Chandramouli, Balasubramanian; Yan, Robert; Brancato, Giuseppe; Pastore, Annalisa

2017-01-01

The bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic FeS cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear. We carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface. We show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow FeS cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation. We conclude that the observed 'fluid' IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements. Our study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils.

PubMed

Roberts, Christina A; Al-Tameemi, Hassan M; Mashruwala, Ameya A; Rosario-Cruz, Zuelay; Chauhan, Unnati; Sause, William E; Torres, Victor J; Belden, William J; Boyd, Jeffrey M

2017-06-01

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf ( su l f ur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD ( sufD *), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD * strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD * strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development. Copyright © 2017 American Society for Microbiology.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils

PubMed Central

Roberts, Christina A.; Al-Tameemi, Hassan M.; Mashruwala, Ameya A.; Rosario-Cruz, Zuelay; Chauhan, Unnati; Sause, William E.; Torres, Victor J.; Belden, William J.

2017-01-01

ABSTRACT Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus. We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB. Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development. PMID:28320837

The ErpA/NfuA complex builds an oxidation-resistant Fe-S cluster delivery pathway.

PubMed

Py, Béatrice; Gerez, Catherine; Huguenot, Allison; Vidaud, Claude; Fontecave, Marc; Ollagnier de Choudens, Sandrine; Barras, Frédéric

2018-05-18

Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both in vitro and in vivo methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable ( t ½ = 100 min) than those bound to SufA ( t ½ = 55 min), ErpA ( t ½ = 54 min), or IscA ( t ½ = 45 min). Surprisingly, the presence of NfuA further enhanced stability of the ErpA-bound cluster to t ½ = 90 min. Using genetic and plasmon surface resonance analyses, we showed that NfuA and ErpA interacted directly with client proteins, whereas IscA or SufA did not. Moreover, NfuA and ErpA interacted with one another. Given all of these observations, we propose an architecture of the Fe-S delivery network in which ErpA is the last factor that delivers cluster directly to most if not all client proteins. NfuA is proposed to assist ErpA under severely unfavorable conditions. A comparison with the strategy employed in yeast and eukaryotes is discussed. © 2018 Py et al.

Mechanisms of iron sensing and regulation in the yeast Saccharomyces cerevisiae.

PubMed

Martínez-Pastor, María Teresa; Perea-García, Ana; Puig, Sergi

2017-04-01

Iron is a redox active element that functions as an essential cofactor in multiple metabolic pathways, including respiration, DNA synthesis and translation. While indispensable for eukaryotic life, excess iron can lead to oxidative damage of macromolecules. Therefore, living organisms have developed sophisticated strategies to optimally regulate iron acquisition, storage and utilization in response to fluctuations in environmental iron bioavailability. In the yeast Saccharomyces cerevisiae, transcription factors Aft1/Aft2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to producing and assembling iron cofactors, mitochondrial iron-sulfur (Fe/S) cluster biogenesis has emerged as a central player in iron sensing. A mitochondrial signal derived from Fe/S synthesis is exported and converted into an Fe/S cluster that interacts directly with Aft1/Aft2 and Yap5 proteins to regulate their transcriptional function. Various conserved proteins, such as ABC mitochondrial transporter Atm1 and, for Aft1/Aft2, monothiol glutaredoxins Grx3 and Grx4 are implicated in this iron-signaling pathway. The analysis of a wide range of S. cerevisiae strains of different geographical origins and sources has shown that yeast strains adapted to high iron display growth defects under iron-deficient conditions, and highlighted connections that exist in the response to both opposite conditions. Changes in iron accumulation and gene expression profiles suggest differences in the regulation of iron homeostasis genes.

Posttranslational control of the scaffold for Fe-S cluster biogenesis as a compensatory regulatory mechanism.

PubMed

Ciesielski, Szymon J; Craig, Elizabeth A

2017-02-01

Though toxic in excess, iron is vital for life. Thus, its use in all cells is tightly regulated. Analysis of Saccharomyces cerevisiae, which has been used extensively as a model system, has revealed layers of regulation of cellular iron trafficking and utilization. This regulation is based on the availability of both elemental iron and functionality of the Fe-S cluster biogenesis system. Here, we discuss a possible "first responder" regulatory mechanism centered on the stability of the scaffold protein on which Fe-S clusters are built.

Photoelectron Spectroscopy and Density Functional Theory Studies of Iron Sulfur (FeS)m- (m = 2-8) Cluster Anions: Coexisting Multiple Spin States.

PubMed

Yin, Shi; Bernstein, Elliot R

2017-10-05

Iron sulfur cluster anions (FeS) m - (m = 2-8) are studied by photoelectron spectroscopy (PES) at 3.492 eV (355 nm) and 4.661 eV (266 nm) photon energies, and by density functional theory (DFT) calculations. The most probable structures and ground state spin multiplicities for (FeS) m - (m = 2-8) clusters are tentatively assigned through a comparison of their theoretical and experiment first vertical detachment energy (VDE) values. Many spin states lie within 0.5 eV of the ground spin state for the larger (FeS) m - (m ≥ 4) clusters. Theoretical VDEs of these low lying spin states are in good agreement with the experimental VDE values. Therefore, multiple spin states of each of these iron sulfur cluster anions probably coexist under the current experimental conditions. Such available multiple spin states must be considered when evaluating the properties and behavior of these iron sulfur clusters in real chemical and biological systems. The experimental first VDEs of (FeS) m - (m = 1-8) clusters are observed to change with the cluster size (number m). The first VDE trends noted can be related to the different properties of the highest singly occupied molecular orbitals (NBO, HSOMOs) of each cluster anion. The changing nature of the NBO/HSOMO of these (FeS) m - (m = 1-8) clusters from a p orbital on S, to a d orbital on Fe, and to an Fe-Fe bonding orbital is probably responsible for the observed increasing trend for their first VDEs with respect to m.

The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria

PubMed Central

Yokoyama, Kenichi; Leimkühler, Silke

2016-01-01

Molybdenum is the only second row transition metal essential for biological systems, which is biologically available as molybdate ion. In eukarya, bacteria and archaea, molybdenum is bound to either to a tricyclic pyranopterin, thereby forming the molybdenum cofactor (Moco), or in some bacteria to the FeS cluster based iron-molybdenum cofactor (FeMoco), which forms the active site of nitrogenase. To date more than 50 Moco-containing enzymes have been purified and biochemically or structurally characterized. The physiological role of molybdenum in these enzymes is fundamental to organisms, since the reactions include the catalysis of key steps in carbon, nitrogen and sulfur metabolism. The catalyzed reactions are in most cases oxo-transfer reactions or the hydroxylation of carbon centers. The biosynthesis of Moco has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the biosynthesis and maturation of molybdoenzymes and the biosynthesis and distribution of FeS clusters has been identified in the last years: 1) The synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) The sulfurtransferase for the dithiolene group in Moco is common also for the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the modification of the active site with a sulfur atom additionally involves a sulfurtransferase, 4) most molybdoenzymes in bacteria require FeS clusters as additional redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. PMID:25268953

The Relationship between Environmental Dioxygen and Iron-Sulfur Proteins Explored at the Genome Level

PubMed Central

Andreini, Claudia; Rosato, Antonio; Banci, Lucia

2017-01-01

About 2 billion years ago, the atmosphere of the Earth experienced a great change due to the buildup of dioxygen produced by photosynthetic organisms. This transition caused a reduction of iron bioavailability and at the same time exposed living organisms to the threat of oxidative stress. Iron-sulfur (Fe-S) clusters require iron ions for their biosynthesis and are labile if exposed to reactive oxygen species. To assess how the above transition influenced the usage of Fe-S clusters by organisms, we compared the distribution of the Fe-S proteins encoded by the genomes of more than 400 prokaryotic organisms as a function of their dioxygen requirements. Aerobic organisms use less Fe-S proteins than the majority of anaerobic organisms with a similar genome size. Furthermore, aerobes have evolved specific Fe-S proteins that bind the less iron-demanding and more chemically stable Fe2S2 clusters while reducing the number of Fe4S4-binding proteins in their genomes. However, there is a shared core of Fe-S protein families composed mainly by Fe4S4-binding proteins. Members of these families are present also in humans. The distribution of human Fe-S proteins within cell compartments shows that mitochondrial proteins are inherited from prokaryotic proteins of aerobes, whereas nuclear and cytoplasmic Fe-S proteins are inherited from anaerobic organisms. PMID:28135316

A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery.

PubMed

Mandin, Pierre; Chareyre, Sylvia; Barras, Frédéric

2016-09-20

Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs). Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA) RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability. Regulatory small RNAs (sRNAs) have emerged as major actors in the control of gene expression in the last few decades. Relatively little is known about how these regulators interact with classical transcription factors to coordinate genetic responses. We show here how an sRNA, RyhB, and a transcription factor, IscR, regulate expression of an essential gene, erpA, in the bacterium E. coli ErpA is involved in the biogenesis of Fe-S clusters, an important class of cofactors involved in a plethora of cellular reactions. Interestingly, we show that RyhB and IscR repress expression of erpA under opposite conditions in regard to iron concentration, forming a regulatory circuit called an "incoherent network." This incoherent network serves to maximize expression of erpA at iron concentrations where it is most needed. Altogether, our study paves the way for a better understanding of mixed regulatory networks composed of RNAs and transcription factors. Copyright © 2016 Mandin et al.

Turning Escherichia coli into a Frataxin-Dependent Organism

PubMed Central

Roche, Béatrice; Agrebi, Rym; Huguenot, Allison; Ollagnier de Choudens, Sandrine; Barras, Frédéric; Py, Béatrice

2015-01-01

Fe-S bound proteins are ubiquitous and contribute to most basic cellular processes. A defect in the ISC components catalyzing Fe-S cluster biogenesis leads to drastic phenotypes in both eukaryotes and prokaryotes. In this context, the Frataxin protein (FXN) stands out as an exception. In eukaryotes, a defect in FXN results in severe defects in Fe-S cluster biogenesis, and in humans, this is associated with Friedreich’s ataxia, a neurodegenerative disease. In contrast, prokaryotes deficient in the FXN homolog CyaY are fully viable, despite the clear involvement of CyaY in ISC-catalyzed Fe-S cluster formation. The molecular basis of the differing importance in the contribution of FXN remains enigmatic. Here, we have demonstrated that a single mutation in the scaffold protein IscU rendered E. coli viability strictly dependent upon a functional CyaY. Remarkably, this mutation changed an Ile residue, conserved in prokaryotes at position 108, into a Met residue, conserved in eukaryotes. We found that in the double mutant IscUIM ΔcyaY, the ISC pathway was completely abolished, becoming equivalent to the ΔiscU deletion strain and recapitulating the drastic phenotype caused by FXN deletion in eukaryotes. Biochemical analyses of the “eukaryotic-like” IscUIM scaffold revealed that it exhibited a reduced capacity to form Fe-S clusters. Finally, bioinformatic studies of prokaryotic IscU proteins allowed us to trace back the source of FXN-dependency as it occurs in present-day eukaryotes. We propose an evolutionary scenario in which the current mitochondrial Isu proteins originated from the IscUIM version present in the ancestor of the Rickettsiae. Subsequent acquisition of SUF, the second Fe-S cluster biogenesis system, in bacteria, was accompanied by diminished contribution of CyaY in prokaryotic Fe-S cluster biogenesis, and increased tolerance to change in the amino acid present at the 108th position of the scaffold. PMID:25996492

Function and maturation of the Fe-S center in dihydroxyacid dehydratase from Arabidopsis.

PubMed

Gao, Huanyao; Azam, Tamanna; Randeniya, Sajini; Couturier, Jérémy; Rouhier, Nicolas; Johnson, Michael K

2018-03-23

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe 4 S 4 ] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe 2 S 2 ] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism and resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from Arabidopsis thaliana ( At ). Our results indicated that At DHAD can accommodate [Fe 2 S 2 ] and [Fe 4 S 4 ] clusters. However, only the [Fe 2 S 2 ] cluster-bound form is catalytically active. We found that the [Fe 2 S 2 ] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. In vitro cluster transfer and reconstitution reactions revealed that [Fe 2 S 2 ] cluster-containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of At DHAD. In summary, At DHAD binds either one [Fe 4 S 4 ] or one [Fe 2 S 2 ] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe 2 S 2 ] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant At DHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells.

PubMed

Fosset, Cédric; Chauveau, Marie-Jeanne; Guillon, Blanche; Canal, Frédéric; Drapier, Jean-Claude; Bouton, Cécile

2006-09-01

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.

A VTVH MCD and EPR Spectroscopic Study of the Maturation of the "Second" Nitrogenase P-Cluster.

PubMed

Rupnik, Kresimir; Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W; Hales, Brian J

2018-04-16

The P-cluster of the nitrogenase MoFe protein is a [ Fe 8 S 7 ] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [ Fe 4 S 4 ] + clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [ Fe 4 S 4 ] + clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [ Fe 4 S 4 ] + clusters into the integer spin cluster [ Fe 4 S 3-4 ] α , a process aided by the assembly protein NifZ, whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [ Fe 8 S 7-8 ] β , which is eventually converted into the P-cluster.

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)-Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway.

PubMed

Wachnowsky, Christine; Wesley, Nathaniel A; Fidai, Insiya; Cowan, J A

2017-03-24

Iron-sulfur (Fe/S)-cluster-containing proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an Fe/S scaffold protein, via an in vitro investigation into the structural and functional consequences. Analysis of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure composition. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an Fe/S cluster from physiologically relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change. Copyright © 2017 Elsevier Ltd. All rights reserved.

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster*

PubMed Central

Golinelli-Cohen, Marie-Pierre; Lescop, Ewen; Mons, Cécile; Gonçalves, Sergio; Clémancey, Martin; Santolini, Jérôme; Guittet, Eric; Blondin, Geneviève; Latour, Jean-Marc; Bouton, Cécile

2016-01-01

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S]+ with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the “active state,” which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a “dormant form.” Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury. PMID:26887944

Primer synthesis by a eukaryotic-like archaeal primase is independent of its Fe-S cluster.

PubMed

Holzer, Sandro; Yan, Jiangyu; Kilkenny, Mairi L; Bell, Stephen D; Pellegrini, Luca

2017-11-23

DNA replication depends on primase, the specialised polymerase responsible for synthesis of the RNA primers that are elongated by the replicative DNA polymerases. In eukaryotic and archaeal replication, primase is a heterodimer of two subunits, PriS and PriL. Recently, a third primase subunit named PriX was identified in the archaeon Sulfolobus solfataricus. PriX is essential for primer synthesis and is structurally related to the Fe-S cluster domain of eukaryotic PriL. Here we show that PriX contains a nucleotide-binding site required for primer synthesis, and demonstrate equivalence of nucleotide-binding residues in PriX with eukaryotic PriL residues that are known to be important for primer synthesis. A primase chimera, where PriX is fused to a truncated version of PriL lacking the Fe-S cluster domain retains wild-type levels of primer synthesis. Our evidence shows that PriX has replaced PriL as the subunit that endows primase with the unique ability to initiate nucleic acid synthesis. Importantly, our findings reveal that the Fe-S cluster is not required for primer synthesis.

In vivo [Fe-S] cluster acquisition by IscR and NsrR, two stress regulators in Escherichia coli.

PubMed

Vinella, Daniel; Loiseau, Laurent; Ollagnier de Choudens, Sandrine; Fontecave, Marc; Barras, Frédéric

2013-02-01

The multi-proteins Isc and Suf systems catalyse the biogenesis of [Fe-S] proteins. Here we investigate how NsrR and IscR, transcriptional regulators that sense NO and [Fe-S] homeostasis, acquire their [Fe-S] clusters under both normal and iron limitation conditions. Clusters directed at the apo-NsrR and apo-IscR proteins are built on either of the two scaffolds, IscU or SufB. However, differences arise in [Fe-S] delivery steps. In the case of NsrR, scaffolds deliver clusters to either one of the two ATCs, IscA and SufA, and, subsequently, to the 'non-Isc non-Suf' ATC, ErpA. Nevertheless, a high level of SufA can bypass the requirement for ErpA. In the case of IscR, several routes occur. One does not include assistance of any ATC. Others implicate ATCs IscA or ErpA, but, surprisingly, SufA was totally absent from any IscR maturation pathways. Both IscR and NsrR have the intrinsic capacity to sense iron limitation. However, NsrR appeared to be efficiently matured by Isc and Suf, thereby preventing NsrR to act as a physiologically relevant iron sensor. This work emphasizes that different maturation pathways arise as a function of the apo-target considered, possibly in relation with the type of cluster, [2Fe-2S] versus [4Fe-4S], it binds. © 2013 Blackwell Publishing Ltd.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase

PubMed Central

2014-01-01

Background In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. Results Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme. Conclusions We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme. PMID:24934472

Ligand Rearrangements at Fe/S Cofactors: Slow Isomerization of a Biomimetic [2Fe-2S] Cluster.

PubMed

Bergner, Marie; Roy, Lisa; Dechert, Sebastian; Neese, Frank; Ye, Shengfa; Meyer, Franc

2017-04-18

Ligand exchange plays an important role in the biogenesis of Fe/S clusters, most prominently during cluster transfer from a scaffold protein to its target protein. Although in vivo and in vitro studies have provided some insight into this process, the microscopic details of the ligand exchange steps are mostly unknown. In this work, the kinetics of the ligand rearrangement in a biomimetic [2Fe-2S] cluster with mixed S/N capping ligands have been studied. Two geometrical isomers of the cluster are present in solution, and mechanistic insight into the isomerization process was obtained by variable-temperature 1 H NMR spectroscopy. Combined experimental and computational results reveal that this is an associative process that involves the coordination of a solvent molecule to one of the ferric ions. The cluster isomerizes at least two orders of magnitude faster in its protonated and mixed-valent states. These findings may contribute to a deeper understanding of cluster transfer and sensing processes occurring in Fe/S cluster biogenesis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mitochondria dysfunctions under Fe and S deficiency: is citric acid involved in the regulation of adaptive responses?

PubMed

Vigani, Gianpiero; Pii, Youry; Celletti, Silvia; Maver, Mauro; Mimmo, Tanja; Cesco, Stefano; Astolfi, Stefania

2018-05-01

Within the last years, extensive information has been accumulated on the reciprocal influence between S and Fe nutrition at both physiological and molecular level in several plant species, but the mechanisms regulating S and Fe sensing and signaling are not fully understood. Fe and S interact for the building of Fe-S clusters, and mitochondria is one of the cellular compartments where Fe-S cluster assembly takes place. Therefore, it would be expected that mitochondria might play a central role in the regulation of Fe and S interaction. The Fe deficiency-induced alteration in the synthesis of mitochondria-derived carboxylic acids, such as citric acid, and the evidence that such molecules have already been identified as important players of metabolite signaling in several organisms, further support this hypothesis. Tomato plants were grown under single or combined Fe and S deficiency with the aim of verifying whether mitochondria activities played a role in Fe/S interaction. Both Fe and S deficiencies determined similar alteration of respiratory chain activity: a general decrease of Fe-S containing complexes as well as an increase of alternative NAD(P)H activities was observed in both Fe and S deficient-plants. However, the content of Krebs cycle-related organic acids in roots was substantially different in response to treatments, being the accumulation of citric acid always increased, while the others (i.e. succinic, malic, fumaric acids) always decreased. Interestingly, citric acid levels significantly correlated with the expression of some Fe and S deficiency induced genes. Our results contribute to existing knowledge on the complexity of the S/Fe interaction, suggesting a model in which endogenous alteration of citric acid content in plant tissues might act as signal molecule for the regulation of some nuclear-encoded and nutrient-responsive genes and also provide a basis for further study of the mechanism underlying S and Fe sensing and signalling. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses.

PubMed

Vuorijoki, Linda; Tiwari, Arjun; Kallio, Pauli; Aro, Eva-Mari

2017-05-01

Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. The approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level. The results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR. The study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism. The study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe-S cluster status and Mg²⁺: implication for ribosomal function.

PubMed

Sims, Lynn M; Igarashi, Robert Y

2012-08-15

Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe₄S₄ clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²⁺. Typically ATPases require Mg²⁺, as is true for PabABCE1, but Mg²⁺ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²⁺] inhibits the PabABCE1 ATPase (K(i) of ∼1 μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²⁺ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from ∼30 μM to over 1 mM with M²⁺. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²⁺]. These findings uncover intricate overlapping effects by both [Mg²⁺] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function. Copyright © 2012 Elsevier Inc. All rights reserved.

The role of the Fe-S cluster in the sensory domain of nitrogenase transcriptional activator VnfA from Azotobacter vinelandii.

PubMed

Nakajima, Hiroshi; Takatani, Nobuyuki; Yoshimitsu, Kyohei; Itoh, Mitsuko; Aono, Shigetoshi; Takahashi, Yasuhiro; Watanabe, Yoshihito

2010-02-01

Transcriptional activator VnfA is required for the expression of a second nitrogenase system encoded in the vnfH and vnfDGK operons in Azotobacter vinelandii. In the present study, we have purified full-length VnfA produced in E. coli as recombinant proteins (Strep-tag attached and tag-less proteins), enabling detailed characterization of VnfA for the first time. The EPR spectra of whole cells producing tag-less VnfA (VnfA) show distinctive signals assignable to a 3Fe-4S cluster in the oxidized form ([Fe(3)S(4)](+)). Although aerobically purified VnfA shows no vestiges of any Fe-S clusters, enzymatic reconstitution under anaerobic conditions reproduced [Fe(3)S(4)](+) dominantly in the protein. Additional spectroscopic evidence of [Fe(3)S(4)](+)in vitro is provided by anaerobically purified Strep-tag attached VnfA. Thus, spectroscopic studies both in vivo and in vitro indicate the involvement of [Fe(3)S(4)](+) as a prosthetic group in VnfA. Molecular mass analyses reveal that VnfA is a tetramer both in the presence and absence of the Fe-S cluster. Quantitative data of iron and acid-labile sulfur in reconstituted VnfA are fitted with four 3Fe-4S clusters per a tetramer, suggesting that one subunit bears one cluster. In vivobeta-gal assays reveal that the Fe-S cluster which is presumably anchored in the GAF domain by the N-terminal cysteine residues is essential for VnfA to exert its transcription activity on the target nitrogenase genes. Unlike the NifAL system of A. vinelandii, O(2) shows no effect on the transcriptional activity of VnfA but reactive oxygen species is reactive to cause disassembly of the Fe-S cluster and turns active VnfA inactive.

Photoelectron spectroscopy and density functional theory studies of (FeS)mH- (m = 2-4) cluster anions: effects of the single hydrogen.

PubMed

Yin, Shi; Bernstein, Elliot R

2017-12-20

Single hydrogen containing iron hydrosulfide cluster anions (FeS) m H - (m = 2-4) are studied by photoelectron spectroscopy (PES) at 3.492 eV (355 nm) and 4.661 eV (266 nm) photon energies, and by Density Functional Theory (DFT) calculations. The structural properties, relative energies of different spin states and isomers, and the first calculated vertical detachment energies (VDEs) of different spin states for these (FeS) m H - (m = 2-4) cluster anions are investigated at various reasonable theory levels. Two types of structural isomers are found for these (FeS) m H - (m = 2-4) clusters: (1) the single hydrogen atom bonds to a sulfur site (SH-type); and (2) the single hydrogen atom bonds to an iron site (FeH-type). Experimental and theoretical results suggest such available different SH- and FeH-type structural isomers should be considered when evaluating the properties and behavior of these single hydrogen containing iron sulfide clusters in real chemical and biological systems. Compared to their related, respective pure iron sulfur (FeS) m - clusters, the first VDE trend of the diverse type (FeS) m H 0,1 - (m = 1-4) clusters can be understood through (1) the different electron distribution properties of their highest singly occupied molecular orbital employing natural bond orbital analysis (NBO/HSOMO), and (2) the partial charge distribution on the NBO/HSOMO localized sites of each cluster anion. Generally, the properties of the NBO/HSOMOs play the principal role with regard to the physical and chemical properties of all the anions. The change of cluster VDE from low to high is associated with the change in nature of their NBO/HSOMO from a dipole bound and valence electron mixed character, to a valence p orbital on S, to a valence d orbital on Fe, and to a valence p orbital on Fe or an Fe-Fe delocalized valence bonding orbital. For clusters having the same properties for NBO/HSOMOs, the partial charge distributions at the NBO/HSOMO localized sites additionally affect their VDEs: a more negative or less positive localized charge distribution is correlated with a lower first VDE. The single hydrogen in these (FeS) m H - (m = 2-4) cluster anions is suggested to affect their first VDEs through the different structure types (SH- or FeH-), the nature of the NBO/HSOMOs at the local site, and the value of partial charge number at the local site of the NBO/HSOMO.

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron-Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis.

PubMed

Artz, Jacob H; Mulder, David W; Ratzloff, Michael W; Lubner, Carolyn E; Zadvornyy, Oleg A; LeVan, Axl X; Williams, S Garrett; Adams, Michael W W; Jones, Anne K; King, Paul W; Peters, John W

2017-07-19

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentials for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (∼ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox /Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd red that accumulates during fermentation. Subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.

The role of the GAF and central domains of the transcriptional activator VnfA in Azotobacter vinelandii.

PubMed

Yoshimitsu, Kyohei; Takatani, Nobuyuki; Miura, Yukio; Watanabe, Yoshihito; Nakajima, Hiroshi

2011-09-01

VnfA is a transcriptional activator that is required for the expression of the structural genes encoding nitrogenase-2 in Azotobacter vinelandii. VnfA consists of three domains: an N-terminal regulatory domain termed GAF, including a Cys-rich motif; a central domain from the AAA+ family; and a C-terminal domain for DNA binding. Previously, we reported that transcriptionally active VnfA harboring an Fe-S cluster (presumably of the 3Fe-4S type) as a prosthetic group and the Cys-rich motif were possibly associated with coordination of the Fe-S cluster. In the present study, we have investigated the roles of the GAF and central domains in the regulatory function of VnfA using truncated variants: ΔN15(VnfA) and ΔGAF(VnfA) that lack the N-terminal 15 residues and whole GAF domain, respectively, and GAF(VnfA) consisting of only the GAF domain. ΔN15(VnfA) and ΔGAF(VnfA) lost the ability to bind the Fe-S cluster, whereas GAF(VnfA) was still able to bind to the cluster, consistent with the hypothesis that the Cys-rich motif is essential for Fe-S cluster binding. The GAF domain showed an inhibitory effect on the transcriptional activity of VnfA, which was reversed in the presence of the Fe-S cluster, and reactivated upon disassembly of the cluster. The inhibitory activity of the GAF domain acts on the NTPase activity of the central domain, whereas the binding ability of VnfA to DNA was not significantly affected, when VnfA retains its tetrameric conformation. The results imply that a major pathway, by which VnfA function is regulated, operates via the control of NTPase activity by the GAF domain. © 2011 The Authors Journal compilation © 2011 FEBS.

Interaction of frataxin, an iron binding protein, with IscU of Fe-S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani.

PubMed

Zaidi, Amir; Singh, Krishn Pratap; Anwar, Shadab; Suman, Shashi S; Equbal, Asif; Singh, Kuljit; Dikhit, Manas R; Bimal, Sanjeeva; Pandey, Krishna; Das, Pradeep; Ali, Vahab

2015-08-01

Leishmania donovani is a unicellular protozoon parasite that causes visceral leishmaniasis (VL), which is a fatal disease if left untreated. Certain Fe-S proteins of the TCA cycle and respiratory chain have been found in the Leishmania parasite but the precise mechanisms for their biogenesis and the maturation of Fe-S clusters remains unknown. Fe-S clusters are ubiquitous cofactors of proteins that perform critical cellular functions. The clusters are biosynthesized by the mitochondrial Iron-Sulphur Cluster (ISC) machinery with core protein components that include the catalytic cysteine desulphurase IscS, the scaffold proteins IscU and IscA, and frataxin as an iron carrier/donor. However, no information regarding frataxin, its regulation, or its role in drug resistance is available for the Leishmania parasite. In this study, we characterized Ld-frataxin to investigate its role in the ISC machinery of L. donovani. We expressed and purified the recombinant Ld-frataxin protein and observed its interaction with Ld-IscU by co-purification and pull-down assay. Furthermore, we observed that the cysteine desulphurase activity of the purified Ld-IscS protein was stimulated in the presence of Ld-frataxin and Ld-IscU, particularly in the presence of iron; neither Ld-frataxin nor Ld-IscU alone had significant effects on Ld-IscS activity. Interestingly, RT-PCR and western blotting showed that Ld-frataxin is upregulated in AmpB-resistant isolates compared to sensitive strains, which may support higher Fe-S protein activity in AmpB-resistant L. donovani. Additionally, Ld-frataxin was localized in the mitochondria, as revealed by digitonin fractionation and indirect immunofluorescence. Thus, our results suggest the role of Ld-frataxin as an iron binding/carrier protein for Fe-S cluster biogenesis that physically interacts with other core components of the ISC machinery within the mitochondria. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Glutathione-complexed [2Fe-2S] clusters function in Fe-S cluster storage and trafficking.

PubMed

Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

2016-10-01

Glutathione-coordinated [2Fe-2S] complex is a non-protein-bound [2Fe-2S] cluster that is capable of reconstituting the human iron-sulfur cluster scaffold protein IscU. This complex demonstrates physiologically relevant solution chemistry and is a viable substrate for iron-sulfur cluster transport by Atm1p exporter protein. Herein, we report on some of the possible functional and physiological roles for this novel [2Fe-2S](GS4) complex in iron-sulfur cluster biosynthesis and quantitatively characterize its role in the broader network of Fe-S cluster transfer reactions. UV-vis and circular dichroism spectroscopy have been used in kinetic studies to determine second-order rate constants for [2Fe-2S] cluster transfer from [2Fe-2S](GS4) complex to acceptor proteins, such as human IscU, Schizosaccharomyces pombe Isa1, human and yeast glutaredoxins (human Grx2 and Saccharomyces cerevisiae Grx3), and human ferredoxins. Second-order rate constants for cluster extraction from these holo proteins were also determined by varying the concentration of glutathione, and a likely common mechanism for cluster uptake was determined by kinetic analysis. The results indicate that the [2Fe-2S](GS4) complex is stable under physiological conditions, and demonstrates reversible cluster exchange with a wide range of Fe-S cluster proteins, thereby supporting a possible physiological role for such centers.

Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase.

PubMed

Shomura, Yasuhito; Yoon, Ki-Seok; Nishihara, Hirofumi; Higuchi, Yoshiki

2011-10-16

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H(2)-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H(2) → 2H(+) + 2e(-) (ref. 1). In contrast to the well-studied O(2)-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O(2)-tolerant H(2) oxidation activity; however, the mechanism of O(2) tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues--in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes--and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O(2)-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O(2) tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O(2) in preventing the formation of an unready, inactive state of the enzyme.

Delivery of Iron-Sulfur Clusters to the Hydrogen-Oxidizing [NiFe]-Hydrogenases in Escherichia coli Requires the A-Type Carrier Proteins ErpA and IscA

PubMed Central

Pinske, Constanze; Sawers, R. Gary

2012-01-01

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements. PMID:22363723

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Artz, Jacob H.; Mulder, David W.; Ratzloff, Michael W.

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentialsmore » for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (~ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox/Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd redthat accumulates during fermentation. In conclusion, subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.« less

Reduction Potentials of [FeFe]-Hydrogenase Accessory Iron–Sulfur Clusters Provide Insights into the Energetics of Proton Reduction Catalysis

DOE PAGES

Artz, Jacob H.; Mulder, David W.; Ratzloff, Michael W.; ...

2017-06-21

An [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, is a model system for biological H 2 activation. In addition to the catalytic H-cluster, CpI contains four accessory iron-sulfur [FeS] clusters in a branched series that transfer electrons to and from the active site. In this work, potentiometric titrations have been employed in combination with electron paramagnetic resonance (EPR) spectroscopy at defined electrochemical potentials to gain insights into the role of the accessory clusters in catalysis. EPR spectra collected over a range of potentials were deconvoluted into individual components attributable to the accessory [FeS] clusters and the active site H-cluster, and reduction potentialsmore » for each cluster were determined. The data suggest a large degree of magnetic coupling between the clusters. The distal [4Fe-4S] cluster is shown to have a lower reduction potential (~ < -450 mV) than the other clusters, and molecular docking experiments indicate that the physiological electron donor, ferredoxin (Fd), most favorably interacts with this cluster. The low reduction potential of the distal [4Fe-4S] cluster thermodynamically restricts the Fd ox/Fd red ratio at which CpI can operate, consistent with the role of CpI in recycling Fd redthat accumulates during fermentation. In conclusion, subsequent electron transfer through the additional accessory [FeS] clusters to the H-cluster is thermodynamically favorable.« less

The antimalarial drug primaquine targets Fe-S cluster proteins and yeast respiratory growth.

PubMed

Lalève, Anaïs; Vallières, Cindy; Golinelli-Cohen, Marie-Pierre; Bouton, Cécile; Song, Zehua; Pawlik, Grzegorz; Tindall, Sarah M; Avery, Simon V; Clain, Jérôme; Meunier, Brigitte

2016-04-01

Malaria is a major health burden in tropical and subtropical countries. The antimalarial drug primaquine is extremely useful for killing the transmissible gametocyte forms of Plasmodium falciparum and the hepatic quiescent forms of P. vivax. Yet its mechanism of action is still poorly understood. In this study, we used the yeast Saccharomyces cerevisiae model to help uncover the mode of action of primaquine. We found that the growth inhibitory effect of primaquine was restricted to cells that relied on respiratory function to proliferate and that deletion of SOD2 encoding the mitochondrial superoxide dismutase severely increased its effect, which can be countered by the overexpression of AIM32 and MCR1 encoding mitochondrial enzymes involved in the response to oxidative stress. This indicated that ROS produced by respiratory activity had a key role in primaquine-induced growth defect. We observed that Δsod2 cells treated with primaquine displayed a severely decreased activity of aconitase that contains a Fe-S cluster notoriously sensitive to oxidative damage. We also showed that in vitro exposure to primaquine impaired the activity of purified aconitase and accelerated the turnover of the Fe-S cluster of the essential protein Rli1. It is suggested that ROS-labile Fe-S groups are the primary targets of primaquine. Aconitase activity is known to be essential at certain life-cycle stages of the malaria parasite. Thus primaquine-induced damage of its labile Fe-S cluster - and of other ROS-sensitive enzymes - could inhibit parasite development. Copyright © 2015. Published by Elsevier B.V.

The NMR contribution to protein-protein networking in Fe-S protein maturation.

PubMed

Banci, Lucia; Camponeschi, Francesca; Ciofi-Baffoni, Simone; Piccioli, Mario

2018-03-22

Iron-sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe-S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe-2S], [3Fe-4S] and [4Fe-4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe-S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe-S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of "Fe-S interactomics". This contribution was particularly effective when protein-protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.

First principles studies of electron tunneling in proteins

PubMed Central

Hayashi, Tomoyuki; Stuchebrukhov, Alexei A.

2014-01-01

A first principles study of electronic tunneling along the chain of seven Fe/S clusters in respiratory complex I, a key enzyme in the respiratory electron transport chain, is described. The broken-symmetry states of the Fe/S metal clusters calculated at both DFT and semi-empirical ZINDO levels were utilized to examine both the extremely weak electronic couplings between Fe/S clusters and the tunneling pathways, which provide a detailed atomistic-level description of the charge transfer process in the protein. One-electron tunneling approximation was found to hold within a reasonable accuracy, with only a moderate induced polarization of the core electrons. The method is demonstrated to be able to calculate accurately the coupling matrix elements as small as 10−4 cm−1. A distinct signature of the wave properties of electrons is observed as quantum interferences of multiple tunneling pathways. PMID:25383312

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase.

PubMed

Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD + -reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H 2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function the various metal cofactors present in the enzyme. Here all iron-containing cofactors of the SH were investigated by 57 Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, which is consistent with amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD + -reducing hydrogenases. For the first time, Fe-CO and Fe-CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13 C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe-CO modes. The present approach explores the complex vibrational signature of the Fe-S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.

The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species.

PubMed

Alhebshi, Alawiah; Sideri, Theodora C; Holland, Sara L; Avery, Simon V

2012-09-01

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary "Achilles' heel" of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster-defective Rli1p construct. The protein's FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily (55)FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress-related disease.

The iron uptake repressor Fep1 in the fission yeast binds Fe-S cluster through conserved cysteines.

PubMed

Kim, Hyo-Jin; Lee, Kang-Lok; Kim, Kyoung-Dong; Roe, Jung-Hye

2016-09-09

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. Copyright © 2016 Elsevier Inc. All rights reserved.

Energies and spin states of FeS(0/-), FeS2(0/-), Fe2S2(0/-), Fe3S4(0/-), and Fe4S4(0/-) clusters.

PubMed

Li, Yan-Ni; Wang, Shengguang; Wang, Tao; Gao, Rui; Geng, Chun-Yu; Li, Yong-Wang; Wang, Jianguo; Jiao, Haijun

2013-04-15

The structures and energies of the electronic ground states of the FeS(0/-), FeS2(0/-), Fe2S2(0/-), Fe3S4(0/-), and Fe4S4(0/-) neutral and anionic clusters have been computed systematically with nine computational methods in combination with seven basis sets. The computed adiabatic electronic affinities (AEA) have been compared with available experimental data. Most reasonable agreements between theory and experiment have been found for both hybrid B3LYP and B3PW91 functionals in conjugation with 6-311+G* and QZVP basis sets. Detailed comparisons between the available experimental and computed AEA data at the B3LYP/6-311+G* level identified the electronic ground state of (5)Δ for FeS, (4)Δ for FeS(-), (5)B2 for FeS2, (6)A1 for FeS2(-), (1)A1 for Fe2S2, (8)A' for Fe2S2(-), (5)A'' for Fe3S4, (6)A'' for Fe3S4(-), (1)A1 for Fe4S4, and (1)A2 for Fe4S4(-). In addition, Fe2S2, Fe3S4, Fe3S4(-), Fe4S4, and Fe4S4(-) are antiferromagnetic at the B3LYP/6-311+G* level. The magnetic properties are discussed on the basis of natural bond orbital analysis. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes

PubMed Central

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-01-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. PMID:27037832

Effect of H bond removal and changes in the position of the iron-sulphur head domain on the spin-lattice relaxation properties of the [2Fe-2S](2+) Rieske cluster in cytochrome bc(1).

PubMed

Sarewicz, Marcin; Dutka, Małgorzata; Pietras, Rafał; Borek, Arkadiusz; Osyczka, Artur

2015-10-14

Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex - cytochrome bc1 - are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1(-1)) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1(-1) (T) over the range 5-120 K yielded the values of the Orbach energy (EOrb), Debye temperature θD and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While θD was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin-orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin-orbit coupling but was also influenced by other factors - possibly the modification of protein rigidity and therefore the vibrational modes around the Fe-S cluster that change upon the movement of the iron-sulphur head domain.

Coulomb- and Antiferromagnetic-Induced Fission in Doubly Charged Cubelike Fe-S Clusters

NASA Astrophysics Data System (ADS)

Yang, Xin; Wang, Xue-Bin; Niu, Shuqiang; Pickett, Chris J.; Ichiye, Toshiko; Wang, Lai-Sheng

2002-09-01

We report the observation of symmetric fission in doubly charged Fe-S cluster anions, [Fe4S4X4]2- -->2[Fe2S2X2]- (X=Cl,Br), owing to both Coulomb repulsion and antiferromagnetic coupling. Photoelectron spectroscopy shows that both the parent and the fission fragments have similar electronic structures and confirms the inverted energy schemes due to the strong spin polarization of the Fe 3d levels. The current observation provides direct confirmation for the unusual spin couplings in the [Fe4S4X4]2- clusters, which contain two valent-delocalized and ferromagnetically coupled Fe2S2 subunits.

Regional Gray Matter Volumes Are Related to Concern About Falling in Older People: A Voxel-Based Morphometric Study.

PubMed

Tuerk, Carola; Zhang, Haobo; Sachdev, Perminder; Lord, Stephen R; Brodaty, Henry; Wen, Wei; Delbaere, Kim

2016-01-01

Concern about falling is common in older people. Various related psychological constructs as well as poor balance and slow gait have been associated with decreased gray matter (GM) volume in old age. The current study investigates the association between concern about falling and voxel-wise GM volumes. A total of 281 community-dwelling older people aged 70-90 years underwent structural magnetic resonance imaging. Concern about falling was assessed using Falls Efficacy Scale-International (FES-I). For each participant, voxel-wise GM volumes were generated with voxel-based morphometry and regressed on raw FES-I scores (p < .05 family-wise error corrected on cluster level). FES-I scores were negatively correlated with total brain volume (r = -.212; p ≤ .001), GM volume (r = -.210; p ≤ .001), and white matter volume (r = -.155; p ≤ .001). Voxel-based morphometry analysis revealed significant negative associations between FES-I and GM volumes of (i) left cerebellum and bilateral inferior occipital gyrus (voxels-in-cluster = 2,981; p < .001) and (ii) bilateral superior frontal gyrus and left supplementary motor area (voxels-in-cluster = 1,900; p = .004). Additional adjustment for vision and physical fall risk did not alter these associations. After adjustment for anxiety, only left cerebellum and bilateral inferior occipital gyrus remained negatively associated with FES-I scores (voxels-in-cluster = 2,426; p < .001). Adjustment for neuroticism removed all associations between FES-I and GM volumes. Our study findings show that concern about falling is negatively associated with brain volumes in areas important for emotional control and for motor control, executive functions and visual processing in a large sample of older men and women. Regression analyses suggest that these relationships were primarily accounted for by psychological factors (generalized anxiety and neuroticism) and not by physical fall risk or vision. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling.

PubMed

Hu, Xueyun; Page, Mike T; Sumida, Akihiro; Tanaka, Ayumi; Terry, Matthew J; Tanaka, Ryouichi

2017-03-01

Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation. © 2016 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serrano, Pauline N.; Wang, Hongxin; Crack, Jason C.

The reaction of protein-bound iron–sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE,more » [Fe 2(NO) 4(Cys) 2]) and Roussin's Black Salt (RBS, [Fe 4(NO) 7S 3]. In the latter case, the absence of 32S/ 34S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.« less

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

PubMed Central

Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

2017-01-01

ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes. PMID:28847921

X-ray and EPR Characterization of the Auxiliary Fe-S Clusters in the Radical SAM Enzyme PqqE.

PubMed

Barr, Ian; Stich, Troy A; Gizzi, Anthony S; Grove, Tyler L; Bonanno, Jeffrey B; Latham, John A; Chung, Tyler; Wilmot, Carrie M; Britt, R David; Almo, Steven C; Klinman, Judith P

2018-02-27

The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

Crystal structure of IscA, an iron-sulfur cluster assembly protein from Escherichia coli.

PubMed

Cupp-Vickery, Jill R; Silberg, Jonathan J; Ta, Dennis T; Vickery, Larry E

2004-04-16

IscA, an 11 kDa member of the hesB family of proteins, binds iron and [2Fe-2S] clusters, and participates in the biosynthesis of iron-sulfur proteins. We report the crystal structure of the apo-protein form of IscA from Escherichia coli to a resolution of 2.3A. The crystals belong to the space group P3(2)21 and have unit cell dimensions a=b=66.104 A, c=150.167 A (alpha=beta=90 degrees, gamma=120 degrees ). The structure was solved using single-wavelength anomalous dispersion (SAD) phasing of a selenomethionyl derivative, and the IscA model was refined to R=21.4% (Rfree=25.4%). IscA exists as an (alpha1alpha2)2 homotetramer with the (alpha1alpha2) dimer comprising the asymmetric unit. Cys35, implicated in Fe-S cluster assembly, is located in a central cavity formed at the tetramer interface with the gamma-sulfur atoms of residues from the alpha1 and alpha2' monomers (and alpha1'alpha2) positioned close to one another (approximately equal 7 A). C-terminal residues 99-107 are disordered, and the exact positions of Cys99 and Cys101 could not be determined. However, computer modeling of C-terminal residues in the tetramer suggests that Cys99 and Cys101 in the alpha1 monomer and those of the alpha1' monomer (or alpha2 and alpha2') are positioned sufficiently close to coordinate [2Fe-2S] clusters between the two dimers, whereas this is not possible within the (alpha1alpha2) or (alpha1'alpha2') dimer. This symmetrical arrangement allows for binding of two [2Fe-2S] clusters on opposite sides of the tetramer. Modeling further reveals that Cys101 is positioned sufficiently close to Cys35 to allow Cys35 to participate in cluster assembly, formation, or transfer.

The Basic Leucine Zipper Stress Response Regulator Yap5 Senses High-Iron Conditions by Coordination of [2Fe-2S] Clusters

PubMed Central

Rietzschel, Nicole; Pierik, Antonio J.; Bill, Eckhard; Mühlenhoff, Ulrich

2014-01-01

Iron is an essential, yet at elevated concentrations toxic trace element. To date, the mechanisms of iron sensing by eukaryotic iron-responsive transcription factors are poorly understood. The Saccharomyces cerevisiae transcription factor Yap5, a member of the Yap family of bZIP stress response regulators, administrates the adaptive response to high-iron conditions. Despite the central role of the iron-sensing process for cell viability, the molecule perceived by Yap5 and the underlying regulatory mechanisms are unknown. Here, we show that Yap5 senses high-iron conditions by two Fe/S clusters bound to its activator domain (Yap5-AD). The more stable iron-regulatory Fe/S cluster at the N-terminal cysteine-rich domain (n-CRD) of Yap5 is detected in vivo and in vitro. The second cluster coordinated by the C-terminal CRD can only be shown after chemical reconstitution, since it is bound in a labile fashion. Both clusters are of the [2Fe-2S] type as characterized by UV/visible (UV/Vis), circular dichroism, electron paramagnetic resonance (EPR), and Mössbauer spectroscopy. Fe/S cluster binding to Yap5-AD induces a conformational change that may activate transcription. The cluster-binding motif of the n-CRD domain is highly conserved in HapX-like transcription factors of pathogenic fungi and thus may represent a general sensor module common to many eukaryotic stress response regulators. PMID:25368382

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes.

PubMed

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-06-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

PubMed

Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

2017-01-01

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyo-Jin; Lee, Kang-Lok; Kim, Kyoung-Dong

Iron homeostasis is tightly regulated since iron is an essential but toxic element in the cell. The GATA-type transcription factor Fep1 and its orthologs contribute to iron homeostasis in many fungi by repressing genes for iron uptake when intracellular iron is high. Even though the function and interaction partners of Fep1 have been elucidated extensively In Schizosaccharomyces pombe, the mechanism behind iron-sensing by Fep1 remains elusive. It has been reported that Fep1 interacts with Fe-S-containing monothiol glutaredoxin Grx4 and Grx4-Fra2 complex. In this study, we demonstrate that Fep1 also binds iron, in the form of Fe-S cluster. Spectroscopic and biochemicalmore » analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. The finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1. - Highlights: • Fep1, a prototype fungal iron uptake regulator, was isolated stably from Schizosaccharomyces pombe. • Fep1 exhibits UV–visible absorption spectrum, characteristic of [Fe-S] proteins. • The iron and sulfide contents in purified or reconstituted Fep1 also support [Fe-S]. • The conserved cysteines are critical for [Fe-S]-binding. • EPR spectra at 5 K and 123 K suggest a mixed population of [Fe-S].« less

The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis*

PubMed Central

Dahl, Jan-Ulrik; Radon, Christin; Bühning, Martin; Nimtz, Manfred; Leichert, Lars I.; Denis, Yann; Jourlin-Castelli, Cécile; Iobbi-Nivol, Chantal; Méjean, Vincent; Leimkühler, Silke

2013-01-01

The Escherichia coli l-cysteine desulfurase IscS mobilizes sulfur from l-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes. PMID:23281480

A new method to evaluate the unfolding activity of chaperone unit ClpA based on Fe-S cluster disruption.

PubMed

Ohgita, Takashi; Okuno, Takashi; Hama, Susumu; Tsuchiya, Hiroyuki; Kogure, Kentaro

2011-01-01

ATP-dependent proteases unfold their substrates and then refold (via chaperone activity) or degrade (via protease activity) them. The proteases choose between these two activities by selecting their substrates; however, little is known about their substrate selection mechanism. The present study attempts to clarify this mechanism by investigating the role of the Escherichia coli (E. coli) ATP-dependent protease ClpAP. To address this, a reaction system that can measure both chaperone and protease activities simultaneously must be constructed. However, the chaperone activities cannot be evaluated in the presence of protease units. Green fluorescent protein (GFP) is usually used as a model substrate of ClpAP; the fluorescence decrease reflects the degradation of substrates. However, it is difficult to evaluate the chaperone activity of ClpAP using this system, because it cannot distinguish between intact and refolded substrates. Therefore, it is necessary to evaluate the exact unfolding activity while avoiding restoration of substrate spectroscopic characteristics due to chaperone activity. In this study, E. coli Ferredoxin (Fd) was used as a new model substrate for ClpAP to evaluate its unfolding activity. Intact and refolded substrates may be distinguished by the existence of an Fd Fe-S cluster. To verify this hypothesis, the absorption spectrum of Fd complexed with ClpA, the chaperone unit of ClpAP, was measured. A decrease in two peaks derived from the Fe-S cluster was observed, indicating that the Fe-S cluster of Fd was disrupted by the ClpA chaperone. This reaction system should prove useful to evaluate the exact unfolding activity of ATP-dependent proteases.

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters

PubMed Central

Darash-Yahana, Merav; Pozniak, Yair; Lu, Mingyang; Sohn, Yang-Sung; Karmi, Ola; Tamir, Sagi; Bai, Fang; Song, Luhua; Jennings, Patricia A.; Pikarsky, Eli; Geiger, Tamar; Onuchic, José N.; Mittler, Ron; Nechushtai, Rachel

2016-01-01

Iron–sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression. PMID:27621439

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters.

PubMed

Darash-Yahana, Merav; Pozniak, Yair; Lu, Mingyang; Sohn, Yang-Sung; Karmi, Ola; Tamir, Sagi; Bai, Fang; Song, Luhua; Jennings, Patricia A; Pikarsky, Eli; Geiger, Tamar; Onuchic, José N; Mittler, Ron; Nechushtai, Rachel

2016-09-27

Iron-sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression.

Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity

PubMed Central

Khoroshilova, Natalia; Popescu, Codrina; Münck, Eckard; Beinert, Helmut; Kiley, Patricia J.

1997-01-01

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]2+ cluster (δ = 0.45 mm/s; ΔEQ = 1.22 mm/s) and that the [4Fe-4S]2+ cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow–green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]2+ cluster was converted in about 60% yield to a [2Fe-2S]2+ cluster (δ = 0.28 mm/s; ΔEQ = 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]2+ cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]2+ cluster could be largely restored from the [2Fe-2S]2+ form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]2+ cluster has any biological significance, e.g., as an in vivo intermediate that is more rapidly converted to the active form than the apoprotein. PMID:9177174

Super reduced Fe4S4 cluster of Balch's dithiolene series.

PubMed

Begum, Ameerunisha; Moula, Golam; Bose, Moumita; Sarkar, Sabyasachi

2012-03-28

A super reduced Fe(4)S(4) cluster with a sulfur based radical, [NBu(4)](4)[Fe(3)(III)Fe(II)(μ(3)-S)(4)(mnt)(3)(6-)(mnt)(1-)˙](4-)˙, (1) (mnt, maleonitrile dithiolate) which evolves H(2)S gas on treatment with acid under ambient conditions has been synthesized and structurally characterized. The Fe-S distances in 1 are in the range 2.246-2.383 Å, in stark contrast to that of the known n = -2 member of the series based on the [Fe(4)(μ(3)-S)(4)(S(2)C(2)R(2))(4)](n) unit (R = CF(3), Ph) with Fe-S bond lengths of 2.149-2.186 Å. The EPR of 1 displays very weak signals at g, 4.03 and 2.38 along with a strong S-based radical EPR signal at g, 2.003 associated with five structured components tentatively assigned to hyperfine interaction arising out of the naturally abundant (57)Fe with = 88 G. The EPR profile resembles the reduced Fe-S cluster of CO inhibited Clostridium pasteurianum W5 hydrogenase or the Fe(4)S(4) centers of wild-type enzyme, IspH treated with HMBPP or IPP.

Mitochondrial and Nucleolar Localization of Cysteine Desulfurase Nfs and the Scaffold Protein Isu in Trypanosoma brucei

PubMed Central

Kovářová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie

2014-01-01

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites. PMID:24243795

Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei.

PubMed

Kovárová, Julie; Horáková, Eva; Changmai, Piya; Vancová, Marie; Lukeš, Julius

2014-03-01

Trypanosoma brucei has a complex life cycle during which its single mitochondrion is subjected to major metabolic and morphological changes. While the procyclic stage (PS) of the insect vector contains a large and reticulated mitochondrion, its counterpart in the bloodstream stage (BS) parasitizing mammals is highly reduced and seems to be devoid of most functions. We show here that key Fe-S cluster assembly proteins are still present and active in this organelle and that produced clusters are incorporated into overexpressed enzymes. Importantly, the cysteine desulfurase Nfs, equipped with the nuclear localization signal, was detected in the nucleolus of both T. brucei life stages. The scaffold protein Isu, an interacting partner of Nfs, was also found to have a dual localization in the mitochondrion and the nucleolus, while frataxin and both ferredoxins are confined to the mitochondrion. Moreover, upon depletion of Isu, cytosolic tRNA thiolation dropped in the PS but not BS parasites.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.

Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe-4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe-4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase.more » The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe-S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P21, with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 {angstrom}, = 92.0{sup o}. Native data sets have been collected from several crystals with resolution extending to 2.8 {angstrom} and the structure has been solved by molecular replacement.« less

Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O 2-tolerant NAD +-reducing [NiFe] hydrogenase

DOE PAGES

Lauterbach, Lars; Wang, Hongxin; Horch, Marius; ...

2014-10-30

Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H 2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters,more » which is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.« less

The rice mitochondrial iron transporter is essential for plant growth

PubMed Central

Bashir, Khurram; Ishimaru, Yasuhiro; Shimo, Hugo; Nagasaka, Seiji; Fujimoto, Masaru; Takanashi, Hideki; Tsutsumi, Nobuhiro; An, Gynheung; Nakanishi, Hiromi; Nishizawa, Naoko K.

2011-01-01

In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development. PMID:21610725

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution.

PubMed

Ciesielski, Szymon J; Schilke, Brenda A; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A; Dutkiewicz, Rafal

2012-03-16

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensible in vivo and conserved in evolution

PubMed Central

Ciesielski, Szymon; Schilke, Brenda; Osipiuk, Jerzy; Bigelow, Lance; Mulligan, Rory; Majewska, Julia; Joachimiak, Andrzej; Marszalek, Jaroslaw; Craig, Elizabeth A.; Dutkiewicz, Rafal

2012-01-01

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70’s ATPase activity. However, in apparent contradiction, we previously reported that an 8 fold decrease in Jac1’s affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, a total of eight surface exposed residues were determined to play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype and a reduction in the activity of Fe-S cluster containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensible role in the essential process of mitochondrial Fe-S cluster biogenesis. PMID:22306468

Quantum Electron Tunneling in Respiratory Complex I1

PubMed Central

Hayashi, Tomoyuki; Stuchebrukhov, Alexei A.

2014-01-01

We have simulated the atomistic details of electronic wiring of all Fe/S clusters in complex I, a key enzyme in the respiratory electron transport chain. The tunneling current theory of many-electron systems is applied to the broken-symmetry (BS) states of the protein at the ZINDO level. One-electron tunneling approximation is found to hold in electron tunneling between the anti-ferromagnetic binuclear and tetranuclear Fe/S clusters with moderate induced polarization of the core electrons. Calculated tunneling energy is about 3 eV higher than Fermi level in the band gap of the protein, which supports that the mechanism of electron transfer is quantum mechanical tunneling, as in the rest of electron transport chain. Resulting electron tunneling pathways consist of up to three key contributing protein residues between neighboring Fe/S clusters. A distinct signature of the wave properties of electrons is observed as quantum interferences when multiple tunneling pathways exist. In N6a-N6b, electron tunnels along different pathways depending on the involved BS states, suggesting possible fluctuations of the tunneling pathways driven by the local protein environment. The calculated distance dependence of the electron transfer rates with internal water molecules included are in good agreement with a reported phenomenological relation. PMID:21495666

The role of FeS(aq) molecular clusters in microbial redox cycling and iron mineralization.

NASA Astrophysics Data System (ADS)

Druschel, G.; Oduro, H.; Sperling, J.; Johnson, C.

2008-12-01

Iron sulfide molecular clusters, FeS(aq), are a group of polynuclear Fe-S complexes varying in size between a few and a few hundred molecules that occur in many environments and are critical parts of cycling between soluble iron and iron sulfide minerals. These clusters react uniquely with voltammetric Au-amalgam electrodes, and the signal for these molecules has now been observed in many terrestrial and marine aquatic settings. FeS(aq) clusters form when aqueous sulfide and iron(II) interact, but the source of those ions can come from abiotic or microbial sulfate and iron reduction or from the abiotic non-oxidative dissolution of iron sulfide minerals. Formation of iron sulfide minerals, principally mackinawite as the first solid nanocrystalline phase in many settings, is necessarily preceeded by formation and evolution of these molecular clusters as mineralization proceeds, and the clusters have been suggested to additionally be part of the pyritization process (Rickard and Luther, 1997; Luther and Rickard, 2005). In several systems, we have also observed FeS(aq) clusters to be the link between Fe-S mineral dissolution and oxidation of iron and sulfide, with important implications for changes to the overall oxidation pathway. Microorganisms can clearly be involved in the formation of FeS(aq) through iron and sulfate reduction, but it is not clear to date if organisms can utilize these clusters either as metabolic components or as anabolic 'building blocks' for enzyme production. Cycling of iron in the Fe-S system linked to FeS(aq) would clearly be a critical part of understanding iron isotope dynamics preserved in iron sulfide minerals. We will review ongoing work towards understanding the role of FeS(aq) in iron cycling and isotope fractionation as well as the measurement and characterization of this key class of iron complexes using environmental voltammetry.

Acclimation of Oxygenic Photosynthesis to Iron Starvation Is Controlled by the sRNA IsaR1.

PubMed

Georg, Jens; Kostova, Gergana; Vuorijoki, Linda; Schön, Verena; Kadowaki, Taro; Huokko, Tuomas; Baumgartner, Desirée; Müller, Maximilian; Klähn, Stephan; Allahverdiyeva, Yagut; Hihara, Yukako; Futschik, Matthias E; Aro, Eva-Mari; Hess, Wolfgang R

2017-05-22

Oxygenic photosynthesis crucially depends on proteins that possess Fe 2+ or Fe/S complexes as co-factors or prosthetic groups. Here, we show that the small regulatory RNA (sRNA) IsaR1 (Iron-Stress-Activated RNA 1) plays a pivotal role in acclimation to low-iron conditions. The IsaR1 regulon consists of more than 15 direct targets, including Fe 2+ -containing proteins involved in photosynthetic electron transfer, detoxification of anion radicals, citrate cycle, and tetrapyrrole biogenesis. IsaR1 is essential for maintaining physiological levels of Fe/S cluster biogenesis proteins during iron deprivation. Consequently, IsaR1 affects the acclimation of the photosynthetic apparatus to iron starvation at three levels: (1) directly, via posttranscriptional repression of gene expression; (2) indirectly, via suppression of pigment; and (3) Fe/S cluster biosynthesis. Homologs of IsaR1 are widely conserved throughout the cyanobacterial phylum. We conclude that IsaR1 is a critically important riboregulator. These findings provide a new perspective for understanding the regulation of iron homeostasis in photosynthetic organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biochemistry of Catabolic Reductive Dehalogenation.

PubMed

Fincker, Maeva; Spormann, Alfred M

2017-06-20

A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

Revisiting the Electronic Structure of FeS Monomers Using ab Initio Ligand Field Theory and the Angular Overlap Model.

PubMed

Chilkuri, Vijay Gopal; DeBeer, Serena; Neese, Frank

2017-09-05

Iron-sulfur (FeS) proteins are universally found in nature with actives sites ranging in complexity from simple monomers to multinuclear sites from two up to eight iron atoms. These sites include mononuclear (rubredoxins), dinuclear (ferredoxins and Rieske proteins), trinuclear (e.g., hydrogenases), and tetranuclear (various ferredoxins and high-potential iron-sulfur proteins). The electronic structure of the higher-nuclearity clusters is inherently extremely complex. Hence, it is reasonable to take a bottom-up approach in which clusters of increasing nuclearity are analyzed in terms of the properties of their lower nuclearity constituents. In the present study, the first step is taken by an in-depth analysis of mononuclear FeS systems. Two different FeS molecules with phenylthiolate and methylthiolate as ligands are studied in their oxidized and reduced forms using modern wave function-based ab initio methods. The ab initio electronic spectra and wave function are presented and analyzed in detail. The very intricate electronic structure-geometry relationship in these systems is analyzed using ab initio ligand field theory (AILFT) in conjunction with the angular overlap model (AOM) parametrization scheme. The simple AOM model is used to explain the effect of geometric variations on the electronic structure. Through a comparison of the ab initio computed UV-vis absorption spectra and the available experimental spectra, the low-energy part of the many-particle spectrum is carefully analyzed. We show ab initio calculated magnetic circular dichroism spectra and present a comparison with the experimental spectrum. Finally, AILFT parameters and the ab initio spectra are compared with those obtained experimentally to understand the effect of the increased covalency of the thiolate ligands on the electronic structure of FeS monomers.

Synthetic modeling chemistry of iron-sulfur clusters in nitric oxide signaling.

PubMed

Fitzpatrick, Jessica; Kim, Eunsuk

2015-08-18

Nitric oxide (NO) is an important signaling molecule that is involved in many physiological and pathological functions. Iron-sulfur proteins are one of the main reaction targets for NO, and the [Fe-S] clusters within these proteins are converted to various iron nitrosyl species upon reaction with NO, of which dinitrosyl iron complexes (DNICs) are the most prevalent. Much progress has been made in identifying the origin of cellular DNIC generation. However, it is not well-understood which other products besides DNICs may form during [Fe-S] cluster degradation nor what effects DNICs and other degradation products can have once they are generated in cells. Even more elusive is an understanding of the manner by which cells cope with unwanted [Fe-S] modifications by NO. This Account describes our synthetic modeling efforts to identify cluster degradation products derived from the [2Fe-2S]/NO reaction in order to establish their chemical reactivity and repair chemistry. Our intent is to use the chemical knowledge that we generate to provide insight into the unknown biological consequences of cluster modification. Our recent advances in three different areas are described. First, new reaction conditions that lead to the formation of previously unrecognized products during the reaction of [Fe-S] clusters with NO are identified. Hydrogen sulfide (H2S), a gaseous signaling molecule, can be generated from the reaction between [2Fe-2S] clusters and NO in the presence of acid or formal H• (e(-)/H(+)) donors. In the presence of acid, a mononitrosyl iron complex (MNIC) can be produced as the major iron-containing product. Second, cysteine analogues can efficiently convert MNICs back to [2Fe-2S] clusters without the need for any other reagents. This reaction is possible for cysteine analogues because of their ability to labilize NO from MNICs and their capacity to undergo C-S bond cleavage, providing the necessary sulfide for [2Fe-2S] cluster formation. Lastly, unique dioxygen reactivity of various types of DNICs has been established. N-bound neutral {Fe(NO)2}(10) DNICs react with O2 to generate low-temperature stable peroxynitrite (ONOO(-)) species, which then carry out nitration chemistry in the presence of phenolic substrates, relevant to tyrosine nitration chemistry. The reaction between S-bound anionic {Fe(NO)2}(9) DNICs and O2 results in the formation of Roussin's red esters (RREs) and thiol oxidation products, chemistry that may be important in biological cysteine oxidation. The N-bound cationic {Fe(NO)2}(9) DNICs can spontaneously release NO, and this property can be utilized in developing a new class of NO-donating agents with anti-inflammatory activity.

Quantum electron tunneling in respiratory complex I.

PubMed

Hayashi, Tomoyuki; Stuchebrukhov, Alexei A

2011-05-12

We have simulated the atomistic details of electronic wiring of all Fe/S clusters in complex I, a key enzyme in the respiratory electron transport chain. The tunneling current theory of many-electron systems is applied to the broken-symmetry (BS) states of the protein at the ZINDO level. While the one-electron tunneling approximation is found to hold in electron tunneling between the antiferromagnetic binuclear and tetranuclear Fe/S clusters without major orbital or spin rearrangement of the core electrons, induced polarization of the core electrons contributes significantly to decrease the electron transfer rates to 19-56 %. Calculated tunneling energy is about 3 eV higher than Fermi level in the band gap of the protein, which supports that the mechanism of electron transfer is quantum mechanical tunneling, as in the rest of the electron transport chain. Resulting electron tunneling pathways consist of up to three key contributing protein residues between neighboring Fe/S clusters. A signature of the wave properties of electrons is observed as distinct quantum interferences when multiple tunneling pathways exist. In N6a-N6b, electron tunnels along different pathways depending on the involved BS states, suggesting possible fluctuations of the tunneling pathways driven by the local protein environment. The calculated distance dependence of the electron transfer rates with internal water molecules included is in good agreement with a reported phenomenological relation.

Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

PubMed Central

Thompson, Michael C.; Wheatley, Nicole M.; Jorda, Julien; Sawaya, Michael R.; Gidaniyan, Soheil D.; Ahmed, Hoda; Yang, Zhongyu; McCarty, Krystal N.; Whitelegge, Julian P.; Yeates, Todd O.

2014-01-01

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein, whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date. PMID:25102080

Insight into Environmental Effects on Bonding and Redox Properties of [4Fe-4S] Clusters in Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niu, Shuqiang; Ichiye, Toshiko

The large differences in redox potentials between the HiPIPs and ferredoxins are generally attributed to hydrogen bonds and electrostatic effects from the protein and solvent. Recent ligand K-edge X-ray absorption studies by Solomon and co-workers show that the Fe-S covalencies of [4Fe-4S] clusters in the two proteins differ considerably apparently because of hydrogen bonds from water, indicating electronic effects may be important. However, combined density function theory (DFT) and photoelectron spectroscopy studies by our group and Wang and co-workers indicate that hydrogen bonds tune the potential of [4Fe-4S] clusters by mainly electrostatics. The DFT studies here rationalize both results, namelymore » that the observed change in the Fe-S covalency is due to differences in ligand conformation between the two proteins rather than hydrogen bonds. Moreover, the ligand conformation affects the calculated potentials by 100 mV and, thus, is a heretofore unconsidered means of tuning the potential.« less

Graphene-based copper oxide thin film nanostructures as high-efficiency photocathode for p-type dye-sensitized solar cells

NASA Astrophysics Data System (ADS)

Kilic, Bayram; Turkdogan, Sunay; Astam, Aykut; Baran, Sümeyra Seniha; Asgin, Mansur; Cebeci, Hulya; Urk, Deniz

2017-10-01

Graphene-based p-type dye-sensitized solar cells (p-DSSCs) have been proposed and fabricated using copper oxide urchin-like nanostructures (COUN) as photocathode with an FeS2 counter electrode (CE). COUN composed of Cu2O core sphere and CuO shell nanorods with overall diameters of 2 to 4 μm were grown by a simple hydrothermal method with self-assemble nucleation. It was figured out that the formation of copper oxide core/shell structures could be adjusted by an ammonia additive leading to pH change of the precursor solution. In addition to a photocathode, we also demonstrated FeS2 thin films as an efficient CE material alternative to the conventional Pt CEs in DSSCs. FeS2 nanostructures, with diameters of 50 to 80 nm, were synthesized by a similar hydrothermal approach. FeS2 nanostructures are demonstrated to be an outstanding CE material in p-DSSCs. We report graphene/COUN as photocathode and Pt/FeS2 as CE in p-DSSCs, and results show that the synergetic combination of electrodes in each side (increased interconnectivity between COUN and graphene layer, high surface area, and high catalytic activity of FeS2) increased the power conversion efficiency from 1.56% to 3.14%. The excellent performances of COUN and FeS2 thin film in working and CEs, respectively, make them unique choices among the various photocathode and CE materials studied.

Dynamics of an [Fe4S4(SPh)4]2- cluster explored via IR, Raman, and nuclear resonance vibrational spectroscopy (NRVS)-analysis using 36S substitution, DFT calculations, and empirical force fields.

PubMed

Xiao, Yuming; Koutmos, Markos; Case, David A; Coucouvanis, Dimitri; Wang, Hongxin; Cramer, Stephen P

2006-05-14

We have used four vibrational spectroscopies--FT-IR, FT-Raman, resonance Raman, and 57Fe nuclear resonance vibrational spectroscopy (NRVS)--to study the normal modes of the Fe-S cluster in [(n-Bu)4N]2[Fe4S4(SPh)4]. This [Fe4S4(SR)4]2- complex serves as a model for the clusters in 4Fe ferredoxins and high-potential iron proteins (HiPIPs). The IR spectra exhibited differences above and below the 243 K phase transition. Significant shifts with 36S substitution into the bridging S positions were also observed. The NRVS results were in good agreement with the low temperature data from the conventional spectroscopies. The NRVS spectra were interpreted by normal mode analysis using optimized Urey-Bradley force fields (UBFF) as well as from DFT theory. For the UBFF calculations, the parameters were refined by comparing calculated and observed NRVS frequencies and intensities. The frequency shifts after 36S substitution were used as an additional constraint. A D 2d symmetry Fe4S4S'4 model could explain most of the observed frequencies, but a better match to the observed intensities was obtained when the ligand aromatic rings were included for a D 2d Fe4S4(SPh)4 model. The best results were obtained using the low temperature structure without symmetry constraints. In addition to stretching and bending vibrations, low frequency modes between approximately 50 and 100 cm(-1) were observed. These modes, which have not been seen before, are interpreted as twisting motions with opposing sides of the cube rotating in opposite directions. In contrast with a recent paper on a related Fe4S4 cluster, we find no need to assign a large fraction of the low frequency NRVS intensity to 'rotational lattice modes'. We also reassign the 430 cm(-1) band as primarily an elongation of the thiophenolate ring, with approximately 10% terminal Fe-S stretch character. This study illustrates the benefits of combining NRVS with conventional Raman and IR analysis for characterization of Fe-S centers. DFT theory is shown to provide remarkable agreement with the experimental NRVS data. These results provide a reference point for the analysis of more complex Fe-S clusters in proteins.

Crystal structure of plant photosystem I

NASA Astrophysics Data System (ADS)

Ben-Shem, Adam; Frolow, Felix; Nelson, Nathan

2003-12-01

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4Å resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.

Chapter Eight - Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases

DOE Office of Scientific and Technical Information (OSTI.GOV)

King, Paul W; Mulder, David W; Artz, Jacob H.

The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas-treatments. Other themesmore » presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.« less

A Scalable Strategy To Develop Advanced Anode for Sodium-Ion Batteries: Commercial Fe3O4-Derived Fe3O4@FeS with Superior Full-Cell Performance.

PubMed

Hou, Bao-Hua; Wang, Ying-Ying; Guo, Jin-Zhi; Zhang, Yu; Ning, Qiu-Li; Yang, Yang; Li, Wen-Hao; Zhang, Jing-Ping; Wang, Xin-Long; Wu, Xing-Long

2018-01-31

A novel core-shell Fe 3 O 4 @FeS composed of Fe 3 O 4 core and FeS shell with the morphology of regular octahedra has been prepared via a facile and scalable strategy via employing commercial Fe 3 O 4 as the precursor. When used as anode material for sodium-ion batteries (SIBs), the prepared Fe 3 O 4 @FeS combines the merits of FeS and Fe 3 O 4 with high Na-storage capacity and superior cycling stability, respectively. The optimized Fe 3 O 4 @FeS electrode shows ultralong cycle life and outstanding rate capability. For instance, it remains a capacity retention of 90.8% with a reversible capacity of 169 mAh g -1 after 750 cycles at 0.2 A g -1 and 151 mAh g -1 at a high current density of 2 A g -1 , which is about 7.5 times in comparison to the Na-storage capacity of commercial Fe 3 O 4 . More importantly, the prepared Fe 3 O 4 @FeS also exhibits excellent full-cell performance. The assembled Fe 3 O 4 @FeS//Na 3 V 2 (PO 4 ) 2 O 2 F sodium-ion full battery gives a reversible capacity of 157 mAh g -1 after 50 cycles at 0.5 A g -1 with a capacity retention of 92.3% and the Coulombic efficiency of around 100%, demonstrating its applicability for sodium-ion full batteries as a promising anode. Furthermore, it is also disclosed that such superior electrochemical properties can be attributed to the pseudocapacitive behavior of FeS shell as demonstrated by the kinetics studies as well as the core-shell structure. In view of the large-scale availability of commercial precursor and ease of preparation, this study provide a scalable strategy to develop advanced anode materials for SIBs.

The iron-sulfur cluster assembly network component NARFL is a key element in the cellular defense against oxidative stress.

PubMed

Corbin, Monique V; Rockx, Davy A P; Oostra, Anneke B; Joenje, Hans; Dorsman, Josephine C

2015-12-01

Aim of this study was to explore cellular changes associated with increased resistance to atmospheric oxygen using high-resolution DNA and RNA profiling combined with functional studies. Two independently selected oxygen-resistant substrains of HeLa cells (capable of proliferating at >80% O2, i.e. hyperoxia) were compared with their parental cells (adapted to growth at 20% O2, but unable to grow at >80% O2). A striking consistent alteration found to be associated with the oxygen-resistant state appeared to be an amplified and overexpressed region on chromosome 16p13.3 harboring 21 genes. The driver gene of this amplification was identified by functional studies as NARFL, which encodes a component of the cytosolic iron-sulfur cluster assembly system. In line with this result we found the cytosolic c-aconitase activity as well as the nuclear protein RTEL1, both Fe-S dependent proteins, to be protected by NARFL overexpression under hyperoxia. In addition, we observed a protective effect of NARFL against hyperoxia-induced loss of sister-chromatid cohesion. NARFL thus appeared to be a key factor in the cellular defense against hyperoxia-induced oxidative stress in human cells. Our findings suggest that new insight into age-related degenerative processes may come from studies that specifically address the involvement of iron-sulfur proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution.

PubMed

Yonemoto, Isaac T; Matteri, Christopher W; Nguyen, Thao Amy; Smith, Hamilton O; Weyman, Philip D

2013-07-02

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

PubMed Central

Lange, Heike; Lisowsky, Thomas; Gerber, Jana; Mühlenhoff, Ulrich; Kispal, Gyula; Lill, Roland

2001-01-01

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process. PMID:11493598

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.

PubMed

Lange, H; Lisowsky, T; Gerber, J; Mühlenhoff, U; Kispal, G; Lill, R

2001-08-01

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.

Spectroscopic and Redox Studies of Valence-Delocalized [Fe2S2]+ Centers in Thioredoxin-Like Ferredoxins

PubMed Central

Subramanian, Sowmya; Duin, Evert C.; Fawcett, Sarah E. J.; Armstrong, Fraser A.; Meyer, Jacques; Johnson, Michael K.

2015-01-01

Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2]+ clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2]2+,+ centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2]+ centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, due to protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized S = 9/2 to valence-localized S = 1/2 [Fe2S2]+ clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe2S2]+ centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe−Fe interaction. The assignments provide experimental assessment of the double exchange parameter, B, for valence-delocalized [Fe2S2]+ centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe2S2]+ fragments in higher nuclearity Fe-S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe-S clusters in general is discussed in light of these results. PMID:25790339

Coordinate regulation of the Suf and Isc Fe-S cluster biogenesis pathways by IscR is essential for viability of Escherichia coli.

PubMed

Mettert, Erin L; Kiley, Patricia J

2014-12-01

Fe-S cluster biogenesis is essential for the viability of most organisms. In Escherichia coli, this process requires either the housekeeping Isc or the stress-induced Suf pathway. The global regulator IscR coordinates cluster synthesis by repressing transcription of the isc operon by [2Fe-2S]-IscR and activating expression of the suf operon. We show that either [2Fe-2S]-IscR or apo-IscR can activate suf, making expression sensitive to mainly IscR levels and not the cluster state, unlike isc expression. We also demonstrate that in the absence of isc, IscR-dependent suf activation is essential since strains lacking both the Isc pathway and IscR were not viable unless Suf was expressed ectopically. Similarly, removal of the IscR binding site in the sufA promoter also led to a requirement for isc. Furthermore, suf expression was increased in a Δisc mutant, presumably due to increased IscR levels in this mutant. This was surprising because the iron-dependent repressor Fur, whose higher-affinity binding at the sufA promoter should occlude IscR binding, showed only partial repression. In addition, Fur derepression was not sufficient for viability in the absence of IscR and the Isc pathway, highlighting the importance of direct IscR activation. Finally, a mutant lacking Fur and the Isc pathway increased suf expression to the highest observed levels and nearly restored [2Fe-2S]-IscR activity, providing a mechanism for regulating IscR activity under stress conditions. Together, these findings have enhanced our understanding of the homeostatic mechanism by which cells use one regulator, IscR, to differentially control Fe-S cluster biogenesis pathways to ensure viability. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Oxygen trapped by rare earth tetrahedral clusters in Nd4FeOS6: Crystal structure, electronic structure, and magnetic properties

NASA Astrophysics Data System (ADS)

Lin, Qisheng; Taufour, Valentin; Zhang, Yuemei; Wood, Max; Drtina, Thomas; Bud'ko, Sergey L.; Canfield, Paul C.; Miller, Gordon J.

2015-09-01

Single crystals of Nd4FeOS6 were grown from an Fe-S eutectic solution. Single crystal X-ray diffraction analysis revealed a Nd4MnOSe6-type structure (P63mc, a=9.2693(1) Å, c=6.6650(1)Å, V=495.94(1) Å3, Z=2), featuring parallel chains of face-sharing [FeS6×1/2]4- trigonal antiprisms and interlinked [Nd4OS3]4+ cubane-like clusters. Oxygen atoms were found to be trapped by Nd4 clusters in the [Nd4OS3]4+ chains. Structural differences among Nd4MnOSe6-type Nd4FeOS6 and the related La3CuSiS7- and Pr8CoGa3-type structures have been described. Magnetic susceptibility measurements on Nd4FeOS6 suggested the dominance of antiferromagnetic interactions at low temperature, but no magnetic ordering down to 2 K was observed. Spin-polarized electronic structure calculations revealed magnetic frustration with dominant antiferromagnetic interactions.

Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

PubMed

Selvaraj, Brinda; Buckel, Wolfgang; Golding, Bernard T; Ullmann, G Matthias; Martins, Berta M

2016-01-01

4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs. © 2016 S. Karger AG, Basel.

Cpe1786/IscR of Clostridium perfringens represses expression of genes involved in Fe-S cluster biogenesis.

PubMed

André, Gaelle; Haudecoeur, Elise; Courtois, Emmanuelle; Monot, Marc; Dupuy, Bruno; Rodionov, Dmitry A; Martin-Verstraete, Isabelle

2017-05-01

Cpe1786 of Clostridium perfringens is an Rrf2-type regulator containing the three-cysteine residues coordinating a Fe-S in IscR, the repressor controlling Fe-S homeostasis in enterobacteria. The cpe1786 gene formed an operon with iscSU involved in Fe-S biogenesis and tmrU. This operon was transcribed from a σ A -dependent promoter. We showed that in the heterologous host Bacillus subtilis, Cpe1786, renamed IscR Cp , negatively controlled its own transcription. We constructed an iscR mutant in C. perfringens. We then compared the expression profile of strain 13 and of the iscR mutant. IscR Cp controlled expression of genes involved in Fe-S biogenesis, in amino acid or sugar metabolisms, in fermentation pathways and in host compound utilization. We then demonstrated, using a ChIP-PCR experiment, that IscR Cp interacted with its promoter region in vivo in C. perfringens and with the promoter of cpe2093 encoding an amino acid ABC transporter. We utilized a comparative genomic approach to infer a candidate IscR binding motif and reconstruct IscR regulons in clostridia. We showed that point mutations in the conserved motif of 29 bp identified upstream of iscR decreased the cysteine-dependent repression of iscR mediated by IscR Cp . Copyright © 2016 Institut Pasteur. All rights reserved.

Differential expression of cysteine desulfurases in soybean

PubMed Central

2011-01-01

Background Iron-sulfur [Fe-S] clusters are prosthetic groups required to sustain fundamental life processes including electron transfer, metabolic reactions, sensing, signaling, gene regulation and stabilization of protein structures. In plants, the biogenesis of Fe-S protein is compartmentalized and adapted to specific needs of the cell. Many environmental factors affect plant development and limit productivity and geographical distribution. The impact of these limiting factors is particularly relevant for major crops, such as soybean, which has worldwide economic importance. Results Here we analyze the transcriptional profile of the soybean cysteine desulfurases NFS1, NFS2 and ISD11 genes, involved in the biogenesis of [Fe-S] clusters, by quantitative RT-PCR. NFS1, ISD11 and NFS2 encoding two mitochondrial and one plastid located proteins, respectively, are duplicated and showed distinct transcript levels considering tissue and stress response. NFS1 and ISD11 are highly expressed in roots, whereas NFS2 showed no differential expression in tissues. Cold-treated plants showed a decrease in NFS2 and ISD11 transcript levels in roots, and an increased expression of NFS1 and ISD11 genes in leaves. Plants treated with salicylic acid exhibited increased NFS1 transcript levels in roots but lower levels in leaves. In silico analysis of promoter regions indicated the presence of different cis-elements in cysteine desulfurase genes, in good agreement with differential expression of each locus. Our data also showed that increasing of transcript levels of mitochondrial genes, NFS1/ISD11, are associated with higher activities of aldehyde oxidase and xanthine dehydrogenase, two cytosolic Fe-S proteins. Conclusions Our results suggest a relationship between gene expression pattern, biochemical effects, and transcription factor binding sites in promoter regions of cysteine desulfurase genes. Moreover, data show proportionality between NFS1 and ISD11 genes expression. PMID:22099069

Mapping cellular Fe-S cluster uptake and exchange reactions - divergent pathways for iron-sulfur cluster delivery to human ferredoxins.

PubMed

Fidai, Insiya; Wachnowsky, Christine; Cowan, J A

2016-12-07

Ferredoxins are protein mediators of biological electron-transfer reactions and typically contain either [2Fe-2S] or [4Fe-4S] clusters. Two ferredoxin homologues have been identified in the human genome, Fdx1 and Fdx2, that share 43% identity and 69% similarity in protein sequence and both bind [2Fe-2S] clusters. Despite the high similarity, the two ferredoxins play very specific roles in distinct physiological pathways and cannot replace each other in function. Both eukaryotic and prokaryotic ferredoxins and homologues have been reported to receive their Fe-S cluster from scaffold/delivery proteins such as IscU, Isa, glutaredoxins, and Nfu. However, the preferred and physiologically relevant pathway for receiving the [2Fe-2S] cluster by ferredoxins is subject to speculation and is not clearly identified. In this work, we report on in vitro UV-visible (UV-vis) circular dichroism studies of [2Fe-2S] cluster transfer to the ferredoxins from a variety of partners. The results reveal rapid and quantitative transfer to both ferredoxins from several donor proteins (IscU, Isa1, Grx2, and Grx3). Transfer from Isa1 to Fdx2 was also observed to be faster than that of IscU to Fdx2, suggesting that Fdx2 could receive its cluster from Isa1 instead of IscU. Several other transfer combinations were also investigated and the results suggest a complex, but kinetically detailed map for cellular cluster trafficking. This is the first step toward building a network map for all of the possible iron-sulfur cluster transfer pathways in the mitochondria and cytosol, providing insights on the most likely cellular pathways and possible redundancies in these pathways.

Export Control Requirements for Tritium Processing Design and R&D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollis, William Kirk; Maynard, Sarah-Jane Wadsworth

This document will address requirements of export control associated with tritium plant design and processes. Los Alamos National Laboratory has been working in the area of tritium plant system design and research and development (R&D) since the early 1970’s at the Tritium Systems Test Assembly (TSTA). This work has continued to the current date with projects associated with the ITER project and other Office of Science Fusion Energy Science (OS-FES) funded programs. ITER is currently the highest funding area for the DOE OS-FES. Although export control issues have been integrated into these projects in the past a general guidance documentmore » has not been available for reference in this area. To address concerns with currently funded tritium plant programs and assist future projects for FES, this document will identify the key reference documents and specific sections within related to tritium research. Guidance as to the application of these sections will be discussed with specific detail to publications and work with foreign nationals.« less

Export Control Requirements for Tritium Processing Design and R&D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollis, William Kirk; Maynard, Sarah-Jane Wadsworth

2015-10-30

This document will address requirements of export control associated with tritium plant design and processes. Los Alamos National Laboratory has been working in the area of tritium plant system design and research and development (R&D) since the early 1970’s at the Tritium Systems Test Assembly (TSTA). This work has continued to the current date with projects associated with the ITER project and other Office of Science Fusion Energy Science (OS-FES) funded programs. ITER is currently the highest funding area for the DOE OS-FES. Although export control issues have been integrated into these projects in the past a general guidance documentmore » has not been available for reference in this area. To address concerns with currently funded tritium plant programs and assist future projects for FES, this document will identify the key reference documents and specific sections within related to tritium research. Guidance as to the application of these sections will be discussed with specific detail to publications and work with foreign nationals.« less

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

PubMed Central

2013-01-01

Background Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. Results We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Conclusions Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement. PMID:23819621

Community Resilience, the Foundation for Earth Science and the ESIP Federation: Bouncing Forward with Collective Impact

NASA Astrophysics Data System (ADS)

Robinson, E.

2015-12-01

The Federal Government has a long history of cross-community coordination between the Scientific Research community, and the Earth Observations and Data Provider communities. Since 1998, the Federation of Earth Science Information Partners (ESIP), organically organized using a collective impact approach that fostered these interactions primarily around Earth science interoperability problems. Unlike most collaborations, collective impact initiatives named in 2011 by the Stanford Social Innovation Review, involve a backbone infrastructure, a dedicated staff, and a structured process that leads to a common agenda, shared measurement, continuous communication, and mutually reinforcing activities among all participants. Over the last ten years, the Foundation for Earth Science (FES) has a proven track record of providing backbone support to ESIP. This presentation will cover FES's general approach to providing backbone support that enables communities to define shared agenda and then will show these practices in two case studies: (1) ESIP at-large as a mature network of developed partnerships and (2) a new project, the Local Community Resilience cluster. This new cluster aims to bridge the gap from the established ESIP network to engage local communities in order to equip citizens, professionals, and other decision-makers with the scientific underpinning necessary to make informed decisions (bounce forward) for society by leveraging the strong existing ESIP community, the backbone capabilities of FES and extending Federal Earth Science, Technology and Innovation Investments.

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly*♦

PubMed Central

Pandey, Alok; Gordon, Donna M.; Pain, Jayashree; Stemmler, Timothy L.; Dancis, Andrew; Pain, Debkumar

2013-01-01

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation. PMID:24217246

Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae

DOE PAGES

Sato, Trey K.; Tremaine, Mary; Parreiras, Lucas S.; ...

2016-10-14

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactionsmore » among genes encoding a xylose reductase ( GRE3), a component of MAP Kinase (MAPK) signaling ( HOG1), a regulator of Protein Kinase A (PKA) signaling ( IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis ( ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Lastly, our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.« less

Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

PubMed

Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

2013-12-27

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

PubMed

Sato, Trey K; Tremaine, Mary; Parreiras, Lucas S; Hebert, Alexander S; Myers, Kevin S; Higbee, Alan J; Sardi, Maria; McIlwain, Sean J; Ong, Irene M; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; McGee, Mick A; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L; Zhang, Yaoping; Coon, Joshua J; Hittinger, Chris Todd; Gasch, Audrey P; Landick, Robert

2016-10-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae

PubMed Central

Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert

2016-01-01

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH).

PubMed

Maliandi, Maria V; Busi, Maria V; Clemente, Marina; Zabaleta, Eduardo J; Araya, Alejandro; Gomez-Casati, Diego F

2007-02-01

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.

Cryptococcus neoformans Iron-Sulfur Protein Biogenesis Machinery Is a Novel Layer of Protection against Cu Stress.

PubMed

Garcia-Santamarina, Sarela; Uzarska, Marta A; Festa, Richard A; Lill, Roland; Thiele, Dennis J

2017-10-31

Copper (Cu) ions serve as catalytic cofactors to drive key biochemical processes, and yet Cu levels that exceed cellular homeostatic control capacity are toxic. The underlying mechanisms for Cu toxicity are poorly understood. During pulmonary infection by the fungal pathogen Cryptococcus neoformans , host alveolar macrophages compartmentalize Cu to the phagosome, and the ability to detoxify Cu is critical for its survival and virulence. Here, we report that iron-sulfur (Fe-S) clusters are critical targets of Cu toxicity in both Saccharomyces cerevisiae and C. neoformans in a manner that depends on the accessibility of Cu to the Fe-S cofactor. To respond to this Cu-dependent Fe-S stress, C. neoformans induces the transcription of mitochondrial ABC transporter Atm1, which functions in cytosolic-nuclear Fe-S protein biogenesis in response to Cu and in a manner dependent on the Cu metalloregulatory transcription factor Cuf1. As Atm1 functions in exporting an Fe-S precursor from the mitochondrial matrix to the cytosol, C. neoformans cells depleted for Atm1 are sensitive to Cu even while the Cu-detoxifying metallothionein proteins are highly expressed. We provide evidence for a previously unrecognized microbial defense mechanism to deal with Cu toxicity, and we highlight the importance for C. neoformans of having several distinct mechanisms for coping with Cu toxicity which together could contribute to the success of this microbe as an opportunistic human fungal pathogen. IMPORTANCE C. neoformans is an opportunistic pathogen that causes lethal meningitis in over 650,000 people annually. The severity of C. neoformans infections is further compounded by the use of toxic or poorly effective systemic antifungal agents as well as by the difficulty of diagnosis. Cu is a natural potent antimicrobial agent that is compartmentalized within the macrophage phagosome and used by innate immune cells to neutralize microbial pathogens. While the Cu detoxification machinery of C. neoformans is essential for virulence, little is known about the mechanisms by which Cu kills fungi. Here we report that Fe-S cluster-containing proteins, including members of the Fe-S protein biogenesis machinery itself, are critical targets of Cu toxicity and therefore that this biosynthetic process provides an important layer of defense against high Cu levels. Given the role of Cu ionophores as antimicrobials, understanding how Cu is toxic to microorganisms could lead to the development of effective, broad-spectrum antimicrobials. Moreover, understanding Cu toxicity could provide additional insights into the pathophysiology of human diseases of Cu overload such as Wilson's disease. Copyright © 2017 Garcia-Santamarina et al.

Mackinawite (FeS) Reduces Mercury(II) under Sulfidic Conditions

PubMed Central

2015-01-01

Mercury (Hg) is a toxicant of global concern that accumulates in organisms as methyl Hg. The production of methyl Hg by anaerobic bacteria may be limited in anoxic sediments by the sequestration of divalent Hg [Hg(II)] into a solid phase or by the formation of elemental Hg [Hg(0)]. We tested the hypothesis that nanocrystalline mackinawite (tetragonal FeS), which is abundant in sediments where Hg is methylated, both sorbs and reduces Hg(II). Mackinawite suspensions were equilibrated with dissolved Hg(II) in batch reactors. Examination of the solid phase using Hg LIII-edge extended X-ray absorption fine structure (EXAFS) spectroscopy showed that Hg(II) was indeed reduced in FeS suspensions. Measurement of purgeable Hg using cold vapor atomic fluorescence spectrometry (CVAFS) from FeS suspensions and control solutions corroborated the production of Hg(0) that was observed spectroscopically. However, a fraction of the Hg(II) initially added to the suspensions remained in the divalent state, likely in the form of β-HgS-like clusters associated with the FeS surface or as a mixture of β-HgS and surface-associated species. Complexation by dissolved S(-II) in anoxic sediments hinders Hg(0) formation, but, by contrast, Hg(II)–S(-II) species are reduced in the presence of mackinawite, producing Hg(0) after only 1 h of reaction time. The results of our work support the idea that Hg(0) accounts for a significant fraction of the total Hg in wetland and estuarine sediments. PMID:25180562

Cofactor composition and function of a H2-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy† †Electronic supplementary information (ESI) available: Tables with the simulation parameters and details of the Mössbauer, and EPR spectra (Tables S1–S4). additional EPR and Mössbauer spectra in Fig. S1–S9. See DOI: 10.1039/c5sc01560j Click here for additional data file.

PubMed Central

Roncaroli, Federico; Friedrich, Bärbel; Lenz, Oliver

2015-01-01

The regulatory hydrogenase (RH) from Ralstonia eutropha H16 acts as a sensor for the detection of environmental H2 and regulates gene expression related to hydrogenase-mediated cellular metabolism. In marked contrast to prototypical energy-converting [NiFe] hydrogenases, the RH is apparently insensitive to inhibition by O2 and CO. While the physiological function of regulatory hydrogenases is well established, little is known about the redox cycling of the [NiFe] center and the nature of the iron–sulfur (FeS) clusters acting as electron relay. The absence of any FeS cluster signals in EPR had been attributed to their particular nature, whereas the observation of essentially only two active site redox states, namely Ni-SI and Ni-C, invoked a different operant mechanism. In the present work, we employ a combination of Mössbauer, FTIR and EPR spectroscopic techniques to study the RH, and the results are consistent with the presence of three [4Fe–4S] centers in the small subunit. In the as-isolated, oxidized RH all FeS clusters reside in the EPR-silent 2+ state. Incubation with H2 leads to reduction of two of the [4Fe–4S] clusters, whereas only strongly reducing agents lead to reduction of the third cluster, which is ascribed to be the [4Fe–4S] center in ‘proximal’ position to the [NiFe] center. In the two different active site redox states, the low-spin FeII exhibits distinct Mössbauer features attributed to changes in the electronic and geometric structure of the catalytic center. The results are discussed with regard to the spectral characteristics and physiological function of H2-sensing regulatory hydrogenases. PMID:29142700

ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.

PubMed

Beilschmidt, Lena Kristina; Ollagnier de Choudens, Sandrine; Fournier, Marjorie; Sanakis, Ioannis; Hograindleur, Marc-André; Clémancey, Martin; Blondin, Geneviève; Schmucker, Stéphane; Eisenmann, Aurélie; Weiss, Amélie; Koebel, Pascale; Messaddeq, Nadia; Puccio, Hélène; Martelli, Alain

2017-05-11

Mammalian A-type proteins, ISCA1 and ISCA2, are evolutionarily conserved proteins involved in iron-sulfur cluster (Fe-S) biogenesis. Recently, it was shown that ISCA1 and ISCA2 form a heterocomplex that is implicated in the maturation of mitochondrial Fe 4 S 4 proteins. Here we report that mouse ISCA1 and ISCA2 are Fe 2 S 2 -containing proteins that combine all features of Fe-S carrier proteins. We use biochemical, spectroscopic and in vivo approaches to demonstrate that despite forming a complex, ISCA1 and ISCA2 establish discrete interactions with components of the late Fe-S machinery. Surprisingly, knockdown experiments in mouse skeletal muscle and in primary cultures of neurons suggest that ISCA1, but not ISCA2, is required for mitochondrial Fe 4 S 4 proteins biogenesis. Collectively, our data suggest that cellular processes with different requirements for ISCA1, ISCA2 and ISCA1-ISCA2 complex seem to exist.

The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster.

PubMed

Wiley, Sandra E; Paddock, Mark L; Abresch, Edward C; Gross, Larry; van der Geer, Peter; Nechushtai, Rachel; Murphy, Anne N; Jennings, Patricia A; Dixon, Jack E

2007-08-17

The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252-E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318-5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.

Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.

PubMed

Mapolelo, Daphne T; Zhang, Bo; Naik, Sunil G; Huynh, Boi Hanh; Johnson, Michael K

2012-10-16

The ability of Azotobacter vinelandii(Nif)IscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. (Nif)IscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV-visible absorption, circular dichroism, and variable-temperature magnetic circular dichroism, electron paramagnetic resonance, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5 cm(-1)) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm(-1)) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound (Nif)IscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ± 15 mV at pH 7.8 (versus NHE). l-Cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of (Nif)IscA. Fe(III)-bound (Nif)IscA was also shown to be a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.

Control of the transition between Ni-C and Ni-SI(a) states by the redox state of the proximal Fe-S cluster in the catalytic cycle of [NiFe] hydrogenase.

PubMed

Tai, Hulin; Nishikawa, Koji; Suzuki, Masayuki; Higuchi, Yoshiki; Hirota, Shun

2014-12-08

[NiFe] hydrogenase catalyzes the reversible cleavage of H2. The electrons produced by the H2 cleavage pass through three Fe-S clusters in [NiFe] hydrogenase to its redox partner. It has been reported that the Ni-SI(a), Ni-C, and Ni-R states of [NiFe] hydrogenase are involved in the catalytic cycle, although the mechanism and regulation of the transition between the Ni-C and Ni-SI(a) states remain unrevealed. In this study, the FT-IR spectra under light irradiation at 138-198 K show that the Ni-L state of [NiFe] hydrogenase is an intermediate between the transition of the Ni-C and Ni-SI(a) states. The transition of the Ni-C state to the Ni-SI(a) state occurred when the proximal [Fe4S4]p(2+/+) cluster was oxidized, but not when it was reduced. These results show that the catalytic cycle of [NiFe] hydrogenase is controlled by the redox state of its [Fe4S4]p(2+/+) cluster, which may function as a gate for the electron flow from the NiFe active site to the redox partner. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formaldehyde and methanol formation from reaction of carbon monoxide and hydrogen on neutral Fe2S2 clusters in the gas phase.

PubMed

Yin, Shi; Wang, Zhechen; Bernstein, Elliot R

2013-04-07

Reaction of CO with H2 on neutral FemSn clusters in a fast flow reactor is investigated both experimentally and theoretically. Single photon ionization at 118 nm is used to detect neutral cluster distributions through time of flight mass spectrometry. FemSn clusters are generated through laser ablation of a mixed iron-sulfur target in the presence of a pure helium carrier gas. A strong size dependent reactivity of (FeS)m clusters toward CO is characterized. The reaction FeS + CO → Fe + OCS is found for the FeS cluster, and the association product Fe2S2CO is observed for the Fe2S2 cluster. Products Fe2S2(13)COH2 and Fe2S2(13)COH4 are identified for reactions of (13)CO and H2 on Fe2S2 clusters: this suggests that the Fe2S2 cluster has a high catalytic activity for hydrogenation reactions of CO to form formaldehyde and methanol. Density functional theory (DFT) calculations are performed to explore the potential energy surfaces for the two reactions: Fe2S2 + CO + 2H2 → Fe2S2 + CH3OH; and Fe2S2 + CO + H2 → Fe2S2 + CH2O. A barrierless, thermodynamically favorable pathway is obtained for both catalytic processes. Catalytic cycles for formaldehyde and methanol formation from CO and H2 on a Fe2S2 cluster are proposed based on our experimental and theoretical investigations. The various reaction mechanisms explored by DFT are in good agreement with the experimental results. Condensed phase iron sulfide, which contains exposed Fe2S2 units on its surface, is suggested to be a good catalyst for low temperature formaldehyde/methanol synthesis.

The [4Fe-4S](2+) cluster in reconstituted biotin synthase binds S-adenosyl-L-methionine.

PubMed

Cosper, Michele Mader; Jameson, Guy N L; Davydov, Roman; Eidsness, Marly K; Hoffman, Brian M; Huynh, Boi Hanh; Johnson, Michael K

2002-11-27

The combination of resonance Raman, electron paramagnetic resonance and Mössbauer spectroscopies has been used to investigate the effect of S-adenosyl-l-methionine (SAM) on the spectroscopic properties of the [4Fe-4S]2+ cluster in biotin synthase. The results indicate that SAM interacts directly at a unique iron site of the [4Fe-4S]2+ cluster in BioB and support the hypothesis of a common inner-sphere mechanism for the reductive cleavage of SAM in the radical SAM family of Fe-S enzymes.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nancy Ryan Gray

Iron-sulfur (FeS) centers are essential for biology and inspirational in chemistry. These protein cofactors are broadly defined as active sites in which Fe is coordinated by S-donor ligands, often in combination with extra non-protein components, for example, additional metal atoms such as Mo and Ni, and soft ligands such as CN{sup -} and CO. Iron-sulfur centers are inherently air sensitive: they are found in essentially all organisms and it is possible that they were integral components of the earliest forms of life, well before oxygen (O{sub 2}) appeared. Proteins containing FeS cofactors perform a variety of biological functions ranging acrossmore » electron transfer, acid-base catalysis, and sensing where they are agents for cell regulation through transcription (DNA) or translation (RNA). They are redox catalysts for radical-based reactions and the activation of H{sub 2}, N{sub 2} and CO{sub 2}, processes that offer scientific and economic challenges for industry. Iron-sulfur centers provide the focus for fundamental investigations of chemical bonding, spectroscopy and paramagnetism, and their functions have numerous implications for health and medicine and applications for technology, including renewable energy. The 2010 Iron-Sulfur Enzymes GRC will bring together researchers from different disciplines for in-depth discussions and presentations of the latest developments. There will be sessions on structural and functional analogues of FeS centers, advances in physical methods, roles of FeS centers in energy and technology, catalysis (including radical-based rearrangements and the activation of nitrogen, hydrogen and carbon), long-range electron transfer, FeS centers in health and disease, cellular regulation, cofactor assembly, their relevance in industry, and experiments and hypotheses relating to the origins of life.« less

Combined Mössbauer spectroscopic, multi-edge X-ray absorption spectroscopic, and density functional theoretical study of the radical SAM enzyme spore photoproduct lyase.

PubMed

Silver, Sunshine C; Gardenghi, David J; Naik, Sunil G; Shepard, Eric M; Huynh, Boi Hanh; Szilagyi, Robert K; Broderick, Joan B

2014-03-01

Spore photoproduct lyase (SPL), a member of the radical S-adenosyl-L-methionine (SAM) superfamily, catalyzes the direct reversal of the spore photoproduct, a thymine dimer specific to bacterial spores, to two thymines. SPL requires SAM and a redox-active [4Fe-4S] cluster for catalysis. Mössbauer analysis of anaerobically purified SPL indicates the presence of a mixture of cluster states with the majority (40 %) as [2Fe-2S](2+) clusters and a smaller amount (15 %) as [4Fe-4S](2+) clusters. On reduction, the cluster content changes to primarily (60 %) [4Fe-4S](+). The speciation information from Mössbauer data allowed us to deconvolute iron and sulfur K-edge X-ray absorption spectra to uncover electronic (X-ray absorption near-edge structure, XANES) and geometric (extended X-ray absorption fine structure, EXAFS) structural features of the Fe-S clusters, and their interactions with SAM. The iron K-edge EXAFS data provide evidence for elongation of a [2Fe-2S] rhomb of the [4Fe-4S] cluster on binding SAM on the basis of an Fe···Fe scatterer at 3.0 Å. The XANES spectra of reduced SPL in the absence and presence of SAM overlay one another, indicating that SAM is not undergoing reductive cleavage. The X-ray absorption spectroscopy data for SPL samples and data for model complexes from the literature allowed the deconvolution of contributions from [2Fe-2S] and [4Fe-4S] clusters to the sulfur K-edge XANES spectra. The analysis of pre-edge features revealed electronic changes in the Fe-S clusters as a function of the presence of SAM. The spectroscopic findings were further corroborated by density functional theory calculations that provided insights into structural and electronic perturbations that can be correlated by considering the role of SAM as a catalyst or substrate.

Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

PubMed Central

Schmitz-Abe, Klaus; Ciesielski, Szymon J.; Schmidt, Paul J.; Campagna, Dean R.; Rahimov, Fedik; Schilke, Brenda A.; Cuijpers, Marloes; Rieneck, Klaus; Lausen, Birgitte; Linenberger, Michael L.; Sendamarai, Anoop K.; Guo, Chaoshe; Hofmann, Inga; Newburger, Peter E.; Matthews, Dana; Shimamura, Akiko; Snijders, Pieter J. L. M.; Towne, Meghan C.; Niemeyer, Charlotte M.; Watson, Henry G.; Dziegiel, Morten H.; Heeney, Matthew M.; May, Alison; Bottomley, Sylvia S.; Swinkels, Dorine W.; Markianos, Kyriacos; Craig, Elizabeth A.

2015-01-01

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance. PMID:26491070

Sulfur Adsorption on the Goethite (110) Surface

NASA Astrophysics Data System (ADS)

Simonetti, S.; Damiani, D.; Brizuela, G.; Juan, A.

The electronic structure of S adsorption on goethite (110) surface has been studied by ASED-MO cluster calculations. For S location, the most exposed surface atoms of goethite surface were selected. The calculations show that the surface offers several places for S adsorption. The most energetically stable system corresponds to S location above H atom. We studied in detail the configurations that correspond to the higher OP values. For these configurations, the H-S and Fe-S computed distances are 2.1 and 3.7 Å, respectively. The H-S and Fe-S are mainly bonding interaction with OP values of 0.156 and 0.034, respectively. The Fe-S interaction mainly involves Fe 3dx2-y2 atomic orbitals with lesser participation of Fe 4py and Fe 3dyz atomic orbitals. The O-S interaction shows the same bonding and antibonding contributions giving a small OP value. The O-S interaction involves O 2p orbitals. There is an electron transfer to the Fe atom from the S atom. On the other hand, there is an electron transfer to S atom from the H and O atoms, respectively.

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.

PubMed Central

Martín, A E; Burgess, B K; Stout, C D; Cash, V L; Dean, D R; Jensen, G M; Stephens, P J

1990-01-01

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure. PMID:2153958

Epigenetic role for the conserved Fe-S cluster biogenesis protein AtDRE2 in Arabidopsis thaliana.

PubMed

Buzas, Diana Mihaela; Nakamura, Miyuki; Kinoshita, Tetsu

2014-09-16

On fertilization in Arabidopsis thaliana, one maternal gamete, the central cell, forms a placenta-like tissue, the endosperm. The DNA glycosylase DEMETER (DME) excises 5-methylcytosine via the base excision repair pathway in the central cell before fertilization, creating patterns of asymmetric DNA methylation and maternal gene expression across DNA replications in the endosperm lineage (EDL). Active DNA demethylation in the central cell is essential for transcriptional activity in the EDL of a set of genes, including FLOWERING WAGENINGEN (FWA). A DME-binding motif for iron-sulfur (Fe-S) cluster cofactors is indispensable for its catalytic activity. We used an FWA-GFP reporter to find mutants defective in maternal activation of FWA-GFP in the EDL, and isolated an allele of the yeast Dre2/human antiapoptotic factor CIAPIN1 homolog, encoding an enzyme previously implicated in the cytosolic Fe-S biogenesis pathway (CIA), which we named atdre2-2. We found that AtDRE2 acts in the central cell to regulate genes maternally activated in the EDL by DME. Furthermore, the FWA-GFP expression defect in atdre2-2 was partially suppressed genetically by a mutation in the maintenance DNA methyltransferase MET1; the DNA methylation levels at four DME targets increased in atdre2-2 seeds relative to WT. Although atdre2-2 shares zygotic seed defects with CIA mutants, it also uniquely manifests dme phenotypic hallmarks. These results demonstrate a previously unidentified epigenetic function of AtDRE2 that may be separate from the CIA pathway.

Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

PubMed

Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Powis, Zoe; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

2017-12-15

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. © The Author 2017. Published by Oxford University Press.

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

PubMed Central

Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

2016-01-01

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

Monothiol CGFS Glutaredoxins and BolA-like Proteins: [2Fe-2S] Binding Partners in Iron Homeostasis

PubMed Central

Li, Haoran; Outten, Caryn E.

2012-01-01

Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S]-bridging interaction between CGFS Grxs and a BolA-like protein in S. cerevisiae provided the first molecular-level understanding of the iron regulation mechanism in this model eukaryote, and established the ubiquitous CGFS Grxs and BolA-like proteins as novel Fe-S cluster-binding regulatory partners. Parallel studies focused on E. coli and human homologues for CGFS Grxs and BolA-like proteins have supported the studies in yeast and provided additional clues to their involvement in cellular iron metabolism. Herein we review recent progress in uncovering the cellular and molecular mechanisms by which CGFS Grxs and BolA-like proteins help regulate iron metabolism in both eukaryotic and prokaryotic organisms. PMID:22583368

Stress Response and Virulence Functions of the Acinetobacter baumannii NfuA Fe-S Scaffold Protein

PubMed Central

Zimbler, Daniel L.; Park, Thomas M.; Arivett, Brock A.; Penwell, William F.; Greer, Samuel M.; Woodruff, Tessa M.; Tierney, David L.

2012-01-01

To successfully establish an infection, Acinetobacter baumannii must overcome the iron starvation and oxidative stress imposed by the human host. Although previous studies have shown that ATCC 19606T cells acquire iron via the acinetobactin-mediated siderophore system, little is known about intracellular iron metabolism and its relation to oxidative stress in this pathogen. Screening of an insertion library resulted in the isolation of the ATCC 19606T derivative 1644, which was unable to grow in iron-chelated media. Rescue cloning and DNA sequencing showed that the insertion inactivated a gene coding for an NfuA Fe-S cluster protein ortholog, without any effect on the expression of the acinetobactin system. The nfuA mutant was also more sensitive to hydrogen peroxide and cumene hydroperoxide than the parental strain. The iron chelation- and oxidative-stress-deficient responses of this mutant were corrected when complemented with either the ATCC 19606T parental allele or the Escherichia coli MG1655 nfuA ortholog. Furthermore, electron paramagnetic resonance (EPR) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analyses showed that the ATCC 19606T NfuA ortholog has iron-binding properties compatible with the formation of [Fe-S] cluster protein. Ex vivo and in vivo assays using human epithelial cells and Galleria mellonella, respectively, showed that NfuA is critical for bacterial growth independent of their capacity to acquire iron or the presence of excess of free iron. Taken together, these observations indicate that the A. baumannii NfuA ortholog plays a role in intracellular iron utilization and protection from oxidative-stress responses that this pathogen could encounter during the infection of the human host. PMID:22467784

Ambulatory and Non-Ambulatory Benefits of Lower Limb Exoskeleton Use, with and without FES, in Clinical and Community Settings

DTIC Science & Technology

2016-10-01

15. SUBJECT TERMS spinal cord injury, paraplegia, exoskeleton, physical medicine and rehabilitation, rehabilitation research, legged mobility...2. KEYWORDS • spinal cord injury • paraplegia • exoskeleton • physical medicine and rehabilitation • rehabilitation research • legged mobility...study protocol notebooks and record books have been assembled with session-by-session instructions and data entry. o Electronic data entry forms have

Electrochemical cell and method of assembly

DOEpatents

Shimotake, Hiroshi; Voss, Ernst C. H.; Bartholme, Louis G.

1979-01-01

A method of preparing an electrochemical cell is disclosed which permits the assembly to be accomplished in air. The cell includes a metal sulfide as the positive electrode reactant, lithium alloy as the negative electrode reactant and an alkali metal, molten salt electrolyte. Positive electrode reactant is introduced as Li.sub.2 FeS.sub.2, a single-phase compound produced by the reaction of Li.sub.2 S and FeS. The use of this compound permits introduction of lithium in an oxidized form. Additional lithium can be introduced in the negative electrode structure enclosed within an aluminum foil envelope between layers of porous aluminum. Molten salt electrolyte is added after assembly and evacuation of the cell by including an interelectrode separator that has been prewet with an organic solution of KCl.

Synthesis, structural characterization and conversion of dinuclear iron-sulfur clusters containing the disulfide ligand: [Cp*Fe(μ-η2:η2-bdt)(cis-μ-η1:η1-S2)FeCp*], [Cp*Fe(μ-S(C6H4S2))(cis-μ-η1:η1-S2)FeCp*], and [{Cp*Fe(bdt)}2(trans-μ-η1:η1-S2)].

PubMed

Ji, Xiaoxiao; Tong, Peng; Yang, Dawei; Wang, Baomin; Zhao, Jinfeng; Li, Yang; Qu, Jingping

2017-03-21

The treatment of [Cp*Fe(μ-η 2 :η 4 -bdt)FeCp*] (1, Cp* = η 5 -C 5 Me 5 , bdt = benzene-1,2-dithiolate) with 1/4 equiv. of elemental sulfur (S 8 ) gave a dinuclear iron-sulfur cluster [Cp*Fe(μ-η 2 :η 2 -bdt)(cis-μ-η 1 :η 1 -S 2 )FeCp*] (2), which contains a cis-1,2-disulfide ligand. When complex 2 further interacted with 1/8 equiv. of S 8 , another sulfur atom inserted into an Fe-S bond to give a rare product [Cp*Fe(μ-S(C 6 H 4 S 2 ))(cis-μ-η 1 :η 1 -S 2 )FeCp*] (3). Unexpectedly, a trans-1,2 disulfide-bridged diiron complex [{Cp*Fe(bdt)} 2 (trans-μ-η 1 :η 1 -S 2 )] (4) was isolated from the reaction of complex 1 with 1/2 equiv. of S 8 , which represents a structural isomer of [2Fe-2S] ferredoxin-type clusters. In addition, cis-1,2-disulfide-bridged complex 3 can slowly convert into trans-1,2-disulfide-bridged complex 4 and the complex [Cp*Fe(μ-η 2 :η 2 -S 2 )(cis-μ-η 1 :η 1 -S 2 )FeCp*] (5) by self-assembly reaction at ambient temperature, which is evidenced by time-dependent 1 H NMR spectroscopy.

Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes.

PubMed Central

Lowe, D J; Eady, R R; Thorneley, N F

1978-01-01

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo--Fe protein showed that they arose from Fe--S clusters in the Mo--Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the --1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme--product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe--S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO. PMID:210766

Cysteine and cystine adsorption on FeS2(100)

NASA Astrophysics Data System (ADS)

Suzuki, Teppei; Yano, Taka-aki; Hara, Masahiko; Ebisuzaki, Toshikazu

2018-08-01

Iron pyrite (FeS2) is the most abundant metal sulfide on Earth. Owing to its reactivity and catalytic activity, pyrite has been studied in various research fields such as surface science, geochemistry, and prebiotic chemistry. Importantly, native iron-sulfur clusters are typically coordinated by cysteinyl ligands of iron-sulfur proteins. In the present paper, we study the adsorption of L-cysteine and its oxidized dimer, L-cystine, on the FeS2 surface, using electronic structure calculations based density functional theory and Raman spectroscopy measurements. Our calculations suggest that sulfur-deficient surfaces play an important role in the adsorption of cysteine and cystine. In the thiol headgroup adsorption on the sulfur-vacancy site, dissociative adsorption is found to be energetically favorable compared with molecular adsorption. In addition, the calculations indicate that, in the cystine adsorption on the defective surface under vacuum conditions, the formation of the S-Fe bond is energetically favorable compared with molecular adsorption. Raman spectroscopic measurements suggest the formation of cystine molecules through the S-S bond on the pyrite surface in aqueous solution. Our results might have implications for chemical evolution at mineral surfaces on the early Earth and the origin of iron-sulfur proteins, which are believed to be one of the most ancient families of proteins.

Structural characterization of metal binding to a cold-adapted frataxin.

PubMed

Noguera, Martín E; Roman, Ernesto A; Rigal, Juan B; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2015-06-01

Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

Phosphorylation of Akt by SC79 Prevents Iron Accumulation and Ameliorates Early Brain Injury in a Model of Experimental Subarachnoid Hemorrhage.

PubMed

Hao, Shuangying; Song, Chuanhui; Shang, Longcheng; Yu, Jiang; Qiao, Tong; Li, Kuanyu

2016-03-10

Previous studies have demonstrated that activation of Akt may alleviate early brain injury (EBI) following subarachnoid hemorrhage (SAH). This study is undertaken to determine whether iron metabolism is involved in the beneficial effect of Akt activation after SAH. Therefore, we used a novel molecule, SC79, to activate Akt in an experimental Sprague-Dawley rat model of SAH. Rats were randomly divided into four groups as follows: sham, SAH, SAH + vehicle, SAH + SC79. The results confirmed that SC79 effectively enhanced the defense against oxidative stress and alleviated EBI in the temporal lobe after SAH. Interestingly, we found that phosphorylation of Akt by SC79 reduced cell surface transferrin receptor-mediated iron uptake and promoted ferroportin-mediated iron transport after SAH. As a result, SC79 administration diminished the iron content in the brain tissue. Moreover, the impaired Fe-S cluster biogenesis was recovered and loss of the activities of the Fe-S cluster-containing enzymes were regained, indicating that injured mitochondrial functions are restored to healthy levels. These findings suggest that disrupted iron homeostasis could contribute to EBI and Akt activation may regulate iron metabolism to relieve iron toxicity, further protecting neurons from EBI after SAH.

Two Fe-S clusters catalyse sulfur insertion by Radical-SAM methylthiotransferases

PubMed Central

Forouhar, Farhad; Arragain, Simon; Atta, Mohamed; Gambarelli, Serge; Mouesca, Jean-Marie; Hussain, Munif; Xiao, Rong; Kieffer-Jaquinod, Sylvie; Seetharaman, Jayaraman; Acton, Thomas B.; Montelione, Gaetano T.

2014-01-01

How living organisms create carbon-sulfur bonds during biosynthesis of critical sulphur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a sub-group of Radical-SAM enzymes that bear two [4Fe-4S] clusters. One cluster binds S-Adenosylmethionine and generates an Ado• radical via a well- established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting Radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur co-substrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction. PMID:23542644

Oxygen consumption during functional electrical stimulation-assisted exercise in persons with spinal cord injury: implications for fitness and health.

PubMed

Hettinga, Dries M; Andrews, Brian J

2008-01-01

A lesion in the spinal cord leads in most cases to a significant reduction in active muscle mass, whereby the paralysed muscles cannot contribute to oxygen consumption (VO2) during exercise. Consequently, persons with spinal cord injury (SCI) can only achieve high VO2 values by excessively stressing the upper body musculature, which might increase the risk of musculoskeletal overuse injury. Alternatively, the muscle mass involved may be increased by using functional electrical stimulation (FES). FES-assisted cycling, FES-cycling combined with arm cranking (FES-hybrid exercise) and FES-rowing have all been suggested as candidates for cardiovascular training in SCI. In this article, we review the levels of VO2 (peak [VO2peak] and sub-peak [VO2sub-peak]) that have been reported for SCI subjects using these FES exercise modalities. A systematic literature search in MEDLINE, EMBASE, AMED, CINAHL, SportDiscus and the authors' own files revealed 35 studies that reported on 499 observations of VO2 levels achieved during FES-exercise in SCI. The results show that VO2peak during FES-rowing (1.98 L/min, n = 17; 24.1 mL/kg/min, n = 11) and FES-hybrid exercise (1.78 L/min, n = 67; 26.5 mL/kg/min, n = 35) is considerably higher than during FES-cycling (1.05 L/min, n = 264; 14.3 mL/kg/min, n = 171). VO2sub-peak values during FES-hybrid exercise were higher than during FES-cycling. FES-exercise training can produce large increases in VO2peak; the included studies report average increases of +11% after FES-rowing training, +12% after FES-hybrid exercise training and +28% after FES-cycling training. This review shows that VO2 during FES-rowing or FES-hybrid exercise is considerably higher than during FES-cycling. These observations are confirmed by a limited number of direct comparisons; larger studies to test the differences in effectiveness of the various types of FES-exercise as cardiovascular exercise are needed. The results to date suggest that FES-rowing and FES-hybrid are more suited for high-intensity, high-volume exercise training than FES-cycling. In able-bodied people, such exercise programmes have shown to result in superior health and fitness benefits. Future research should examine whether similar high-intensity and high-volume exercise programmes also give persons with SCI superior fitness and health benefits. This kind of research is very timely given the high incidence of physical inactivity-related health conditions in the aging SCI population.

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from Allochromatium vinosum and some EPR and IR properties of the hydrogenase module.

PubMed

Long, Minnan; Liu, Jingjing; Chen, Zhifeng; Bleijlevens, Boris; Roseboom, Winfried; Albracht, Simon P J

2007-01-01

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M (r) = 52 kDa) and a small (M (r) = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe-S clusters, three [2Fe-2S] clusters and five [4Fe-4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe-S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H(2)-dependent reduction of NAD(+). Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni-Fe active site and a [4Fe-4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni(a)-C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.

Atrophy, ultra-structural disorders, severe atrophy and degeneration of denervated human muscle in SCI and Aging. Implications for their recovery by Functional Electrical Stimulation, updated 2017.

PubMed

Kern, Helmut; Hofer, Cristian; Loefler, Stefan; Zampieri, Sandra; Gargiulo, Paolo; Baba, Alfonc; Marcante, Andrea; Piccione, Francesco; Pond, Amber; Carraro, Ugo

2017-07-01

Long-term lower motor neuron denervation of skeletal muscle is known to result in degeneration of muscle with replacement by adipose and fibrotic tissues. However, long-term survival of a subset of skeletal myofibers also occurs. We performed transverse and longitudinal studies of patients with spinal cord injury (SCI), patients specifically complete Conus and Cauda Equina Syndrome and also of active and sedentary seniors which included analyses of muscle biopsies from the quadriceps m. Surprisingly, we discovered that human denervated myofibers survive years of denervation after full and irreversible disconnection from their motor neurons. We found that atrophic myofibers could be rescued by home-based Functional Electrical Stimulation (h-bFES), using purpose developed stimulators and electrodes. Although denervated myofibers quickly lose the ability to sustain high-frequency contractions, they respond to very long impulses that are able to allow for re-emergence of tetanic contractions. A description of the early muscle changes in humans are hampered by a paucity of patients suffering complete Conus and Cauda Equina Syndrome, but the cohort enrolled in the EU RISE Project has shown that even five years after SCI, severe atrophic myofibers with a peculiar cluster reorganization of myonuclei are present in human muscles and respond to h-bFES. Human myofibers survive permanent denervation longer than generally accepted and they respond to h-bFES beyond the stage of simple atrophy. Furthermore, long-term denervation/reinnervation events occur in elderly people and are part of the mechanisms responsible for muscle aging and again h-bFES was beneficial in delaying aging decay.

Comparison of Gait Aspects According to FES Stimulation Position Applied to Stroke Patients

PubMed Central

Mun, Byeong-mu; Kim, Tae-ho; Lee, Jin-hwan; Lim, Jin-youg; Seo, Dong-kwon; Lee, Dong-jin

2014-01-01

[Purpose] This study sought to identify the gait aspects according to the FES stimulation position in stroke patients during gait training. [Subjects and Methods] To perform gait analysis, ten stroke patients were grouped based on 4 types of gait conditions: gait without FES stimulation (non-FES), gait with FES stimulation on the tibialis anterior (Ta), gait with FES stimulation on the tibialis anterior and quadriceps (TaQ), and gait with FES stimulation on the tibialis anterior and gluteus medius (TaGm). [Results] Based on repeated measures analysis of variance of measurements of gait aspects comprised of gait speed, gait cycle, and step length according to the FES stimulation position, the FES stimulation significantly affected gait aspects. [Conclusion] In conclusion, stimulating the tibialis anterior and quadriceps and stimulating the tibialis anterior and gluteus medius are much more effective than stimulating only the tibialis anterior during gait training in stroke patients using FES. PMID:24764634

Two variants of fat embolism syndrome evolving in a young patient with multiple fractures

PubMed Central

Bajuri, Mohd Yazid; Johan, Rudy Reza; Shukur, Hassan

2013-01-01

Fat embolism syndrome (FES) is a continuum of fat emboli. Variants of FES: acute fulminant form and classic FES are postulated to represent two different pathomechanisms. Acute fulminant FES occurs during the first 24 h. It is attributed to massive mechanical blockage pulmonary vasculature by the fat emboli. The classic FES typically has a latency period of 24–36 h manifestation of respiratory failure and other signs of fat embolism. Progression of asymptomatic fat embolism with FES frequently represents inadequate treatment of hypovolaemic shock. We present a rare case of two variants of FES evolving in a patient with multiple fractures to emphasis the importance of adequate and appropriate treatment of shock in preventing the development of FES. Since supportive therapy which is a ventilatory support remains as the treatment of FES, it is appropriate to treat FES in the intensive care unit setting. PMID:23576653

Two variants of fat embolism syndrome evolving in a young patient with multiple fractures.

PubMed

Bajuri, Mohd Yazid; Johan, Rudy Reza; Shukur, Hassan

2013-04-09

Fat embolism syndrome (FES) is a continuum of fat emboli. Variants of FES: acute fulminant form and classic FES are postulated to represent two different pathomechanisms. Acute fulminant FES occurs during the first 24 h. It is attributed to massive mechanical blockage pulmonary vasculature by the fat emboli. The classic FES typically has a latency period of 24-36 h manifestation of respiratory failure and other signs of fat embolism. Progression of asymptomatic fat embolism with FES frequently represents inadequate treatment of hypovolaemic shock. We present a rare case of two variants of FES evolving in a patient with multiple fractures to emphasis the importance of adequate and appropriate treatment of shock in preventing the development of FES. Since supportive therapy which is a ventilatory support remains as the treatment of FES, it is appropriate to treat FES in the intensive care unit setting.

Growth Mechanism of Cluster-Assembled Surfaces: From Submonolayer to Thin-Film Regime

NASA Astrophysics Data System (ADS)

Borghi, Francesca; Podestà, Alessandro; Piazzoni, Claudio; Milani, Paolo

2018-04-01

Nanostructured films obtained by assembling preformed atomic clusters are of strategic importance for a wide variety of applications. The deposition of clusters produced in the gas phase onto a substrate offers the possibility to control and engineer the structural and functional properties of the cluster-assembled films. To date, the microscopic mechanisms underlying the growth and structuring of cluster-assembled films are poorly understood, and, in particular, the transition from the submonolayer to the thin-film regime is experimentally unexplored. Here we report the systematic characterization by atomic force microscopy of the evolution of the structural properties of cluster-assembled films deposited by supersonic cluster beam deposition. As a paradigm of nanostructured systems, we focus our attention on cluster-assembled zirconia films, investigating the influence of the building block dimensions on the growth mechanisms and roughening of the thin films, following the growth process from the early stages of the submonolayer to the thin-film regime. Our results demonstrate that the growth dynamics in the submonolayer regime determines different morphological properties of the cluster-assembled thin film. The evolution of the roughness with the number of deposited clusters reproduces the growth exponent of the ballistic deposition in the 2 +1 model from the submonolayer to the thin-film regime.

Corneal biomechanical properties in floppy eyelid syndrome.

PubMed

Muniesa, MaJesús; Muniesa Royo, MaJesús; March, Ana; March de Ribot, Ana; Sánchez-de-la-Torre, Manuel; Huerva, Valetín; Huerva Escanilla, Valetín; Jurjo, Carmen; Jurjo Campo, Carmen; Barbé, Ferran; Barbé Illa, Ferran

2015-05-01

To determine corneal biomechanical properties in patients with floppy eyelid syndrome (FES) and to compare them with eyes of controls. This case-control study included 208 eyes (72 eyes with FES and 136 without FES) of 107 patients (37 patients with FES and 70 without FES). Patients underwent a complete clinical eye examination that included corneal biomechanical evaluation carried out with the Reichert Ocular Response Analyzer. Corneal hysteresis (CH), corneal resistance factor (CRF), central corneal thickness (CCT), Goldmann-correlated intraocular pressure (IOPg), and corneal-compensated intraocular pressure (IOPcc) were evaluated. Mean CH was significantly lower in patients with FES than in those without FES (9.51 ± 1.56 vs. 11.66 ± 9.11; P < 0.001). These results remained statistically significant after adjusting for age and apnea-hypoapnea index (AHI) (P = 0.028). Mean CRF was 10.02 ± 2.08 in the group of patients with FES and 11.21 ± 5.36 in the group of patients without FES (P = 0.001). Mean IOPcc was 17.7 ± 4.8 in patients with FES and 16.3 ± 4.4 in those without FES (P = 0.036). After adjusting for age and AHI, these differences in CRF and IOPcc were not statistically significant (P = 0.26 and P = 0.87, respectively). No statistically significant difference was found between patients with and without FES for Goldmann-correlated intraocular pressure or CCT. Patients with FES had statistically lower CH values. Our findings suggest that corneal biomechanical properties could be changed in patients with FES, reflecting additional structural changes in FES.

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

PubMed

Zhang, Peng; Liu, Yu; Wang, Min; Dong, Miren; Liu, Zhaoqun; Jia, Zhihao; Wang, Weilin; Zhang, Anguo; Wang, Lingling; Song, Linsheng

2018-07-01

Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H 2 O 2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

First-principles calculations of the structural, elastic and thermodynamic properties of mackinawite (FeS) and pyrite (FeS2)

NASA Astrophysics Data System (ADS)

Wen, Xiangli; Liang, Yuxuan; Bai, Pengpeng; Luo, Bingwei; Fang, Teng; Yue, Luo; An, Teng; Song, Weiyu; Zheng, Shuqi

2017-11-01

The thermodynamic properties of Fe-S compounds with different crystal structure are very different. In this study, the structural, elastic and thermodynamic properties of mackinawite (FeS) and pyrite (FeS2) were investigated by first-principles calculations. Examination of the electronic density of states shows that mackinawite (FeS) is metallic and that pyrite (FeS2) is a semiconductor with a band gap of Eg = 1.02 eV. Using the stress-strain method, the elastic properties including the bulk modulus and shear modulus were derived from the elastic Cij data. Density functional perturbation theory (DFPT) calculations within the quasi-harmonic approximation (QHA) were used to calculate the thermodynamic properties, and the two Fe-S compounds are found to be dynamically stable. The isothermal bulk modulus, thermal expansion coefficient, heat capacities, Gibbs free energy and entropy of the Fe-S compounds are obtained by first-principles phonon calculations. Furthermore, the temperature of the mackinawite (FeS) ⟶ pyrite (FeS2) phase transition at 0 GPa was predicted. Based on the calculation results, the model for prediction of Fe-S compounds in the Fe-H2S-H2O system was improved.

Effects of brain-computer interface-based functional electrical stimulation on balance and gait function in patients with stroke: preliminary results

PubMed Central

Chung, EunJung; Park, Sang-In; Jang, Yun-Yung; Lee, Byoung-Hee

2015-01-01

[Purpose] The purpose of this study was to determine the effects of brain-computer interface (BCI)-based functional electrical stimulation (FES) on balance and gait function in patients with stroke. [Subjects] Subjects were randomly allocated to a BCI-FES group (n=5) and a FES group (n=5). [Methods] The BCI-FES group received ankle dorsiflexion training with FES according to a BCI-based program for 30 minutes per day for 5 days. The FES group received ankle dorsiflexion training with FES for the same duration. [Results] Following the intervention, the BCI-FES group showed significant differences in Timed Up and Go test value, cadence, and step length on the affected side. The FES group showed no significant differences after the intervention. However, there were no significant differences between the 2 groups after the intervention. [Conclusion] The results of this study suggest that BCI-based FES training is a more effective exercise for balance and gait function than FES training alone in patients with stroke. PMID:25729205

Effects of brain-computer interface-based functional electrical stimulation on brain activation in stroke patients: a pilot randomized controlled trial.

PubMed

Chung, EunJung; Kim, Jung-Hee; Park, Dae-Sung; Lee, Byoung-Hee

2015-03-01

[Purpose] This study sought to determine the effects of brain-computer interface-based functional electrical stimulation (BCI-FES) on brain activation in patients with stroke. [Subjects] The subjects were randomized to in a BCI-FES group (n=5) and a functional electrical stimulation (FES) group (n=5). [Methods] Patients in the BCI-FES group received ankle dorsiflexion training with FES for 30 minutes per day, 5 times under the brain-computer interface-based program. The FES group received ankle dorsiflexion training with FES for the same amount of time. [Results] The BCI-FES group demonstrated significant differences in the frontopolar regions 1 and 2 attention indexes, and frontopolar 1 activation index. The FES group demonstrated no significant differences. There were significant differences in the frontopolar 1 region activation index between the two groups after the interventions. [Conclusion] The results of this study suggest that BCI-FES training may be more effective in stimulating brain activation than only FES training in patients recovering from stroke.

Effects of brain-computer interface-based functional electrical stimulation on balance and gait function in patients with stroke: preliminary results.

PubMed

Chung, EunJung; Park, Sang-In; Jang, Yun-Yung; Lee, Byoung-Hee

2015-02-01

[Purpose] The purpose of this study was to determine the effects of brain-computer interface (BCI)-based functional electrical stimulation (FES) on balance and gait function in patients with stroke. [Subjects] Subjects were randomly allocated to a BCI-FES group (n=5) and a FES group (n=5). [Methods] The BCI-FES group received ankle dorsiflexion training with FES according to a BCI-based program for 30 minutes per day for 5 days. The FES group received ankle dorsiflexion training with FES for the same duration. [Results] Following the intervention, the BCI-FES group showed significant differences in Timed Up and Go test value, cadence, and step length on the affected side. The FES group showed no significant differences after the intervention. However, there were no significant differences between the 2 groups after the intervention. [Conclusion] The results of this study suggest that BCI-based FES training is a more effective exercise for balance and gait function than FES training alone in patients with stroke.

Towards Cluster-Assembled Materials of True Monodispersity in Size and Chemical Environment: Synthesis, Dynamics and Activity

DTIC Science & Technology

2016-10-27

AFRL-AFOSR-UK-TR-2016-0037 Towards cluster-assembled materials of true monodispersity in size and chemical environment: Synthesis, Dynamics and...Towards cluster-assembled materials of true monodispersity in size and chemical environment: synthesis, dynamics and activity 5a.  CONTRACT NUMBER 5b...report Towards cluster-assembled materials of true monodispersity in size and chemical environment: Synthesis, Dynamics and Activity Ulrich Heiz

FES kinase participates in KIT-ligand induced chemotaxis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II

2010-02-26

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less

MetaCAA: A clustering-aided methodology for efficient assembly of metagenomic datasets.

PubMed

Reddy, Rachamalla Maheedhar; Mohammed, Monzoorul Haque; Mande, Sharmila S

2014-01-01

A key challenge in analyzing metagenomics data pertains to assembly of sequenced DNA fragments (i.e. reads) originating from various microbes in a given environmental sample. Several existing methodologies can assemble reads originating from a single genome. However, these methodologies cannot be applied for efficient assembly of metagenomic sequence datasets. In this study, we present MetaCAA - a clustering-aided methodology which helps in improving the quality of metagenomic sequence assembly. MetaCAA initially groups sequences constituting a given metagenome into smaller clusters. Subsequently, sequences in each cluster are independently assembled using CAP3, an existing single genome assembly program. Contigs formed in each of the clusters along with the unassembled reads are then subjected to another round of assembly for generating the final set of contigs. Validation using simulated and real-world metagenomic datasets indicates that MetaCAA aids in improving the overall quality of assembly. A software implementation of MetaCAA is available at https://metagenomics.atc.tcs.com/MetaCAA. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation.

PubMed

Lim, Jong Gyu; Bang, Ye-Ji; Choi, Sang Ho

2014-12-26

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that reduce toxic peroxides. A new Vibrio vulnificus Prx, named Prx3, was identified and characterized in this study. Biochemical and mutational analyses revealed that Prx3 reduces H2O2, utilizing glutaredoxin 3 (Grx3) and glutathione (GSH) as reductants, and requires only N-terminal peroxidatic cysteine for its catalysis. These results, combined with the monomeric size of Prx3 observed under non-reducing conditions, suggested that Prx3 is a Grx3/GSH-dependent 1-Cys Prx and oxidized without forming intermolecular disulfide bonds. The prx3 mutation impaired growth in the medium containing peroxides and reduced virulence in mice, indicating that Prx3 is essential for survival under oxidative stress and pathogenesis of V. vulnificus. The Fe-S cluster regulator IscR activates prx3 by direct binding to a specific binding sequence centered at -44 from the transcription start site. The binding sequence was homologous to the Type 2 IscR-binding sequence, most likely recognized by the Fe-S clusterless apo-IscR in Escherichia coli. The iscR3CA mutant, chromosomally encoding the apo-locked IscR, exhibited 3-fold higher levels of activation of prx3 than the wild type and accumulated more IscR3CA protein in cells. The IscR-dependent activation of prx3 by aerobic growth and iron starvation was also associated with the increase in cellular levels of IscR protein. Taken together, the results suggested that IscR senses iron starvation as well as reactive oxygen species and shifts to the apo-form, which leads to the increase of cellular IscR and in turn prx3 expression, contributing to the survival and virulence of V. vulnificus during pathogenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Creating Quasi Two-Dimensional Cluster-Assembled Materials through Self-Assembly of a Janus Polyoxometalate-Silsesquioxane Co-Cluster.

PubMed

Wu, Han; Zhang, Yu-Qi; Hu, Min-Biao; Ren, Li-Jun; Lin, Yue; Wang, Wei

2017-05-30

Clusters are an important class of nanoscale molecules or superatoms that exhibit an amazing diversity in structure, chemical composition, shape, and functionality. Assembling two types of clusters is creating emerging cluster-assembled materials (CAMs). In this paper, we report an effective approach to produce quasi two-dimensional (2D) CAMs of two types of spherelike clusters, polyhedral oligomeric silsesquioxanes (POSS), and polyoxometalates (POM). To avoid macrophase separation between the two clusters, they are covalently linked to form a POM-POSS cocluster with Janus characteristics and a dumbbell shape. This Janus characteristics enables the cocluster to self-assemble into diverse nanoaggregates, as conventional amphiphilic molecules and macromolecules do, in selective solvents. In our study, we obtained micelles, vesicles, nanosheets, and nanoribbons by tuning the n-hexane content in mixed solvents of acetone and n-hexane. Ordered packing of clusters in the nanosheets and nanoribbons were directly visualized using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) technique. We infer that the increase of packing order results in the vesicle-to-sheet transition and the change in packing mode causes the sheet-to-ribbon transitions. Our findings have verified the effectivity of creating quasi 2D cluster-assembled materials though the cocluster self-assembly as a new approach to produce novel CAMs.

Protonation and Proton-Coupled Electron Transfer at S-Ligated [4Fe-4S] Clusters

PubMed Central

Morris, Wesley D.; Darcy, Julia W.; Mayer, James M.

2015-01-01

Biological [Fe-S] clusters are increasingly recognized to undergo proton-coupled electron transfer (PCET), but the site of protonation, mechanism, and role for PCET remains largely unknown. Here we explore this reactivity with synthetic model clusters. Protonation of the arylthiolate-ligated [4Fe-4S] cluster [Fe4S4(SAr)4]2- (1, SAr = S-2,4-6-(iPr)3C6H2) leads to thiol dissociation, reversibly forming [Fe4S4(SAr)3L]1- (2) + ArSH (L = solvent, and/or conjugate base). Solutions of 2 + ArSH react with the nitroxyl radical TEMPO to give [Fe4S4(SAr)4]1- (1ox) and TEMPOH. This reaction involves PCET coupled to thiolate association and may proceed via the unobserved protonated cluster [Fe4S4(SAr)3(HSAr)]1-(1-H). Similar reactions with this and related clusters proceed comparably. An understanding of the PCET thermochemistry of this cluster system has been developed, encompassing three different redox levels and two protonation states. PMID:25965413

Inorganic mercury binding with different sulfur species in anoxic sediments and their gut juice extractions.

PubMed

Zhong, Huan; Wang, Wen-Xiong

2009-09-01

To investigate the roles of different sulfur (S) species in controlling the partitioning and bioavailability of inorganic mercury (Hg) in anoxic sediments, we examined the differential binding of Hg with three key S species in anoxic sediment (mackinawite [FeS], pyrite [FeS2], and S(2-)) and then quantified their extraction by the gut juice of deposit-feeding sipunculans Sipunculus nudus. A sequential extraction method was simultaneously used to distinguish Hg sorption with different sediment components. All three S-containing sediment components could lead to a high binding of Hg in sediments, but most Hg was sorbed with FeS or FeS2 instead of formation of Hg sulfide despite the presence of S(2-) or humic acid. The gut juice extraction was relatively low and constant whenever FeS and FeS2 were in the sediment, indicating that both FeS and FeS2 controlled the Hg gut juice extraction and thus bioavailability. Mercury sorbed with FeS2 had higher gut juice extraction than that with FeS, while Hg sulfide was not extracted, strongly suggesting that Hg sorbed with FeS2 was more bioavailable than that with other S species. Mercury sorbed with FeS had very low bioavailability to sipunculans at a low Hg:S ratio in the sediment but was more bioavailable with increasing Hg:S ratio up to a maximum (approximately 1:10, mole based). The present study showed that different S species (FeS, FeS2) and Hg:S ratios significantly affected the binding and bioavailability of Hg in anoxic sediments.

Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

PubMed

Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

2016-10-01

Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

Method of preparing a positive electrode for an electrochemical cell

DOEpatents

Tomczuk, Zygmunt

1979-01-01

A method of preparing an electrochemical cell including a metal sulfide as the positive electrode reactant and lithium alloy as the negative electrochemical reactant with an alkali metal, molten salt electrolyte is disclosed which permits the assembly to be accomplished in air. The electrode reactants are introduced in the most part as a sulfide of lithium and the positive electrode metal in a single-phase compound. For instance, Li.sub.2 FeS.sub.2 is a single-phase compound that is produced by the reaction of Li.sub.2 S and FeS. This compound is an intermediate in the positive electrode cycle from FeS.sub.2 to Fe and Li.sub.2 S. Its use minimizes volumetric changes from the assembled to the charged and discharged conditions of the electrode and minimizes electrode material interaction with air and moisture during assembly.

A Phase 2 Study of 16α-[18F]-fluoro-17β-estradiol Positron Emission Tomography (FES-PET) as a Marker of Hormone Sensitivity in Metastatic Breast Cancer (MBC)

PubMed Central

Peterson, Lanell M.; Kurland, Brenda F.; Schubert, Erin K.; Link, Jeanne M.; Gadi, V.K.; Specht, Jennifer M.; Eary, Janet F.; Porter, Peggy; Shankar, Lalitha K.; Mankoff, David A.; Linden, Hannah M.

2014-01-01

Purpose 16α-[18F]-fluoro-17β-estradiol positron emission tomography (FES-PET) quantifies estrogen receptor (ER) expression in tumors and may provide diagnostic benefit. Procedures Women with newly diagnosed metastatic breast cancer (MBC) from an ER-positive primary tumor were imaged before starting endocrine therapy. FES uptake was evaluated qualitatively and quantitatively, and associated with response and with ER expression. Results Nineteen patients underwent FES imaging. Fifteen had a biopsy of a metastasis and 15 were evaluable for response. Five patients had quantitatively low FES uptake, six had at least one site of qualitatively FES-negative disease. All patients with an ER-negative biopsy had both low uptake and at least one site of FES-negative disease. Of response-evaluable patients, 2/2 with low FES standard uptake value tumors had progressive disease within 6 months, as did 2/3 with qualitatively FES-negative tumors. Conclusions Low/absent FES uptake correlates with lack of ER expression. FES-positron emission tomography can help identify patients with endocrine resistant disease and safely measures ER in MBC. PMID:24170452

Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.

PubMed

Rincon-Enriquez, Gabriel; Crété, Patrice; Barras, Frédéric; Py, Béatrice

2008-03-01

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence. IscR was able to suppress all of the phenotypes listed above in a sufC-dependent manner while depletion of the Isc system led to IscR-dependent activation of the suf operon. No virulence defects were found associated with csdA or nifS mutations. Surprisingly, we found that the sufC mutant was virulent against A. thaliana, whereas its virulence had been found altered in Saintpaulia. Collectively, these results lead us to propose that E. chrysanthemi possess the Fe/S biogenesis strategy suited to the physico-chemical conditions encountered in its host upon infection. In this view, the IscR regulator, which controls both Isc and Suf, is predicted to play a major role in the ability of E. chrysanthemi to colonize a wide array of different plants.

Genetics Home Reference: myopathy with deficiency of iron-sulfur cluster assembly enzyme

MedlinePlus

... Myopathy with deficiency of iron-sulfur cluster assembly enzyme Printable PDF Open All Close All Enable Javascript ... Myopathy with deficiency of iron-sulfur cluster assembly enzyme is an inherited disorder that primarily affects muscles ...

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.

PubMed

Hoff, Kevin G; Ta, Dennis T; Tapley, Tim L; Silberg, Jonathan J; Vickery, Larry E

2002-07-26

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

Orientations of Iron-Sulfur Clusters FA and FB in the Homodimeric Type-I Photosynthetic Reaction Center of Heliobacterium modesticaldum.

PubMed

Kondo, Toru; Matsuoka, Masahiro; Azai, Chihiro; Itoh, Shigeru; Oh-Oka, Hirozo

2016-05-12

Orientations of the FA and FB iron-sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium Heliobacterium modesticaldum by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome b/c complex showed a well-oriented EPR signal. Illumination at 14 K induced an FB(-) signal with g-axes of gz = 2.066, gy = 1.937, and gx = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of FA(-), which magnetically interacted with FB(-), with gz = 2.046, gy = 1.942, and gx = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of FA(-) and FB(-) in hRC resemble those of FB(-) and FA(-), respectively, in PS I. Therefore, FA and FB in hRC, named after their g-value similarities, seem to be located like FB and FA, not like FA and FB, respectively, in PS I. The reducing side of hRC could resemble those in PS I, if the names of FA and FB are interchanged with each other.

Real-time optical studies of respiratory Complex I turnover.

PubMed

Belevich, Nikolai; Belevich, Galina; Verkhovskaya, Marina

2014-12-01

Reduction of Complex l (NADH:ubiquinone oxidoreductase l) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.21Jl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 ).IS to seconds. The data acquisition system collected spectra in the visible range every 50 )JS. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of <270 ).IS, 0.45-0.9 ms and 3-6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major ( T = 0.45-0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD+ in the catalytic site. The fast optical changes in the time range of 0.27- 1.5 ms were not altered significantly in the presence of 1 0-fold excess of NAD+ over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.

An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

PubMed Central

Hidalgo, E; Demple, B

1994-01-01

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress. Images PMID:8306957

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase.

PubMed

Jameson, Guy N L; Cosper, Michele Mader; Hernández, Heather L; Johnson, Michael K; Huynh, Boi Hanh

2004-02-24

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, Mössbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.

Transcriptome analysis of filling stage seeds among three buckwheat species with emphasis on rutin accumulation

PubMed Central

Wang, Tingting; Liu, Minxuan; Liu, Jing; Zhang, Zongwen

2017-01-01

Buckwheat is an important minor crop with pharmaceutical functions due to rutin enrichment in the seed. Seeds of common buckwheat cultivars (Fagopyrum esculentum, Fes) usually have much lower rutin content than tartary buckwheat (F. tartaricum, Ft). We previously found a wild species of common buckwheat (F. esculentum ssp. ancestrale, Fea), with seeds that are high in rutin, similar to Ft. In the present study, we investigated the mechanism by which rutin production varies among different buckwheat cultivars, Fea, a Ft variety (Xide) and a Fes variety (No.2 Pingqiao) using RNA sequencing of filling stage seeds. Sequencing data generated approximately 43.78-Gb of clean bases, all these data were pooled together and assembled 180,568 transcripts, and 109,952 unigenes. We established seed gene expression profiles of each buckwheat sample and assessed genes involved in flavonoid biosynthesis, storage proteins production, CYP450 family, starch and sucrose metabolism, and transcription factors. Differentially expressed genes between Fea and Fes were further analyzed due to their close relationship than with Ft. Expression levels of flavonoid biosynthesis gene FLS1 (Flavonol synthase 1) were similar in Fea and Ft, and much higher than in Fes, which was validated by qRT-PCR. This suggests that FLS1 transcript levels may be associated with rutin accumulation in filling stage seeds of buckwheat species. Further, we explored transcription factors by iTAK, and multiple gene families were identified as being involved in the coordinate regulation of metabolism and development. Our extensive transcriptomic data sets provide a complete description of metabolically related genes that are differentially expressed in filling stage buckwheat seeds and suggests that FLS1 is a key controller of rutin synthesis in buckwheat species. FLS1 can effectively convert dihydroflavonoids into flavonol products. These findings provide a basis for further studies of flavonoid biosynthesis in buckwheat breeding to help accelerate flavonoid metabolic engineering that would increase rutin content in cultivars of common buckwheat. PMID:29261741

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes.

PubMed

Zhong, Xingyu; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

2013-07-01

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

Clinical usefulness of brain-computer interface-controlled functional electrical stimulation for improving brain activity in children with spastic cerebral palsy: a pilot randomized controlled trial.

PubMed

Kim, Tae-Woo; Lee, Byoung-Hee

2016-09-01

[Purpose] Evaluating the effect of brain-computer interface (BCI)-based functional electrical stimulation (FES) training on brain activity in children with spastic cerebral palsy (CP) was the aim of this study. [Subjects and Methods] Subjects were randomized into a BCI-FES group (n=9) and a functional electrical stimulation (FES) control group (n=9). Subjects in the BCI-FES group received wrist and hand extension training with FES for 30 minutes per day, 5 times per week for 6 weeks under the BCI-based program. The FES group received wrist and hand extension training with FES for the same amount of time. Sensorimotor rhythms (SMR) and middle beta waves (M-beta) were measured in frontopolar regions 1 and 2 (Fp1, Fp2) to determine the effects of BCI-FES training. [Results] Significant improvements in the SMR and M-beta of Fp1 and Fp2 were seen in the BCI-FES group. In contrast, significant improvement was only seen in the SMR and M-beta of Fp2 in the control group. [Conclusion] The results of the present study suggest that BCI-controlled FES training may be helpful in improving brain activity in patients with cerebral palsy and may be applied as effectively as traditional FES training.

The effects of electromyography-controlled functional electrical stimulation on upper extremity function and cortical perfusion in stroke patients.

PubMed

Hara, Yukihiro; Obayashi, Shigeru; Tsujiuchi, Kazuhito; Muraoka, Yoshihiro

2013-10-01

The relation was investigated between hemiparetic arm function improvement and brain cortical perfusion (BCP) change during voluntary muscle contraction (VOL), EMG-controlled FES (EMG-FES) and simple electrical muscle stimulation (ES) before and after EMG-FES therapy in chronic stroke patients. Sixteen chronic stroke patients with moderate residual hemiparesis underwent 5 months of task-orientated EMG-FES therapy of the paretic arm once or twice a week. Before and after treatment, arm function was clinically evaluated and BCP during VOL, ES and EMG-FES were assessed using multi-channel near-infrared spectroscopy. BCP in the ipsilesional sensory-motor cortex (SMC) was greater during EMG-FES than during VOL or ES; therefore, EMG-FES caused a shift in the dominant BCP from the contralesional to ipsilesional SMC. After EMG-FES therapy, arm function improved in most patients, with some individual variability, and there was significant improvement in Fugl-Meyer (FM) score and maximal grip strength (GS). Clinical improvement was accompanied by an increase in ipsilesional SMC activation during VOL and EMG-FES condition. The EMG-FES may have more influence on ipsilesional BCP than VOL or ES alone. The sensory motor integration during EMG-FES therapy might facilitate BCP of the ipsilesional SMC and result in functional improvement of hemiparetic upper extremity. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

Emergency management of fat embolism syndrome

PubMed Central

Shaikh, Nissar

2009-01-01

Fat emboli occur in all patients with long-bone fractures, but only few patients develop systemic dysfunction, particularly the triad of skin, brain, and lung dysfunction known as the fat embolism syndrome (FES). Here we review the FES literature under different subheadings. The incidence of FES varies from 1–29%. The etiology may be traumatic or, rarely, nontraumatic. Various factors increase the incidence of FES. Mechanical and biochemical theories have been proposed for the pathophysiology of FES. The clinical manifestations include respiratory and cerebral dysfunction and a petechial rash. Diagnosis of FES is difficult. The other causes for the above-mentioned organ dysfunction have to be excluded. The clinical criteria along with imaging studies help in diagnosis. FES can be detected early by continuous pulse oximetry in high-risk patients. Treatment of FES is essentially supportive. Medications, including steroids, heparin, alcohol, and dextran, have been found to be ineffective. PMID:19561953

Density Functional Investigation of the Inclusion of Gold Clusters on a CH 3 S Self-Assembled Lattice on Au(111)

DOE PAGES

Allen, Darnel J.; Archibald, Wayne E.; Harper, John A.; ...

2016-01-01

We employ first-principles density functional theoretical calculations to address the inclusion of gold (Au) clusters in a well-packed CH 3 S self-assembled lattice. We compute CH 3 S adsorption energies to quantify the energetic stability of the self-assembly and gold adsorption and dissolution energies to characterize the structural stability of a series of Au clusters adsorbed at the SAM-Au interface. Our results indicate that the inclusion of Au clusters with less than four Au atoms in the SAM-Au interface enhances the binding of CH 3 S species. In contrast, larger Au clusters destabilize the self-assembly. We attribute this effect tomore » the low-coordinated gold atoms in the cluster. For small clusters, these low-coordinated sites have significantly different electronic properties compared to larger islands, which makes the binding with the self-assembly energetically more favorable. Our results further indicate that Au clusters in the SAM-Au interface are thermodynamically unstable and they will tend to dissolve, producing Au adatoms incorporated in the self-assembly in the form of CH 3 S-Au-SCH 3 species. This is due to the strong S-Au bond which stabilizes single Au adatoms in the self-assembly. Our results provide solid insight into the impact of adatom islands at the CH 3 S-Au interface.« less

De Novo Construction of Redox Active Proteins.

PubMed

Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

2016-01-01

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. © 2016 Elsevier Inc. All rights reserved.

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells.

PubMed

Dumitrache, Alexandru; Klingeman, Dawn M; Natzke, Jace; Rodriguez, Miguel; Giannone, Richard J; Hettich, Robert L; Davison, Brian H; Brown, Steven D

2017-02-27

Clostridium (Ruminiclostridium) thermocellum is a model organism for its ability to deconstruct plant biomass and convert the cellulose into ethanol. The bacterium forms biofilms adherent to lignocellulosic feedstocks in a continuous cell-monolayer in order to efficiently break down and uptake cellulose hydrolysates. We developed a novel bioreactor design to generate separate sessile and planktonic cell populations for omics studies. Sessile cells had significantly greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division consistent with substrate replete conditions. Planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. This study demonstrates that microbial attachment to cellulose substrate produces widespread gene expression changes for critical functions of this organism and provides physiological insights for two cells populations relevant for engineering of industrially-ready phenotypes.

Hydrogenases and H(+)-reduction in primary energy conservation.

PubMed

Vignais, Paulette M

2008-01-01

Hydrogenases are metalloenzymes subdivided into two classes that contain iron-sulfur clusters and catalyze the reversible oxidation of hydrogen gas (H(2)[Symbol: see text]left arrow over right arrow[Symbol: see text]2H(+)[Symbol: see text]+[Symbol: see text]2e(-)). Two metal atoms are present at their active center: either a Ni and an Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. They are phylogenetically distinct classes of proteins. The catalytic core of [NiFe]hydrogenases is a heterodimeric protein associated with additional subunits in many of these enzymes. The catalytic core of [FeFe]hydrogenases is a domain of about 350 residues that accommodates the active site (H cluster). Many [FeFe]hydrogenases are monomeric but possess additional domains that contain redox centers, mostly Fe-S clusters. A third class of hydrogenase, characterized by a specific iron-containing cofactor and by the absence of Fe-S cluster, is found in some methanogenic archaea; this Hmd hydrogenase has catalytic properties different from those of [NiFe]- and [FeFe]hydrogenases. The [NiFe]hydrogenases can be subdivided into four subgroups: (1) the H(2) uptake [NiFe]hydrogenases (group 1); (2) the cyanobacterial uptake hydrogenases and the cytoplasmic H(2) sensors (group 2); (3) the bidirectional cytoplasmic hydrogenases able to bind soluble cofactors (group 3); and (4) the membrane-associated, energy-converting, H(2) evolving hydrogenases (group 4). Unlike the [NiFe]hydrogenases, the [FeFe]hydrogenases form a homogeneous group and are primarily involved in H(2) evolution. This review recapitulates the classification of hydrogenases based on phylogenetic analysis and the correlation with hydrogenase function of the different phylogenetic groupings, discusses the possible role of the [FeFe]hydrogenases in the genesis of the eukaryotic cell, and emphasizes the structural and functional relationships of hydrogenase subunits with those of complex I of the respiratory electron transport chain.

Probing Ligand Effects on the Redox Energies of [4Fe-4S] Clusters Using Broken-Symmetry Density Functional Theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niu, Shuqiang; Ichiye, Toshiko

A central issue in understanding redox properties of iron-sulfur proteins is determining the factors that tune the reduction potentials of the Fe-S clusters. Recently, Solomon and coworkers have shown that the Fe-S bond covalency of protein analogs measured by %L, the percent ligand character of the Fe 3d orbitals, from ligand K-edge X-ray absorption spectroscopy (XAS) correlates with the electrochemical redox potentials. Also, Wang and coworkers have measured electron detachment energies for iron-sulfur clusters without environmental perturbations by gas-phase photoelectron spectroscopy (PES). Here the correlations of the ligand character with redox energy and %L character are examined in [Fe₄S₄L₄]2⁻ clustersmore » with different ligands by broken symmetry density functional theory (BS-DFT) calculations using the B3LYP functional together with PES and XAS experimental results. These gas-phase studies assess ligand effects independently of environmental perturbations and thus provide essential information for computational studies of iron-sulfur proteins. The B3LYP oxidation energies agree well with PES data, and the %L character obtained from natural bond orbital analysis correlates with XAS values, although it systematically underestimates them because of basis set effects. The results show that stronger electron-donating terminal ligands increase %Lt, the percent ligand character from terminal ligands, but decrease %Sb, the percent ligand character from the bridging sulfurs. Because the oxidized orbital has significant Fe-Lt antibonding character, the oxidation energy correlates well with %Lt. However, because the reduced orbital has varying contributions of both Fe-Lt and Fe-Sb antibonding character, the reduction energy does not correlate with either %Lt or %Sb. Overall, BSDFT calculations together with XAS and PES experiments can unravel the complex underlying factors in the redox energy and chemical bonding of the [4Fe-4S] clusters in iron-sulfur proteins.« less

The Fe-S cluster-containing NEET proteins mitoNEET and NAF-1 as chemotherapeutic targets in breast cancer.

PubMed

Bai, Fang; Morcos, Faruck; Sohn, Yang-Sung; Darash-Yahana, Merav; Rezende, Celso O; Lipper, Colin H; Paddock, Mark L; Song, Luhua; Luo, Yuting; Holt, Sarah H; Tamir, Sagi; Theodorakis, Emmanuel A; Jennings, Patricia A; Onuchic, José N; Mittler, Ron; Nechushtai, Rachel

2015-03-24

Identification of novel drug targets and chemotherapeutic agents is a high priority in the fight against cancer. Here, we report that MAD-28, a designed cluvenone (CLV) derivative, binds to and destabilizes two members of a unique class of mitochondrial and endoplasmic reticulum (ER) 2Fe-2S proteins, mitoNEET (mNT) and nutrient-deprivation autophagy factor-1 (NAF-1), recently implicated in cancer cell proliferation. Docking analysis of MAD-28 to mNT/NAF-1 revealed that in contrast to CLV, which formed a hydrogen bond network that stabilized the 2Fe-2S clusters of these proteins, MAD-28 broke the coordinative bond between the His ligand and the cluster's Fe of mNT/NAF-1. Analysis of MAD-28 performed with control (Michigan Cancer Foundation; MCF-10A) and malignant (M.D. Anderson-metastatic breast; MDA-MB-231 or MCF-7) human epithelial breast cells revealed that MAD-28 had a high specificity in the selective killing of cancer cells, without any apparent effects on normal breast cells. MAD-28 was found to target the mitochondria of cancer cells and displayed a surprising similarity in its effects to the effects of mNT/NAF-1 shRNA suppression in cancer cells, causing a decrease in respiration and mitochondrial membrane potential, as well as an increase in mitochondrial iron content and glycolysis. As expected, if the NEET proteins are targets of MAD-28, cancer cells with suppressed levels of NAF-1 or mNT were less susceptible to the drug. Taken together, our results suggest that NEET proteins are a novel class of drug targets in the chemotherapeutic treatment of breast cancer, and that MAD-28 can now be used as a template for rational drug design for NEET Fe-S cluster-destabilizing anticancer drugs.

Site specific ligand substitution in cubane-type Mo3FeS(4)(4+) clusters: kinetics and mechanism of reaction and isolation of mixed ligand Cl/SPh complexes.

PubMed

Algarra, Andrés G; Basallote, Manuel G; Fernandez-Trujillo, M J; Llusar, Rosa; Pino-Chamorro, Jose A; Sorribes, Ivan; Vicent, Cristian

2010-04-21

The synthesis, crystal structure and solution characterization of the cubane-type [Mo(3)(FeCl)S(4)(dmpe)(3)Cl(3)] (1) (dmpe = 1,2-bis(dimethylphophane-ethane)) cluster are reported and the ligand substitution processes of chloride by thiophenolate investigated. The kinetics and the intimate mechanism of these substitutions reveal that compound 1 undergoes a number of Fe and Mo site specific ligand substitution reactions in acetonitrile solutions. In particular, PhS(-) coordination at the tetrahedral Fe site proceeds in a single resolved kinetic step whereas such substitutions at the Mo sites proceed more slowly. The effect of the presence of acids in the reaction media is also investigated and reveals that an acid excess hinders substitution reactions both at the Fe and Mo sites; however, an acid-promoted solvolysis of the Fe-Cl bonds is observed. Electrospray ionization (ESI) and tandem (ESI-MS/MS) mass spectrometry allow the identification of all the reaction intermediates proposed on the basis of stopped-flow measurements. The distinctive site specific reactivity made it possible to isolate two new clusters of the Mo(3)FeS(4)(4+) family featuring mixed chlorine/thiophenolate ligands, namely Mo(3)S(4)(FeSPh)(dmpe)(3)Cl(3) (2) and [Mo(3)S(4)(FeSPh)(dmpe)(3)(SPh)(3)] (3). A detailed computational study has also been carried out to understand the details of the mechanism of substitution at the M-Cl (M = Mo and Fe) bonds as well as the solvolysis at the Fe-Cl sites, with particular emphasis on the role of acids on the substitution process. The results of the calculations are in agreement with the experimental observations, thus justifying the non-existence of an accelerating effect of acids on the thiophenolate substitution reaction, which differs from previous proposals for the Fe(4)S(4) and MoFe(3)S(4) clusters and some related compounds.

Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcɛRI Pathway in Mast Cells▿ †

PubMed Central

McPherson, Victor A.; Everingham, Stephanie; Karisch, Robert; Smith, Julie A.; Udell, Christian M.; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W. B.

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcɛRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcɛRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcɛRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcɛRI signaling and potential regulation the actin reorganization in mast cells. PMID:19001085

Contributions of F-BAR and SH2 domains of Fes protein tyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.

PubMed

McPherson, Victor A; Everingham, Stephanie; Karisch, Robert; Smith, Julie A; Udell, Christian M; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W B

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.

Autopsy diagnosis of fat embolism syndrome.

PubMed

Miller, Peter; Prahlow, Joseph A

2011-09-01

The fat embolism syndrome (FES) is considered a clinical diagnosis. It typically occurs within several days following major traumatic injury, usually involving fractures of the pelvis and/or lower extremities. Fat embolism syndrome is characterized by the onset of respiratory, neurological, cutaneous, and hematologic manifestations and is thought to be related to intravascular embolization of fat, presumably arising from within the fractured bone marrow space. In its most severe form, FES can be lethal. The presence of fat emboli within the microvasculature of the lungs, brain, and sometimes other organs verifies the clinical impression of FES. Despite its relatively well-known clinical characterization, debate exists within the clinical literature regarding the most appropriate diagnostic criteria for FES. Given this fact, along with the fact that FES is a clinical diagnosis, it is not surprising that forensic pathologists may be somewhat reluctant to make a postmortem diagnosis of FES, especially in cases where insufficient clinical information is available. A case of fatal FES is presented in which rapid clinical deterioration occurred, followed by death, such that a clinical diagnosis of FES was never rendered. We propose that, given the correct circumstances, clinical scenario, and autopsy findings, it is appropriate and acceptable to make a postmortem diagnosis of FES. A multitiered approach to the postmortem diagnosis of FES is presented.

Rehabilitation of hand in subacute tetraplegic patients based on brain computer interface and functional electrical stimulation: a randomised pilot study

NASA Astrophysics Data System (ADS)

Osuagwu, Bethel C. A.; Wallace, Leslie; Fraser, Mathew; Vuckovic, Aleksandra

2016-12-01

Objective. To compare neurological and functional outcomes between two groups of hospitalised patients with subacute tetraplegia. Approach. Seven patients received 20 sessions of brain computer interface (BCI) controlled functional electrical stimulation (FES) while five patients received the same number of sessions of passive FES for both hands. The neurological assessment measures were event related desynchronization (ERD) during movement attempt, Somatosensory evoked potential (SSEP) of the ulnar and median nerve; assessment of hand function involved the range of motion (ROM) of wrist and manual muscle test. Main results. Patients in both groups initially had intense ERD during movement attempt that was not restricted to the sensory-motor cortex. Following the treatment, ERD cortical activity restored towards the activity in able-bodied people in BCI-FES group only, remaining wide-spread in FES group. Likewise, SSEP returned in 3 patients in BCI-FES group, having no changes in FES group. The ROM of the wrist improved in both groups. Muscle strength significantly improved for both hands in BCI-FES group. For FES group, a significant improvement was noticed for right hand flexor muscles only. Significance. Combined BCI-FES therapy results in better neurological recovery and better improvement of muscle strength than FES alone. For spinal cord injured patients, BCI-FES should be considered as a therapeutic tool rather than solely a long-term assistive device for the restoration of a lost function.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry

PubMed Central

2016-01-01

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5′-deoxyadenosyl 5′-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation. PMID:27598886

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

PubMed

Molle, Thibaut; Moreau, Yohann; Clemancey, Martin; Forouhar, Farhad; Ravanat, Jean-Luc; Duraffourg, Nicolas; Fourmond, Vincent; Latour, Jean-Marc; Gambarelli, Serge; Mulliez, Etienne; Atta, Mohamed

2016-10-18

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C 3 methylthiolation of the D89 residue in the ribosomal S 12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS - ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

The validity and reliability of the Finnish Family Empowerment Scale (FES): a survey of parents with small children.

PubMed

Vuorenmaa, M; Halme, N; Åstedt-Kurki, P; Kaunonen, M; Perälä, M-L

2014-07-01

The Family Empowerment Scale (FES) is a widely used instrument which measures the parents' own sense of their empowerment at the level of the family, service system and community. It was originally developed for parents of children with emotional disabilities. The aims of this study were to evaluate the validity and reliability of the Finnish FES and to examine its responsiveness in measuring the empowerment of parents with small children. The English FES was translated into Finnish using back translation and modified so as to be generic and convenient for all families. The construct, convergent, discriminant and concurrent validities, reliability and responsiveness of the Finnish FES were examined. Participants (n = 955) were the parents of children aged 0-9 years who had been selected using stratified random sampling. Confirmatory factor analysis proved that the Finnish FES had three subscales based on the original FES. Convergent and discriminant validities confirmed and supported the same construct. The relationship between parents' participation and empowerment was tested for concurrent validity. As in previous FES studies, the participating parents were more empowered, which supported the concurrent validity. The reliability of the Finnish FES proved acceptable for both parents. The Finnish FES could also discriminate the responses of the parents. Participation in the activities organized by the family service system influenced parents' perceptions of empowerment more than did their background characteristics. The Finnish FES is a valid and reliable instrument and it is suitable for measuring the empowerment of parents. However, it is necessary to consider how the FES would identify in the best way the parents who perhaps need some help. © 2013 John Wiley & Sons Ltd.

Downregulation of the c-Fes protein-tyrosine kinase inhibits the proliferation of human renal carcinoma cells

PubMed Central

Kanda, Shigeru; Miyata, Yasuyoshi; Kanetake, Hiroshi; Smithgall, Thomas E.

2009-01-01

The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKα on T23, and cyclin D1 expression, enhanced the expression of IκBα, and prevented the nuclear localization of NFκB. Treatment with an NFκB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFκB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFκB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells. PMID:19082481

Integrating Fat Embolism Syndrome Scoring Indices in Sickle Cell Disease: A Practice Management Review.

PubMed

Bailey, Keneisha; Wesley, Jagila; Adeyinka, Adebayo; Pierre, Louisdon

2017-01-01

Fat embolism syndrome (FES) has been described in the literature as a rare complication of sickle cell disease (SCD). A review article published in 2005 reported 24 cases of FES associated with SCD. In many cases, a definitive diagnosis of FES in SCD is made on autopsy because of the lack of early recognition and the paucity of sensitive and specific testing for this syndrome. Patients with FES usually have a fulminant, rapidly deteriorating clinical course with mortality occurring within the first 24 hours. We postulate that FES is not well recognized in SCD and that FES scores are useful diagnostic tools in patients with SCD. We queried the electronic medical records with the diagnostic codes for SCD with acute chest syndrome (ACS), pulmonary embolism, or acute respiratory distress syndrome admitted to our hospital from 2008 to 2016 to identify patients suspected of having FES. In addition, we performed an extensive literature review to evaluate the management practice of pediatric patients with FES and SCD from 1966 to 2016. Six patients met our selection criteria from the hospital records, and 4 case reports from the literature search were also included. We applied the Gurd and Wilson criteria and the Schonfeld Fat Embolism Index to identify patients who met the criteria for FES. Nine patients fulfilled Gurd and Wilson criteria, and 9 patients who were evaluable met the Schonfeld criteria for FES. A rapidly deteriorating clinical course in a patient with SCD presenting with ACS or severe vaso-occlusive crisis should trigger a high index of suspicion for FES. Gurd and Wilson criteria or the Schonfeld Fat Embolism Index are useful diagnostic tools for FES in SCD.

Preparation and development of FeS2 Quantum Dots on SiO2 nanostructures immobilized in biopolymers and synthetic polymers as nanoparticles and nanofibers catalyst for antibiotic degradation.

PubMed

Gao, Wei; Razavi, Razieh; Fakhri, Ali

2018-07-15

The FeS 2 Quantum Dots (QDs) decorated SiO 2 nanostructure were prepared by hydrothermal synthesis method. Chitosan and polypyrrole as polymers were used for the immobilization process. The characteristic structure of prepared samples was analyzed using several techniques such as X-ray diffraction, scanning and transmittance electron microscopy, photoluminescence and UV-vis spectroscopy. The mean crystallite sizes of FeS 2 QDs/SiO 2 nanocomposites, FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids are 56.12, 76.38, and 83.24nm, respectively. The band gap energy of FeS 2 QDs/SiO 2 nanocomposites, FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids were found out to be 3.0, 2.8, and 2.7eV, respectively. The photocatalysis properties were investigated by degradation of ampicillin under UV light illumination. The effect of experimental variables, such as, pH and time, on photo-degradation efficiency was studied. The results show that the three prepared samples nanopowders under UV light was in pH3 at 60min. As it could be seen that the amount of ampicillin degradation was increased with the loading of FeS 2 QDs on SiO 2 and FeS 2 QDs/SiO 2 on chitosan nanoparticles and polypyrrole nanofiber. The antibacterial experiment was investigated under visible light illumination and the FeS 2 QDs/SiO 2 -chitosan nanocomposites and FeS 2 QDs/SiO 2 -polypyrrole nanohybrids demonstrate good antibacterial compared to FeS 2 QDs/SiO 2 nanocomposites. Copyright © 2018 Elsevier B.V. All rights reserved.

Event related desynchronization-modulated functional electrical stimulation system for stroke rehabilitation: a feasibility study.

PubMed

Takahashi, Mitsuru; Takeda, Kotaro; Otaka, Yohei; Osu, Rieko; Hanakawa, Takashi; Gouko, Manabu; Ito, Koji

2012-08-16

We developed an electroencephalogram-based brain computer interface system to modulate functional electrical stimulation (FES) to the affected tibialis anterior muscle in a stroke patient. The intensity of FES current increased in a stepwise manner when the event-related desynchronization (ERD) reflecting motor intent was continuously detected from the primary cortical motor area. We tested the feasibility of the ERD-modulated FES system in comparison with FES without ERD modulation. The stroke patient who presented with severe hemiparesis attempted to perform dorsiflexion of the paralyzed ankle during which FES was applied either with or without ERD modulation. After 20 minutes of training, the range of movement at the ankle joint and the electromyography amplitude of the affected tibialis anterior muscle were significantly increased following the ERD-modulated FES compared with the FES alone. The proposed rehabilitation technique using ERD-modulated FES for stroke patients was feasible. The system holds potentials to improve the limb function and to benefit stroke patients.

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

Effects of Brain-Computer Interface-controlled Functional Electrical Stimulation Training on Shoulder Subluxation for Patients with Stroke: A Randomized Controlled Trial.

PubMed

Jang, Yun Young; Kim, Tae Hoon; Lee, Byoung Hee

2016-06-01

The purpose of this study was to investigate the effects of brain-computer interface (BCI)-controlled functional electrical stimulation (FES) training on shoulder subluxation of patients with stroke. Twenty subjects were randomly divided into two groups: the BCI-FES group (n = 10) and the FES group (n = 10). Patients in the BCI-FES group were administered conventional therapy with the BCI-FES on the shoulder subluxation area of the paretic upper extremity, five times per week during 6 weeks, while the FES group received conventional therapy with FES only. All patients were assessed for shoulder subluxation (vertical distance, VD; horizontal distance, HD), pain (visual analogue scale, VAS) and the Manual Function Test (MFT) at the time of recruitment to the study and after 6 weeks of the intervention. The BCI-FES group demonstrated significant improvements in VD, HD, VAS and MFT after the intervention period, while the FES group demonstrated significant improvements in HD, VAS and MFT. There were also significant differences in the VD and two items (shoulder flexion and abduction) of the MFT between the two groups. The results of this study suggest that BCI-FES training may be effective in improving shoulder subluxation of patients with stroke by facilitating motor recovery. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Self-assembled clusters of spheres related to spherical codes.

PubMed

Phillips, Carolyn L; Jankowski, Eric; Marval, Michelle; Glotzer, Sharon C

2012-10-01

We consider the thermodynamically driven self-assembly of spheres onto the surface of a central sphere. This assembly process forms self-limiting, or terminal, anisotropic clusters (N-clusters) with well-defined structures. We use Brownian dynamics to model the assembly of N-clusters varying in size from two to twelve outer spheres and free energy calculations to predict the expected cluster sizes and shapes as a function of temperature and inner particle diameter. We show that the arrangements of outer spheres at finite temperatures are related to spherical codes, an ideal mathematical sequence of points corresponding to the densest possible sphere packings. We demonstrate that temperature and the ratio of the diameters of the inner and outer spheres dictate cluster morphology. We present a surprising result for the equilibrium structure of a 5-cluster, for which the square pyramid arrangement is preferred over a more symmetric structure. We show this result using Brownian dynamics, a Monte Carlo simulation, and a free energy approximation. Our results suggest a promising way to assemble anisotropic building blocks from constituent colloidal spheres.

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn 4CaO 5-cluster in photosystem II

DOE PAGES

Zhang, Miao; Bommer, Martin; Chatterjee, Ruchira; ...

2017-07-18

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn 4CaO 5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn 4CaO 5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water moleculesmore » largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn 4CaO 5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.« less

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II.

PubMed

Zhang, Miao; Bommer, Martin; Chatterjee, Ruchira; Hussein, Rana; Yano, Junko; Dau, Holger; Kern, Jan; Dobbek, Holger; Zouni, Athina

2017-07-18

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn 4 CaO 5 -cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn 4 CaO 5 -cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn 4 CaO 5 -cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn 4 CaO 5 -cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

First assembly times and equilibration in stochastic coagulation-fragmentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

D’Orsogna, Maria R.; Department of Mathematics, CSUN, Los Angeles, California 91330-8313; Lei, Qi

2015-07-07

We develop a fully stochastic theory for coagulation and fragmentation (CF) in a finite system with a maximum cluster size constraint. The process is modeled using a high-dimensional master equation for the probabilities of cluster configurations. For certain realizations of total mass and maximum cluster sizes, we find exact analytical results for the expected equilibrium cluster distributions. If coagulation is fast relative to fragmentation and if the total system mass is indivisible by the mass of the largest allowed cluster, we find a mean cluster-size distribution that is strikingly broader than that predicted by the corresponding mass-action equations. Combinations ofmore » total mass and maximum cluster size under which equilibration is accelerated, eluding late-stage coarsening, are also delineated. Finally, we compute the mean time it takes particles to first assemble into a maximum-sized cluster. Through careful state-space enumeration, the scaling of mean assembly times is derived for all combinations of total mass and maximum cluster size. We find that CF accelerates assembly relative to monomer kinetic only in special cases. All of our results hold in the infinite system limit and can be only derived from a high-dimensional discrete stochastic model, highlighting how classical mass-action models of self-assembly can fail.« less

Combined functional electrical stimulation (FES) and robotic system for wrist rehabiliation after stroke.

PubMed

Hu, Xiaoling; Tong, K Y; Li, R; Chen, M; Xue, J J; Ho, S K; Chen, P N

2010-01-01

Functional electrical stimulation (FES) and rehabilitation robots are techniques used to assist in post-stroke rehabilitation. However, FES and rehabilitation robots are still separate systems currently; and their combined training effects on persons after experiencing a stroke have not been well studied yet. In this work, a new combined FES-robot system driven by user's voluntary intention was developed for wrist joint training after stroke. The performance of the FES-robot assisted wrist tracking was evaluated on five subjects with chronic stroke. With simultaneous assistance from both the FES and robot parts of the system, the motion accuracy was improved and excessive activation in elbow flexor was reduced during wrist tracking.

Anti-friction performance of FeS nanoparticle synthesized by biological method

NASA Astrophysics Data System (ADS)

Zhou, Lu Hai; Wei, Xi Cheng; Ma, Zi Jian; Mei, Bin

2017-06-01

FeS nanoparticle is prepared by a biological method. The size, morphology and structure of the FeS nanoparticle are characterized by the means of X-ray diffraction and transmission electron microscopy. The anti-friction behavior of the FeS nanoparticle as a lubricating oil additive is evaluated in the engine oil by using a face-to-face contact mode. The worn surface is characterized by using the scanning electron microscopy and secondary ion mass spectroscopy in order to find the reasons resulting in the reduction of friction coefficient due to the addition of the FeS nanoparticle. The anti-friction mechanism of the FeS nanoparticle is elucidated based on the experimental results.

Are large-scale flow experiments informing the science and management of freshwater ecosystems?

USGS Publications Warehouse

Olden, Julian D.; Konrad, Christopher P.; Melis, Theodore S.; Kennard, Mark J.; Freeman, Mary C.; Mims, Meryl C.; Bray, Erin N.; Gido, Keith B.; Hemphill, Nina P.; Lytle, David A.; McMullen, Laura E.; Pyron, Mark; Robinson, Christopher T.; Schmidt, John C.; Williams, John G.

2013-01-01

Greater scientific knowledge, changing societal values, and legislative mandates have emphasized the importance of implementing large-scale flow experiments (FEs) downstream of dams. We provide the first global assessment of FEs to evaluate their success in advancing science and informing management decisions. Systematic review of 113 FEs across 20 countries revealed that clear articulation of experimental objectives, while not universally practiced, was crucial for achieving management outcomes and changing dam-operating policies. Furthermore, changes to dam operations were three times less likely when FEs were conducted primarily for scientific purposes. Despite the recognized importance of riverine flow regimes, four-fifths of FEs involved only discrete flow events. Over three-quarters of FEs documented both abiotic and biotic outcomes, but only one-third examined multiple taxonomic responses, thus limiting how FE results can inform holistic dam management. Future FEs will present new opportunities to advance scientifically credible water policies.

Resilient carbon encapsulation of iron pyrite (FeS2) cathodes in lithium ion batteries

NASA Astrophysics Data System (ADS)

Yoder, Tara S.; Tussing, Matthew; Cloud, Jacqueline E.; Yang, Yongan

2015-01-01

Converting iron pyrite (FeS2) from a non-cyclable to a cyclable cathode material for lithium ion batteries has been an ongoing challenge in recent years. Herein we report a promising mitigation strategy: wet-chemistry based conformal encapsulation of synthetic FeS2 nanocrystals in a resilient carbon (RC) matrix (FeS2@RC). The FeS2@RC composite was fabricated by dispersing autoclave-synthesized FeS2 nanocrystals in an aqueous glucose solution, polymerizing the glucose in a hydrothermal reactor, and finally heating the polymer/FeS2 composite in a tube furnace to partially carbonize the polymer. The FeS2@RC electrodes showed superior cyclability compared with the FeS2 electrodes, that is, 25% versus 1% of retention at the 20th cycle. Based on electrochemical analysis, XRD study, and SEM characterization, the performance enhancement was attributed to RC's ability to accommodate volume fluctuation, enhance charge transfer, alleviate detrimental side reactions, and suppress loss of the active material. Furthermore, the remaining issues associated with the current system were identified and future research directions were proposed.

Photoemission and Auger-electron spectroscopic study of the Chevrel-phase compound FexMo6S8

NASA Astrophysics Data System (ADS)

Fujimori, A.; Sekita, M.; Wada, H.

1986-05-01

The electronic structure of the Chevrel-phase compound FexMo6S8 has been studied by photoemission and Auger-electron spectroscopy. Core-level shifts suggest a large charge transfer from the Fe atoms to the Mo6S8 clusters and a small Mo-to-S charge transfer within the cluster. Line-shape asymmetry in the core levels indicates that the density of states (DOS) at the Fermi level has a finite S 3p component as well as the dominant Mo 3d character. Satellite structure and exchange splitting in the Fe core levels point to weak Fe 3d-S 3p hybridization in spite of the short Fe-S distances comparable to that in FeS. The x-ray and ultraviolet valence-band photoemission spectra and the Mo 4d partial DOS obtained by deconvoluting the Mo M4,5VV Auger spectrum are compared with existing band-structure calculations, and the Mo 4d-S 3p bonding character, the structure of the Mo 4d-derived conduction band etc., are discussed. In particular, it is shown that the conduction-band structure is sensitive to the noncubic distortion of the crystal through changes in the intercluster Mo 4d-S 3p hybridization. A pronounced final-state effect is found in the Mo M4,5N2,3V Auger spectrum and is attributed to strong 4p-4d intershell coupling.

[Study on structure and phase transformation laws of natural FeS2 whisker by Raman spectroscopy].

PubMed

Huang, Fei; Kou, Da-Ming; Yao, Yu-Zeng; Ni, Pei; Ding, Jun-Ying

2009-08-01

FeS2 belongs to sulfide, including pyrite of isometric system and marcasite of orthorhombic system. The FeS2 discovered in Gengzhuang, Shanxi Province, was growing in the form of whisker. The study with scanning electron microscopy and electron probe show that the mineral components of FeS2 vary regularly. The structure of natural nano-micron FeS2 whisker was determined by micro-Raman spectroscopy. The results show that there exist two types of structure in FeS2 whiskers: pyrite and marcasite. Marcasite presents irregular shapes, such as coarse lotus root joints, crude columnar or beaded. Pyrite exists in the shape of straight line and smooth surface. In the early growing stage, Gengzhuang FeS2 whisker was mainly marcasite-type structure; in the middle stage it was coexistent structure of pyrite- and marcasite-type; in the late stage it was mainly pyrite-type. The growing stages of the whisker FeS2 show the phase transformation laws. Moreover, during the growing process marcasite was growing with pyrite coated on. Study on FeS2 whisker structure shows that there are correlations between phase transformation laws of the structure and forms, and between the forming time and the composition characteristics.

Can FES-rowing mediate bone mineral density in SCI: a pilot study.

PubMed

Gibbons, R S; McCarthy, I D; Gall, A; Stock, C G; Shippen, J; Andrews, B J

2014-11-01

A single case study. To compare proximal tibia trabecular bone mineral density (BMD) of a participant with complete spinal cord injury (SCI), long-termed functional electrical stimulation-rowing (FES-R) trained, with previously reported SCI and non-SCI group norms. To estimate lower limb joint contact forces (JCFs) in the FES-R trained participant. UK University and orthopaedic hospital research centre. Bilateral proximal tibial trabecular BMD of the FES-R trained participant was measured using peripheral quantitative computerised tomography, and the data were compared with SCI and non-SCI groups. An instrumented four-channel FES-R system was used to measure the lower limb JCFs in the FES-R trained participant. Structurally, proximal tibial trabecular BMD was higher in the FES-R trained participant compared with the SCI group, but was less than the non-SCI group. Furthermore, left (184.7 mg cm(-3)) and right (160.7 mg cm(-3)) BMD were well above the threshold associated with non-traumatic fracture. The knee JCFs were above the threshold known to mediate BMD in SCI, but below threshold at the hip and ankle. As pathological fractures predominate in the distal femur and proximal tibia in chronic SCI patients, the fact that the FES-R trained participant's knee JCFs were above those known to partially prevent bone loss, suggests that FES-R training may provide therapeutic benefit. Although the elevated bilateral proximal tibial BMD of the FES-R participant provides circumstantial evidence of osteogenesis, this single case precludes any statement on the clinical significance. Further investigations are required involving larger numbers and additional channels of FES to increase loading at the hip and ankle.

Funktionelle Elektrostimulation Paraplegischer Patienten.

PubMed

Kern, Helmut

2014-07-08

Functional Electrical Stimulation on Paraplegic Patients. We report on clinical and physiological effects of 8 months Functional Electrical Stimulation (FES) of quadriceps femoris muscle on 16 paraplegic patients. Each patient had muscle biopsies, CT-muscle diameter measurements, knee extension strength testing carried out before and after 8 months FES training. Skin perfusion was documented through infrared telethermography and xenon clearance, muscle perfusion was recorded through thallium scintigraphy. After 8 months FES training baseline skin perfusion showed 86 % increase, muscle perfusion was augmented by 87 %. Muscle fiber diameters showed an average increase of 59 % after 8 months FES training. Muscles in patients with spastic paresis as well as in patients with denervation showed an increase in aerob and anaerob muscle enzymes up to the normal range. Even without axonal neurotropic substances FES was able to demonstrate fiberhypertrophy, enzyme adaptation and intracellular structural benefits in denervated muscles. The increment in muscle area as visible on CT-scans of quadriceps femoris was 30 % in spastic paraplegia and 10 % in denervated patients respectively. FES induced changes were less in areas not directly underneath the surface electrodes. We strongly recommend the use of Kern's current for FES in denervated muscles to induce tetanic muscle contractions as we formed a very critical opinion of conventional exponential current. In patients with conus-cauda-lesions FES must be integrated into modern rehabilitation to prevent extreme muscle degeneration and decubital ulcers. Using FES we are able to improve metabolism and induce positive trophic changes in our patients lower extremities. In spastic paraplegics the functions "rising and walking" achieved through FES are much better training than FES ergometers. Larger muscle masses are activated and an increased heart rate is measured, therefore the impact on cardiovascular fitness and metabolism is much greater. This effectively addresses and prevents all problems which result from inactivity in paraplegic patients.

Upper-extremity functional electric stimulation-assisted exercises on a workstation in the subacute phase of stroke recovery.

PubMed

Kowalczewski, Jan; Gritsenko, Valeriya; Ashworth, Nigel; Ellaway, Peter; Prochazka, Arthur

2007-07-01

To test the efficacy of functional electric stimulation (FES)-assisted exercise therapy (FES-ET) on a workstation in the subacute phase of recovery from a stroke. Single-blind, randomly controlled comparison of high- and low-intensity treatment. Laboratory in a rehabilitation hospital. Nineteen stroke survivors (10 men, 9 women; mean age +/- standard deviation, 60.6+/-5.8y), with upper-extremity hemiplegia (mean poststroke time, 48+/-17d). The main inclusion criteria were: stroke occurred within 3 months of onset of trial and resulted in severe upper-limb dysfunction, and FES produced adequate hand opening. An FES stimulator and an exercise workstation with instrumented objects were used by 2 groups to perform specific motor tasks with their affected upper extremity. Ten subjects in the high-intensity FES-ET group received FES-ET for 1 hour a day on 15 to 20 consecutive workdays. Nine subjects in the low-intensity FES-ET group received 15 minutes of sensory electric stimulation 4 days a week and on the fifth day they received 1 hour of FES-ET. Primary outcome measure included the Wolf Motor Function Test (WMFT). Secondary outcome measures included the Motor Activity Log (MAL), the upper-extremity portion of the Fugl-Meyer Assessment (FMA), and the combined kinematic score (CKS) derived from workstation measurements. The WMFT, MAL, and FMA were used to assess function in the absence of FES whereas CKS was used to evaluate function assisted by FES. Improvements in the WMFT and CKS were significantly greater in the high-intensity group (post-treatment effect size, .95) than the low-intensity group (post-treatment effect size, 1.3). The differences in MAL and FMA were not statistically significant. Subjects performing high-intensity FES-ET showed significantly greater improvements on the WMFT than those performing low-intensity FES-ET. However, this was not reflected in subjects' self-assessments (MAL) or in their FMA scores, so the clinical significance of the result is open to debate. The CKS data suggest that high-intensity FES-ET may be advantageous in neuroprosthetic applications.

Auxiliary iron-sulfur cofactors in radical SAM enzymes.

PubMed

Lanz, Nicholas D; Booker, Squire J

2015-06-01

A vast number of enzymes are now known to belong to a superfamily known as radical SAM, which all contain a [4Fe-4S] cluster ligated by three cysteine residues. The remaining, unligated, iron ion of the cluster binds in contact with the α-amino and α-carboxylate groups of S-adenosyl-l-methionine (SAM). This binding mode facilitates inner-sphere electron transfer from the reduced form of the cluster into the sulfur atom of SAM, resulting in a reductive cleavage of SAM to methionine and a 5'-deoxyadenosyl radical. The 5'-deoxyadenosyl radical then abstracts a target substrate hydrogen atom, initiating a wide variety of radical-based transformations. A subset of radical SAM enzymes contains one or more additional iron-sulfur clusters that are required for the reactions they catalyze. However, outside of a subset of sulfur insertion reactions, very little is known about the roles of these additional clusters. This review will highlight the most recent advances in the identification and characterization of radical SAM enzymes that harbor auxiliary iron-sulfur clusters. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Nitrogenase assembly

PubMed Central

Hu, Yilin; Ribbe, Markus W.

2013-01-01

Nitrogenase contains two unique metalloclusters: the P-cluster and the M-cluster. The assembly processes of P- and M-clusters are arguably the most complicated processes in bioinorganic chemistry. There is considerable interest in decoding the biosynthetic mechanisms of the P- and M-clusters, because these clusters are not only biologically important, but also chemically unprecedented. Understanding the assembly mechanisms of these unique metalloclusters is crucial for understanding the structure-function relationship of nitrogenase. Here, we review the recent advances in this research area, with an emphasis on our work that provide important insights into the biosynthetic pathways of these high-nuclearity metal centers. PMID:23232096

Structural insights into the light-driven auto-assembly process of the water-oxidizing Mn4CaO5-cluster in photosystem II

PubMed Central

Zhang, Miao; Bommer, Martin; Chatterjee, Ruchira; Hussein, Rana; Yano, Junko; Dau, Holger; Kern, Jan; Dobbek, Holger; Zouni, Athina

2017-01-01

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate. DOI: http://dx.doi.org/10.7554/eLife.26933.001 PMID:28718766

Functional electrical stimulation: cardiorespiratory adaptations and applications for training in paraplegia.

PubMed

Deley, Gaëlle; Denuziller, Jérémy; Babault, Nicolas

2015-01-01

Regular exercise can be broadly beneficial to health and quality of life in humans with spinal cord injury (SCI). However, exercises must meet certain criteria, such as the intensity and muscle mass involved, to induce significant benefits. SCI patients can have difficulty achieving these exercise requirements since the paralysed muscles cannot contribute to overall oxygen consumption. One solution is functional electrical stimulation (FES) and, more importantly, hybrid training that combines volitional arm and electrically controlled contractions of the lower limb muscles. However, it might be rather complicated for therapists to use FES because of the wide variety of protocols that can be employed, such as stimulation parameters or movements induced. Moreover, although the short-term physiological and psychological responses during different types of FES exercises have been extensively reported, there are fewer data regarding the long-term effects of FES. Therefore, the purpose of this brief review is to provide a critical appraisal and synthesis of the literature on the use of FES for exercise in paraplegic individuals. After a short introduction underlying the importance of exercise for SCI patients, the main applications and effects of FES are reviewed and discussed. Major findings reveal an increased physiological demand during FES hybrid exercises as compared with arms only exercises. In addition, when repeated within a training period, FES exercises showed beneficial effects on muscle characteristics, force output, exercise capacity, bone mineral density and cardiovascular parameters. In conclusion, there appears to be promising evidence of beneficial effects of FES training, and particularly FES hybrid training, for paraplegic individuals.

Event-Related Beta EEG Changes During Active, Passive Movement and Functional Electrical Stimulation of the Lower Limb.

PubMed

Qiu, Shuang; Yi, Weibo; Xu, Jiapeng; Qi, Hongzhi; Du, Jingang; Wang, Chunfang; He, Feng; Ming, Dong

2016-02-01

A number of electroencephalographic (EEG) studies have reported on event-related desynchronization/synchronization (ERD/ERS) during active movements, passive movements, and the movements induced by functional electrical stimulation (FES). However, the quantitative differences in ERD values and affected frequency bands associated with the lower limb have not been discussed. The goal of this paper was to quantitatively compare the ERD patterns during active movement, passive movement and FES-induced movement of the lower limb. 64-channel EEG signals were recorded to investigate the brain oscillatory patterns during active movement, passive movement and FES-induced movement of the lower limb in twelve healthy subjects. And passive movement and FES-induced movement were also performed in a hemiplegic stroke patient. For healthy subjects, FES-induced movement presented significantly higher characteristic frequency of central beta ERD while there was no significant difference in ERD values compared with active or passive movement. Meanwhile, beta ERD values of FES-induced movement were significantly correlated with those of active movement, and spatial distribution of beta ERD pattern for FES-induced movement was more correlated with that for active movement. In addition, the stroke patient presented central ERD patterns during FES-induced movement, while no ERD with similar frequencies could be found during passive movement. This work implies that the EEG oscillatory pattern under FES-induced movement tends more towards active movement instead of passive movement. The quantification of ERD patterns could be expected as a potential technique to evaluate the brain response during FES-induced movement.

Femoral artery blood flow and microcirculatory perfusion during acute, low-level functional electrical stimulation in spinal cord injury.

PubMed

Barton, Thomas J; Low, David A; Janssen, Thomas W J; Sloots, Maurits; Smit, Christof A J; Thijssen, Dick H J

2018-04-19

Functional electrical stimulation (FES) may help to reduce the risk of developing macro- and microvascular complications in people with SCI. Low-intensity FES has significant clinical potential since this can be applied continuously throughout the day. This study examines the acute effects of low intensity FES using wearable clothing garment on vascular blood flow and oxygen consumption in people with SCI. Cross-sectional observation study METHODS: Eight participants with a motor complete SCI received 4x3 minutes of unilateral FES to the gluteal and hamstring muscles. Skin and deep femoral artery blood flow and oxygen consumption were measured at baseline and during each bout of stimulation. Femoral artery blood flow increased by 18.1% with the application of FES (P=0.02). Moreover, femoral artery blood flow increased further during each subsequent block of FES (P=0.004). Skin perfusion did not change during an individual block of stimulation (P=0.66). Skin perfusion progressively increased with each subsequent bout (P<0.001). There was no change in femoral or skin perfusion across time in the non-stimulated leg (all P>0.05). Low-intensity FES acutely increased blood flow during stimulation, with a progressive increase across subsequent FES bouts. These observations suggest continuous, low-intensity FES may represent a practical and effective strategy to improve perfusion and reduce the risk of vascular complications.

Magnetic assembly of 3D cell clusters: visualizing the formation of an engineered tissue.

PubMed

Ghosh, S; Kumar, S R P; Puri, I K; Elankumaran, S

2016-02-01

Contactless magnetic assembly of cells into 3D clusters has been proposed as a novel means for 3D tissue culture that eliminates the need for artificial scaffolds. However, thus far its efficacy has only been studied by comparing expression levels of generic proteins. Here, it has been evaluated by visualizing the evolution of cell clusters assembled by magnetic forces, to examine their resemblance to in vivo tissues. Cells were labeled with magnetic nanoparticles, then assembled into 3D clusters using magnetic force. Scanning electron microscopy was used to image intercellular interactions and morphological features of the clusters. When cells were held together by magnetic forces for a single day, they formed intercellular contacts through extracellular fibers. These kept the clusters intact once the magnetic forces were removed, thus serving the primary function of scaffolds. The cells self-organized into constructs consistent with the corresponding tissues in vivo. Epithelial cells formed sheets while fibroblasts formed spheroids and exhibited position-dependent morphological heterogeneity. Cells on the periphery of a cluster were flattened while those within were spheroidal, a well-known characteristic of connective tissues in vivo. Cells assembled by magnetic forces presented visual features representative of their in vivo states but largely absent in monolayers. This established the efficacy of contactless assembly as a means to fabricate in vitro tissue models. © 2016 John Wiley & Sons Ltd.

The immitigable nature of assembly bias: the impact of halo definition on assembly bias

NASA Astrophysics Data System (ADS)

Villarreal, Antonio S.; Zentner, Andrew R.; Mao, Yao-Yuan; Purcell, Chris W.; van den Bosch, Frank C.; Diemer, Benedikt; Lange, Johannes U.; Wang, Kuan; Campbell, Duncan

2017-11-01

Dark matter halo clustering depends not only on halo mass, but also on other properties such as concentration and shape. This phenomenon is known broadly as assembly bias. We explore the dependence of assembly bias on halo definition, parametrized by spherical overdensity parameter, Δ. We summarize the strength of concentration-, shape-, and spin-dependent halo clustering as a function of halo mass and halo definition. Concentration-dependent clustering depends strongly on mass at all Δ. For conventional halo definitions (Δ ∼ 200 - 600 m), concentration-dependent clustering at low mass is driven by a population of haloes that is altered through interactions with neighbouring haloes. Concentration-dependent clustering can be greatly reduced through a mass-dependent halo definition with Δ ∼ 20 - 40 m for haloes with M200 m ≲ 1012 h-1M⊙. Smaller Δ implies larger radii and mitigates assembly bias at low mass by subsuming altered, so-called backsplash haloes into now larger host haloes. At higher masses (M200 m ≳ 1013 h-1M⊙) larger overdensities, Δ ≳ 600 m, are necessary. Shape- and spin-dependent clustering are significant for all halo definitions that we explore and exhibit a relatively weaker mass dependence. Generally, both the strength and the sense of assembly bias depend on halo definition, varying significantly even among common definitions. We identify no halo definition that mitigates all manifestations of assembly bias. A halo definition that mitigates assembly bias based on one halo property (e.g. concentration) must be mass dependent. The halo definitions that best mitigate concentration-dependent halo clustering do not coincide with the expected average splashback radii at fixed halo mass.

Fat embolism syndrome in an adolescent before surgical treatment of an isolated closed tibial shaft fracture.

PubMed

Nawaf, Cayce B; Kelly, Derek M; Warner, William C; Beaty, James H; Rhodes, Leslie; Sawyer, Jeffrey R

2012-12-01

Fat embolism syndrome (FES) occurs most commonly in adults with high-energy trauma, especially fractures of the long-bones and pelvis. Because of unique age-related physiologic differences in the immature skeleton, as well as differences in fracture management in pediatric patients, FES is rare in children. To our knowledge, this is the first case report of FES occurring before surgical fixation of a closed tibial shaft fracture in an adolescent. A 16-year-old, 109 kg, Caucasian adolescent boy developed FES after closed diaphyseal fractures of the distal tibia and fibula, showing signs of respiratory distress and mental status changes. The FES resolved with supportive respiratory care and intramedullary nailing of the fracture was done without further respiratory compromise. FES is uncommon in children and adolescents. A high index of suspicion is required to make the diagnosis promptly and institute appropriate treatment. Intramedullary nailing of a long-bone fracture can be done safely and successfully after resolution of the FES.

Funktionelle Elektrostimulation Paraplegischer Patienten

PubMed Central

2014-01-01

Functional Electrical Stimulation on Paraplegic Patients. We report on clinical and physiological effects of 8 months Functional Electrical Stimulation (FES) of quadriceps femoris muscle on 16 paraplegic patients. Each patient had muscle biopsies, CT-muscle diameter measurements, knee extension strength testing carried out before and after 8 months FES training. Skin perfusion was documented through infrared telethermography and xenon clearance, muscle perfusion was recorded through thallium scintigraphy. After 8 months FES training baseline skin perfusion showed 86 % increase, muscle perfusion was augmented by 87 %. Muscle fiber diameters showed an average increase of 59 % after 8 months FES training. Muscles in patients with spastic paresis as well as in patients with denervation showed an increase in aerob and anaerob muscle enzymes up to the normal range. Even without axonal neurotropic substances FES was able to demonstrate fiberhypertrophy, enzyme adaptation and intracellular structural benefits in denervated muscles. The increment in muscle area as visible on CT-scans of quadriceps femoris was 30 % in spastic paraplegia and 10 % in denervated patients respectively. FES induced changes were less in areas not directly underneath the surface electrodes. We strongly recommend the use of Kern’s current for FES in denervated muscles to induce tetanic muscle contractions as we formed a very critical opinion of conventional exponential current. In patients with conus-cauda-lesions FES must be integrated into modern rehabilitation to prevent extreme muscle degeneration and decubital ulcers. Using FES we are able to improve metabolism and induce positive trophic changes in our patients lower extremities. In spastic paraplegics the functions „rising and walking“ achieved through FES are much better training than FES ergometers. Larger muscle masses are activated and an increased heart rate is measured, therefore the impact on cardiovascular fitness and metabolism is much greater. This effectively addresses and prevents all problems which result from inactivity in paraplegic patients. PMID:26913132

Validity and sensitivity to change of the falls efficacy scales international to assess fear of falling in older adults with and without cognitive impairment.

PubMed

Hauer, Klaus A; Kempen, Gertrudis I J M; Schwenk, Michael; Yardley, Lucy; Beyer, Nina; Todd, Chris; Oster, Peter; Zijlstra, G A Rixt

2011-01-01

Measures of fear of falling have not yet been validated in patients with dementia, leaving a methodological gap that limits research in a population at high risk of falling and fall-related consequences. The objectives of this study are to determine: (1) the validity of the 7-item Short Falls Efficacy Scale International (Short FES-I) in geriatric patients with and without cognitive impairment, and (2) the sensitivity to change of the 10-item Falls Efficacy Scale (FES), the 16-item FES-I and the 7-item Short FES-I in geriatric patients with dementia. Cross-sectional data of community-dwelling older adults and geriatric rehabilitation patients (n = 284) collected during face-to-face interviews were used to determine construct and discriminant validity by testing for differences within variables related to fear of falling. Sensitivity to change was studied in an intervention study including patients with mild to moderate dementia (n = 130) as determined by standard response means (SRMs). The Short FES-I showed excellent construct and discriminant validity in the total group and subsamples according to cognitive status. Sensitivity to change was adequate to good in the FES (range SRM: 0.18-0.77) and FES-I (range SRM: 0.21-0.74), with the Short FES-I showing the highest peak sensitivity to change (range SRM: 0.18-0.91). The Short FES-I is a valid measure to assess fear of falling in frail older adults with and without cognitive impairment, yet it may show floor effects in higher functioning older people. All scales, including the Short FES-I, were sensitive to detecting intervention-induced changes in concerns about falling in geriatric patients with dementia. Copyright © 2010 S. Karger AG, Basel.

Stepwise Assembly and Characterization of DNA Linked Two-Color Quantum Dot Clusters.

PubMed

Coopersmith, Kaitlin; Han, Hyunjoo; Maye, Mathew M

2015-07-14

The DNA-mediated self-assembly of multicolor quantum dot (QD) clusters via a stepwise approach is described. The CdSe/ZnS QDs were synthesized and functionalized with an amphiphilic copolymer, followed by ssDNA conjugation. At each functionalization step, the QDs were purified via gradient ultracentrifugation, which was found to remove excess polymer and QD aggregates, allowing for improved conjugation yields and assembly reactivity. The QDs were then assembled and disassembled in a stepwise manner at a ssDNA functionalized magnetic colloid, which provided a convenient way to remove unreacted QDs and ssDNA impurities. After assembly/disassembly, the clusters' optical characteristics were studied by fluorescence spectroscopy and the assembly morphology and stoichiometry was imaged via electron microscopy. The results indicate that a significant amount of QD-to-QD energy transfer occurred in the clusters, which was studied as a function of increasing acceptor-to-donor ratios, resulting in increased QD acceptor emission intensities compared to controls.

Iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis

PubMed Central

Landry, Aaron P.; Cheng, Zishuo; Ding, Huangen

2013-01-01

Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by L-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis. PMID:23258274

Iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.

PubMed

Landry, Aaron P; Cheng, Zishuo; Ding, Huangen

2013-03-07

Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by l-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.

Prospective memory impairments in heavy social drinkers are partially overcome by future event simulation.

PubMed

Platt, Bradley; Kamboj, Sunjeev K; Italiano, Tommaso; Rendell, Peter G; Curran, H Valerie

2016-02-01

Recent research suggests that alcohol acutely impairs prospective memory (PM), and this impairment can be overcome using a strategy called 'future event simulation' (FES). Impairment in event-based PM found in detoxifying alcohol-dependent participants is reversed through FES. However, the impact of the most common problematic drinking patterns that do not involve alcohol dependence on PM remains unclear. Here, we examine the impact of frequent heavy drinking on PM and the degree to which any impairments can be reversed through FES. PM was assessed in 19 heavy drinkers (AUDIT scores ≥ 15) and 18 matched control participants (AUDIT scores ≤ 7) using the 'Virtual Week' task both at baseline and again following FES. Heavy drinkers performed significantly worse than controls on regular and irregular time-based PM tasks. FES improved the performance of controls but not of heavy drinkers on time-based tasks. In contrast, FES improved heavy drinkers' performance on event-based PM tasks. These findings suggest that heavy drinkers experience deficits in strategic monitoring processing associated with time-based PM tasks which do not abate after FES. That the same strategy improves their event-based PM suggests that FES may be helpful for individuals with problematic drinking patterns in improving their prospective memory.

Rigid-Cluster Models of Conformational Transitions in Macromolecular Machines and Assemblies

PubMed Central

Kim, Moon K.; Jernigan, Robert L.; Chirikjian, Gregory S.

2005-01-01

We present a rigid-body-based technique (called rigid-cluster elastic network interpolation) to generate feasible transition pathways between two distinct conformations of a macromolecular assembly. Many biological molecules and assemblies consist of domains which act more or less as rigid bodies during large conformational changes. These collective motions are thought to be strongly related with the functions of a system. This fact encourages us to simply model a macromolecule or assembly as a set of rigid bodies which are interconnected with distance constraints. In previous articles, we developed coarse-grained elastic network interpolation (ENI) in which, for example, only Cα atoms are selected as representatives in each residue of a protein. We interpolate distance differences of two conformations in ENI by using a simple quadratic cost function, and the feasible conformations are generated without steric conflicts. Rigid-cluster interpolation is an extension of the ENI method with rigid-clusters replacing point masses. Now the intermediate conformations in an anharmonic pathway can be determined by the translational and rotational displacements of large clusters in such a way that distance constraints are observed. We present the derivation of the rigid-cluster model and apply it to a variety of macromolecular assemblies. Rigid-cluster ENI is then modified for a hybrid model represented by a mixture of rigid clusters and point masses. Simulation results show that both rigid-cluster and hybrid ENI methods generate sterically feasible pathways of large systems in a very short time. For example, the HK97 virus capsid is an icosahedral symmetric assembly composed of 60 identical asymmetric units. Its original Hessian matrix size for a Cα coarse-grained model is >(300,000)2. However, it reduces to (84)2 when we apply the rigid-cluster model with icosahedral symmetry constraints. The computational cost of the interpolation no longer scales heavily with the size of structures; instead, it depends strongly on the minimal number of rigid clusters into which the system can be decomposed. PMID:15833998

Vortex pinning and irreversibility fields in FeS1-xSex (x = 0, 0.06)

NASA Astrophysics Data System (ADS)

Wang, Aifeng; Petrovic, C.

2017-06-01

We report strong vortex pinning and large irreversibility fields in single crystals of tetragonal FeS1-xSex (x = 0, 0.06). Vortex dynamics is characterized by crossover in field dependence of the depinning energy U0, indicative of single flux surface pinning to the region of collective flux pinning on point-like defects. The close proximity of the irreversibility lines to the upper critical field (Hc2) is consistent with strong pinning in FeS and FeS0.94Se0.06, pointing that new materials with building-blocks of FeS4 tetrahedra are likely to host high critical currents.

Adjustable metal-semiconductor transition of FeS thin films by thermal annealing

NASA Astrophysics Data System (ADS)

Fu, Ganhua; Polity, Angelika; Volbers, Niklas; Meyer, Bruno K.; Mogwitz, Boris; Janek, Jürgen

2006-12-01

FeS polycrystalline thin films were prepared on float glass at 500°C by radio-frequency reactive sputtering. The influence of vacuum annealing on the metal-semiconductor transition of FeS films was investigated. It has been found that with the increase of the annealing temperature from 360to600°C, the metal-semiconductor transition temperature of FeS films first decreases and then increases, associated with first a reduction and then an enhancement of hysteresis width. The thermal stress is considered to give rise to the abnormal change of the metal-semiconductor transition of the FeS film during annealing.

Stepwise assembly of a semiconducting coordination polymer [Cd8S(SPh)14(DMF)(bpy)]n and its photodegradation of organic dyes.

PubMed

Xu, Chao; Hedin, Niklas; Shi, Hua-Tian; Xin, ZhiFeng; Zhang, Qian-Feng

2015-04-14

Chalcogenolate clusters can be interlinked with organic linkers into semiconducting coordination polymers with photocatalytic properties. Here, discrete clusters of Cd8S(SPh)14(DMF)3 were interlinked with 4,4'-bipyridine into a one dimensional coordination polymer of [Cd8S(SPh)14(DMF)(bpy)]n with helical chains. A stepwise mechanism for the assembly of the coordination polymer in DMF was revealed by an ex situ dynamic light scattering study. The cluster was electrostatically neutral and showed a penta-supertetrahedral structure. During the assembly each cluster was interlinked with two 4,4'-bipyridine molecules, which replaced the two terminal DMF molecules of the clusters. In their solid-state forms, the cluster and the coordination polymer were semiconductors with wide band gaps of 3.08 and 2.80 ev. They photocatalytically degraded rhodamine B and methylene blue in aqueous solutions. The moderate conditions used for the synthesis could allow for further in situ studies of the reaction-assembly of related clusters and coordination polymers.

Monoatomic and cluster beam effect on ToF-SIMS spectra of self-assembled monolayers on gold

NASA Astrophysics Data System (ADS)

Tuccitto, N.; Torrisi, V.; Delfanti, I.; Licciardello, A.

2008-12-01

Self-assembled monolayers represent well-defined systems that is a good model surface to study the effect of primary ion beams used in secondary ion mass spectrometry. The effect of polyatomic primary beams on both aliphatic and aromatic self-assembled monolayers has been studied. In particular, we analysed the variation of the relative secondary ion yield of both substrate metal-cluster (Au n-) in comparison with the molecular ions (M -) and clusters (M xAu y-) by using Bi +, Bi 3+, Bi 5+ beams. Moreover, the differences in the secondary ion generation efficiency are discussed. The main effect of the cluster beams is related to an increased formation of low-mass fragments and to the enhancement of the substrate related gold-clusters. The results show that, at variance of many other cases, the static SIMS of self-assembled monolayers does not benefit of the use of polyatomic primary ions.

FeS/S/FeS2 Redox System and Its Oxidoreductase-like Chemistry in the Iron-Sulfur World

NASA Astrophysics Data System (ADS)

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS2 redox system on the interconversion between several pairs of ±-hydroxy acids and ±-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized ±-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS2 redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world.

Pyrite (FeS2) nanocrystals as inexpensive high-performance lithium-ion cathode and sodium-ion anode materials

NASA Astrophysics Data System (ADS)

Walter, Marc; Zünd, Tanja; Kovalenko, Maksym V.

2015-05-01

In light of the impeding depletion of fossil fuels and necessity to lower carbon dioxide emissions, economically viable high-performance batteries are urgently needed for numerous applications ranging from electric cars to stationary large-scale electricity storage. Due to its low raw material cost, non-toxicity and potentially high charge-storage capacity pyrite (FeS2) is a highly promising material for such next-generation batteries. In this work we present the electrochemical performance of FeS2 nanocrystals (NCs) as lithium-ion and sodium-ion storage materials. First, we show that nanoscopic FeS2 is a promising lithium-ion cathode material, delivering a capacity of 715 mA h g-1 and average energy density of 1237 Wh kg-1 for 100 cycles, twice higher than for commonly used LiCoO2 cathodes. Then we demonstrate, for the first time, that FeS2 NCs can serve as highly reversible sodium-ion anode material with long cycling life. As sodium-ion anode material, FeS2 NCs provide capacities above 500 mA h g-1 for 400 cycles at a current rate of 1000 mA g-1. In all our tests and control experiments, the performance of chemically synthesized nanoscale FeS2 clearly surpasses bulk FeS2 as well as large number of other nanostructured metal sulfides.In light of the impeding depletion of fossil fuels and necessity to lower carbon dioxide emissions, economically viable high-performance batteries are urgently needed for numerous applications ranging from electric cars to stationary large-scale electricity storage. Due to its low raw material cost, non-toxicity and potentially high charge-storage capacity pyrite (FeS2) is a highly promising material for such next-generation batteries. In this work we present the electrochemical performance of FeS2 nanocrystals (NCs) as lithium-ion and sodium-ion storage materials. First, we show that nanoscopic FeS2 is a promising lithium-ion cathode material, delivering a capacity of 715 mA h g-1 and average energy density of 1237 Wh kg-1 for 100 cycles, twice higher than for commonly used LiCoO2 cathodes. Then we demonstrate, for the first time, that FeS2 NCs can serve as highly reversible sodium-ion anode material with long cycling life. As sodium-ion anode material, FeS2 NCs provide capacities above 500 mA h g-1 for 400 cycles at a current rate of 1000 mA g-1. In all our tests and control experiments, the performance of chemically synthesized nanoscale FeS2 clearly surpasses bulk FeS2 as well as large number of other nanostructured metal sulfides. Electronic supplementary information (ESI) available: Materials and methods, additional structural and electrochemical characterization. See DOI: 10.1039/c5nr00398a

Cluster-assembled metallic glasses

PubMed Central

2013-01-01

A bottom-up approach to nanofabricate metallic glasses from metal clusters as building blocks is presented. Considering metallic glasses as a subclass of cluster-assembled materials, the relation between the two lively fields of metal clusters and metallic glasses is pointed out. Deposition of selected clusters or collections of them, generated by state-of-the-art cluster beam sources, could lead to the production of a well-defined amorphous material. In contrast to rapidly quenched glasses where only the composition of the glass can be controlled, in cluster-assembled glasses, one can precisely control the structural building blocks. Comparing properties of glasses with similar compositions but differing in building blocks and therefore different in structure will facilitate the study of structure–property correlation in metallic glasses. This bottom-up method provides a novel alternative path to the synthesis of glassy alloys and will contribute to improving fundamental understanding in the field of metallic glasses. It may even permit the production of glassy materials for alloys that cannot be quenched rapidly enough to circumvent crystallization. Additionally, gaining deeper insight into the parameters governing the structure–property relation in metallic glasses can have a great impact on understanding and design of other cluster-assembled materials. PMID:23899019

Observation of Superconductivity in Tetragonal FeS.

PubMed

Lai, Xiaofang; Zhang, Hui; Wang, Yingqi; Wang, Xin; Zhang, Xian; Lin, Jianhua; Huang, Fuqiang

2015-08-19

The possibility of superconductivity in tetragonal FeS has attracted considerable interest because of its similarities to the FeSe superconductor. However, all efforts made to pursue superconductivity in tetragonal FeS have failed so far, and it remains controversial whether tetragonal FeS is metallic or semiconducting. Here we report the observation of superconductivity at 5 K in tetragonal FeS that is synthesized by the hydrothermal reaction of iron powder with sulfide solution. The obtained samples are highly crystalline and less air-sensitive, in contrast to those reported in the literature, which are meta-stable and air-sensitive. Magnetic and electrical properties measurements show that the samples behave as a paramagnetic metal in the normal state and exhibit superconductivity below 5 K. The high crystallinity and the stoichiometry of the samples play important roles in the observation of superconductivity. The present results demonstrate that tetragonal FeS is a promising new platform to realize high-temperature superconductors.

Activation Thermodynamics and H/D Kinetic Isotope Effect of the Hox to HredH+ Transition in [FeFe] Hydrogenase.

PubMed

Ratzloff, Michael W; Wilker, Molly B; Mulder, David W; Lubner, Carolyn E; Hamby, Hayden; Brown, Katherine A; Dukovic, Gordana; King, Paul W

2017-09-20

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox →H red H + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ∼2.5-fold kinetic isotope effect. Overall, these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox →H red H + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.

Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

PubMed

Feliciano, Patricia R; Drennan, Catherine L; Nonato, M Cristina

2016-08-30

Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases.

Real-time electron transfer in respiratory complex I

PubMed Central

Verkhovskaya, Marina L.; Belevich, Nikolai; Euro, Liliya; Wikström, Mårten; Verkhovsky, Michael I.

2008-01-01

Electron transfer in complex I from Escherichia coli was investigated by an ultrafast freeze-quench approach. The reaction of complex I with NADH was stopped in the time domain from 90 μs to 8 ms and analyzed by electron paramagnetic resonance (EPR) spectroscopy at low temperatures. The data show that after binding of the first molecule of NADH, two electrons move via the FMN cofactor to the iron–sulfur (Fe/S) centers N1a and N2 with an apparent time constant of ≈90 μs, implying that these two centers should have the highest redox potential in the enzyme. The rate of reduction of center N2 (the last center in the electron transfer sequence) is close to that predicted by electron transfer theory, which argues for the absence of coupled proton transfer or conformational changes during electron transfer from FMN to N2. After fast reduction of N1a and N2, we observe a slow, ≈1-ms component of reduction of other Fe/S clusters. Because all elementary electron transfer rates between clusters are several orders of magnitude higher than this observed rate, we conclude that the millisecond component is limited by a single process corresponding to dissociation of the oxidized NAD+ molecule from its binding site, where it prevents entry of the next NADH molecule. Despite the presence of approximately one ubiquinone per enzyme molecule, no transient semiquinone formation was observed, which has mechanistic implications, suggesting a high thermodynamic barrier for ubiquinone reduction to the semiquinone radical. Possible consequences of these findings for the proton translocation mechanism are discussed. PMID:18316732

Global Transcriptional Profiles of the Copper Responses in the Cyanobacterium Synechocystis sp. PCC 6803

PubMed Central

Giner-Lamia, Joaquin; López-Maury, Luis; Florencio, Francisco J.

2014-01-01

Copper is an essential element involved in fundamental processes like respiration and photosynthesis. However, it becomes toxic at high concentration, which has forced organisms to control its cellular concentration. We have recently described a copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803, which is mediated by the two-component system, CopRS, a RND metal transport system, CopBAC and a protein of unknown function, CopM. Here, we report the transcriptional responses to copper additions at non-toxic (0.3 µM) and toxic concentrations (3 µM) in the wild type and in the copper sensitive copR mutant strain. While 0.3 µM copper slightly stimulated metabolism and promoted the exchange between cytochrome c6 and plastocyanin as soluble electron carriers, the addition of 3 µM copper catalyzed the formation of ROS, led to a general stress response and induced expression of Fe-S cluster biogenesis genes. According to this, a double mutant strain copRsufR, which expresses constitutively the sufBCDS operon, tolerated higher copper concentration than the copR mutant strain, suggesting that Fe-S clusters are direct targets of copper toxicity in Synechocystis. In addition we have also demonstrated that InrS, a nickel binding transcriptional repressor that belong to the CsoR family of transcriptional factor, was involved in heavy metal homeostasis, including copper, in Synechocystis. Finally, global gene expression analysis of the copR mutant strain suggested that CopRS only controls the expression of copMRS and copBAC operons in response to copper. PMID:25268225

Mitochondrial Cochaperone Mge1 Is Involved in Regulating Susceptibility to Fluconazole in Saccharomyces cerevisiae and Candida Species.

PubMed

Demuyser, Liesbeth; Swinnen, Erwin; Fiori, Alessandro; Herrera-Malaver, Beatriz; Vestrepen, Kevin; Van Dijck, Patrick

2017-07-18

MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker's yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans . Copyright © 2017 Demuyser et al.

Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase*

PubMed Central

Fritsch, Johannes; Siebert, Elisabeth; Priebe, Jacqueline; Zebger, Ingo; Lendzian, Friedhelm; Teutloff, Christian; Friedrich, Bärbel; Lenz, Oliver

2014-01-01

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster. PMID:24448806

Self-Assembly of Octopus Nanoparticles into Pre-Programmed Finite Clusters

NASA Astrophysics Data System (ADS)

Halverson, Jonathan; Tkachenko, Alexei

2012-02-01

The precise control of the spatial arrangement of nanoparticles (NP) is often required to take full advantage of their novel optical and electronic properties. NPs have been shown to self-assemble into crystalline structures using either patchy surface regions or complementary DNA strands to direct the assembly. Due to a lack of specificity of the interactions these methods lead to only a limited number of structures. An emerging approach is to bind ssDNA at specific sites on the particle surface making so-called octopus NPs. Using octopus NPs we investigate the inverse problem of the self-assembly of finite clusters. That is, for a given target cluster (e.g., arranging the NPs on the vertices of a dodecahedron) what are the minimum number of complementary DNA strands needed for the robust self-assembly of the cluster from an initially homogeneous NP solution? Based on the results of Brownian dynamics simulations we have compiled a set of design rules for various target clusters including cubes, pyramids, dodecahedrons and truncated icosahedrons. Our approach leads to control over the kinetic pathway and has demonstrated nearly perfect yield of the target.

Strategies for Rapid Muscle Fatigue Reduction during FES Exercise in Individuals with Spinal Cord Injury: A Systematic Review.

PubMed

Ibitoye, Morufu Olusola; Hamzaid, Nur Azah; Hasnan, Nazirah; Abdul Wahab, Ahmad Khairi; Davis, Glen M

2016-01-01

Rapid muscle fatigue during functional electrical stimulation (FES)-evoked muscle contractions in individuals with spinal cord injury (SCI) is a significant limitation to attaining health benefits of FES-exercise. Delaying the onset of muscle fatigue is often cited as an important goal linked to FES clinical efficacy. Although the basic concept of fatigue-resistance has a long history, recent advances in biomedical engineering, physiotherapy and clinical exercise science have achieved improved clinical benefits, especially for reducing muscle fatigue during FES-exercise. This review evaluated the methodological quality of strategies underlying muscle fatigue-resistance that have been used to optimize FES therapeutic approaches. The review also sought to synthesize the effectiveness of these strategies for persons with SCI in order to establish their functional impacts and clinical relevance. Published scientific literature pertaining to the reduction of FES-induced muscle fatigue was identified through searches of the following databases: Science Direct, Medline, IEEE Xplore, SpringerLink, PubMed and Nature, from the earliest returned record until June 2015. Titles and abstracts were screened to obtain 35 studies that met the inclusion criteria for this systematic review. Following the evaluation of methodological quality (mean (SD), 50 (6) %) of the reviewed studies using the Downs and Black scale, the largest treatment effects reported to reduce muscle fatigue mainly investigated isometric contractions of limited functional and clinical relevance (n = 28). Some investigations (n = 13) lacked randomisation, while others were characterised by small sample sizes with low statistical power. Nevertheless, the clinical significance of emerging trends to improve fatigue-resistance during FES included (i) optimizing electrode positioning, (ii) fine-tuning of stimulation patterns and other FES parameters, (iii) adjustments to the mode and frequency of exercise training, and (iv) biofeedback-assisted FES-exercise to promote selective recruitment of fatigue-resistant motor units. Although the need for further in-depth clinical trials (especially RCTs) was clearly warranted to establish external validity of outcomes, current evidence was sufficient to support the validity of certain techniques for rapid fatigue-reduction in order to promote FES therapy as an integral part of SCI rehabilitation. It is anticipated that this information will be valuable to clinicians and other allied health professionals administering FES as a treatment option in rehabilitation and aid the development of effective rehabilitation interventions.

Strategies for Rapid Muscle Fatigue Reduction during FES Exercise in Individuals with Spinal Cord Injury: A Systematic Review

PubMed Central

Ibitoye, Morufu Olusola; Hamzaid, Nur Azah; Hasnan, Nazirah; Abdul Wahab, Ahmad Khairi; Davis, Glen M.

2016-01-01

Background Rapid muscle fatigue during functional electrical stimulation (FES)-evoked muscle contractions in individuals with spinal cord injury (SCI) is a significant limitation to attaining health benefits of FES-exercise. Delaying the onset of muscle fatigue is often cited as an important goal linked to FES clinical efficacy. Although the basic concept of fatigue-resistance has a long history, recent advances in biomedical engineering, physiotherapy and clinical exercise science have achieved improved clinical benefits, especially for reducing muscle fatigue during FES-exercise. This review evaluated the methodological quality of strategies underlying muscle fatigue-resistance that have been used to optimize FES therapeutic approaches. The review also sought to synthesize the effectiveness of these strategies for persons with SCI in order to establish their functional impacts and clinical relevance. Methods Published scientific literature pertaining to the reduction of FES-induced muscle fatigue was identified through searches of the following databases: Science Direct, Medline, IEEE Xplore, SpringerLink, PubMed and Nature, from the earliest returned record until June 2015. Titles and abstracts were screened to obtain 35 studies that met the inclusion criteria for this systematic review. Results Following the evaluation of methodological quality (mean (SD), 50 (6) %) of the reviewed studies using the Downs and Black scale, the largest treatment effects reported to reduce muscle fatigue mainly investigated isometric contractions of limited functional and clinical relevance (n = 28). Some investigations (n = 13) lacked randomisation, while others were characterised by small sample sizes with low statistical power. Nevertheless, the clinical significance of emerging trends to improve fatigue-resistance during FES included (i) optimizing electrode positioning, (ii) fine-tuning of stimulation patterns and other FES parameters, (iii) adjustments to the mode and frequency of exercise training, and (iv) biofeedback-assisted FES-exercise to promote selective recruitment of fatigue-resistant motor units. Conclusion Although the need for further in-depth clinical trials (especially RCTs) was clearly warranted to establish external validity of outcomes, current evidence was sufficient to support the validity of certain techniques for rapid fatigue-reduction in order to promote FES therapy as an integral part of SCI rehabilitation. It is anticipated that this information will be valuable to clinicians and other allied health professionals administering FES as a treatment option in rehabilitation and aid the development of effective rehabilitation interventions. PMID:26859296

Timing of definitive fixation of major long bone fractures: Can fat embolism syndrome be prevented?

PubMed

Blokhuis, Taco J; Pape, Hans-Christoph; Frölke, Jan-Paul

2017-06-01

Fat embolism is common in patients with major fractures, but leads to devastating consequences, named fat embolism syndrome (FES) in some. Despite advances in treatment strategies regarding the timing of definitive fixation of major fractures, FES still occurs in patients. In this overview, current literature is reviewed and optimal treatment strategies for patients with multiple traumatic injuries, including major fractures, are discussed. Considering the multifactorial etiology of FES, including mechanical and biochemical pathways, FES cannot be prevented in all patients. However, screening for symptoms of FES should be standard in the pre-operative work-up of these patients, prior to definitive fixation of major fractures. Copyright © 2017. Published by Elsevier Ltd.

Beyond assembly bias: exploring secondary halo biases for cluster-size haloes

NASA Astrophysics Data System (ADS)

Mao, Yao-Yuan; Zentner, Andrew R.; Wechsler, Risa H.

2018-03-01

Secondary halo bias, commonly known as `assembly bias', is the dependence of halo clustering on a halo property other than mass. This prediction of the Λ Cold Dark Matter cosmology is essential to modelling the galaxy distribution to high precision and interpreting clustering measurements. As the name suggests, different manifestations of secondary halo bias have been thought to originate from halo assembly histories. We show conclusively that this is incorrect for cluster-size haloes. We present an up-to-date summary of secondary halo biases of high-mass haloes due to various halo properties including concentration, spin, several proxies of assembly history, and subhalo properties. While concentration, spin, and the abundance and radial distribution of subhaloes exhibit significant secondary biases, properties that directly quantify halo assembly history do not. In fact, the entire assembly histories of haloes in pairs are nearly identical to those of isolated haloes. In general, a global correlation between two halo properties does not predict whether or not these two properties exhibit similar secondary biases. For example, assembly history and concentration (or subhalo abundance) are correlated for both paired and isolated haloes, but follow slightly different conditional distributions in these two cases. This results in a secondary halo bias due to concentration (or subhalo abundance), despite the lack of assembly bias in the strict sense for cluster-size haloes. Due to this complexity, caution must be exercised in using any one halo property as a proxy to study the secondary bias due to another property.

Inverted Pendulum Standing Apparatus for Investigating Closed-Loop Control of Ankle Joint Muscle Contractions during Functional Electrical Stimulation.

PubMed

Tan, John F; Masani, Kei; Vette, Albert H; Zariffa, José; Robinson, Mark; Lynch, Cheryl; Popovic, Milos R

2014-01-01

The restoration of arm-free standing in individuals with paraplegia can be facilitated via functional electrical stimulation (FES). In developing adequate control strategies for FES systems, it remains challenging to test the performance of a particular control scheme on human subjects. In this study, we propose a testing platform for developing effective control strategies for a closed-loop FES system for standing. The Inverted Pendulum Standing Apparatus (IPSA) is a mechanical inverted pendulum, whose angular position is determined by the subject's ankle joint angle as controlled by the FES system while having the subject's body fixed in a standing frame. This approach provides a setup that is safe, prevents falling, and enables a research and design team to rigorously test various closed-loop controlled FES systems applied to the ankle joints. To demonstrate the feasibility of using the IPSA, we conducted a case series that employed the device for studying FES closed-loop controllers for regulating ankle joint kinematics during standing. The utilized FES system stimulated, in able-bodied volunteers, the plantarflexors as they prevent toppling during standing. Four different conditions were compared, and we were able to show unique performance of each condition using the IPSA. We concluded that the IPSA is a useful tool for developing and testing closed-loop controlled FES systems for regulating ankle joint position during standing.

Inverted Pendulum Standing Apparatus for Investigating Closed-Loop Control of Ankle Joint Muscle Contractions during Functional Electrical Stimulation

PubMed Central

Tan, John F.; Masani, Kei; Vette, Albert H.; Zariffa, José; Robinson, Mark; Lynch, Cheryl; Popovic, Milos R.

2014-01-01

The restoration of arm-free standing in individuals with paraplegia can be facilitated via functional electrical stimulation (FES). In developing adequate control strategies for FES systems, it remains challenging to test the performance of a particular control scheme on human subjects. In this study, we propose a testing platform for developing effective control strategies for a closed-loop FES system for standing. The Inverted Pendulum Standing Apparatus (IPSA) is a mechanical inverted pendulum, whose angular position is determined by the subject's ankle joint angle as controlled by the FES system while having the subject's body fixed in a standing frame. This approach provides a setup that is safe, prevents falling, and enables a research and design team to rigorously test various closed-loop controlled FES systems applied to the ankle joints. To demonstrate the feasibility of using the IPSA, we conducted a case series that employed the device for studying FES closed-loop controllers for regulating ankle joint kinematics during standing. The utilized FES system stimulated, in able-bodied volunteers, the plantarflexors as they prevent toppling during standing. Four different conditions were compared, and we were able to show unique performance of each condition using the IPSA. We concluded that the IPSA is a useful tool for developing and testing closed-loop controlled FES systems for regulating ankle joint position during standing. PMID:27350992

A generic model of real-world non-ideal behaviour of FES-induced muscle contractions: simulation tool

NASA Astrophysics Data System (ADS)

Lynch, Cheryl L.; Graham, Geoff M.; Popovic, Milos R.

2011-08-01

Functional electrical stimulation (FES) applications are frequently evaluated in simulation prior to testing in human subjects. Such simulations are usually based on the typical muscle responses to electrical stimulation, which may result in an overly optimistic assessment of likely real-world performance. We propose a novel method for simulating FES applications that includes non-ideal muscle behaviour during electrical stimulation resulting from muscle fatigue, spasms and tremors. A 'non-idealities' block that can be incorporated into existing FES simulations and provides a realistic estimate of real-world performance is described. An implementation example is included, showing how the non-idealities block can be incorporated into a simulation of electrically stimulated knee extension against gravity for both a proportional-integral-derivative controller and a sliding mode controller. The results presented in this paper illustrate that the real-world performance of a FES system may be vastly different from the performance obtained in simulation using nominal muscle models. We believe that our non-idealities block should be included in future simulations that involve muscle response to FES, as this tool will provide neural engineers with a realistic simulation of the real-world performance of FES systems. This simulation strategy will help engineers and organizations save time and money by preventing premature human testing. The non-idealities block will become available free of charge at www.toronto-fes.ca in late 2011.

Evaluation of Functional Electrical Stimulation to Assist Cycling in Four Adolescents with Spastic Cerebral Palsy

PubMed Central

Harrington, Ann Tokay; McRae, Calum G. A.; Lee, Samuel C. K.

2012-01-01

Introduction. Adolescents with cerebral palsy (CP) often have difficulty participating in exercise at intensities necessary to improve cardiovascular fitness. Functional electrical stimulation- (FES-) assisted cycling is proposed as a form of exercise for adolescents with CP. The aims of this paper were to adapt methods and assess the feasibility of applying FES cycling technology in adolescents with CP, determine methods of performing cycling tests in adolescents with CP, and evaluate the immediate effects of FES assistance on cycling performance. Materials/Methods. Four participants (12–14 years old; GMFCS levels III-IV) participated in a case-based pilot study of FES-assisted cycling in which bilateral quadriceps muscles were activated using surface electrodes. Cycling cadence, power output, and heart rate were collected. Results. FES-assisted cycling was well tolerated (n = 4) and cases are presented demonstrating increased cadence (2–43 rpm), power output (19–70%), and heart rates (4-5%) and decreased variability (8–13%) in cycling performance when FES was applied, compared to volitional cycling without FES assistance. Some participants (n = 2) required the use of an auxiliary hub motor for assistance. Conclusions. FES-assisted cycling is feasible for individuals with CP and may lead to immediate improvements in cycling performance. Future work will examine the potential for long-term fitness gains using this intervention. PMID:22685479

Reversible Self-Assembly of Glutathione-Coated Gold Nanoparticle Clusters via pH-Tunable Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moaseri, Ehsan; Bollinger, Jonathan A.; Changalvaie, Behzad

In this study, nanoparticle (NP) clusters with diameters ranging from 20 to 100 nm are reversibly assembled from 5 nm gold (Au) primary particles coated with glutathione (GSH) in aqueous solution as a function of pH in the range of 5.4 to 3.8. As the pH is lowered, the GSH surface ligands become partially zwitterionic and form interparticle hydrogen bonds that drive the self-limited assembly of metastable clusters in <1 min. Whereas clusters up to 20 nm in size are stable against cluster–cluster aggregation for up to 1 day, clusters up to 80 nm in size can be stabilized overmore » this period via the addition of citrate to the solution in equal molarity with GSH molecules. The cluster diameter may be cycled reversibly by tuning pH to manipulate the colloidal interactions; however, modest background cluster–cluster aggregation occurs during cycling. Cluster sizes can be stabilized for at least 1 month via the addition of PEG-thiol as a grafted steric stabilizer, where PEG-grafted clusters dissociate back to starting primary NPs at pH 7 in fewer than 3 days. Whereas the presence of excess citrate has little effect on the initial size of the metastable clusters, it is necessary for both the cycling and dissociation to mediate the GSH–GSH hydrogen bonds. In conclusion, these metastable clusters exhibit significant characteristics of equilibrium self-limited assembly between primary particles and clusters on time scales where cluster–cluster aggregation is not present.« less

Reversible Self-Assembly of Glutathione-Coated Gold Nanoparticle Clusters via pH-Tunable Interactions

DOE PAGES

Moaseri, Ehsan; Bollinger, Jonathan A.; Changalvaie, Behzad; ...

2017-10-06

In this study, nanoparticle (NP) clusters with diameters ranging from 20 to 100 nm are reversibly assembled from 5 nm gold (Au) primary particles coated with glutathione (GSH) in aqueous solution as a function of pH in the range of 5.4 to 3.8. As the pH is lowered, the GSH surface ligands become partially zwitterionic and form interparticle hydrogen bonds that drive the self-limited assembly of metastable clusters in <1 min. Whereas clusters up to 20 nm in size are stable against cluster–cluster aggregation for up to 1 day, clusters up to 80 nm in size can be stabilized overmore » this period via the addition of citrate to the solution in equal molarity with GSH molecules. The cluster diameter may be cycled reversibly by tuning pH to manipulate the colloidal interactions; however, modest background cluster–cluster aggregation occurs during cycling. Cluster sizes can be stabilized for at least 1 month via the addition of PEG-thiol as a grafted steric stabilizer, where PEG-grafted clusters dissociate back to starting primary NPs at pH 7 in fewer than 3 days. Whereas the presence of excess citrate has little effect on the initial size of the metastable clusters, it is necessary for both the cycling and dissociation to mediate the GSH–GSH hydrogen bonds. In conclusion, these metastable clusters exhibit significant characteristics of equilibrium self-limited assembly between primary particles and clusters on time scales where cluster–cluster aggregation is not present.« less

Elimination-Fusion Self-Assembly of a Nanometer-Scale 72-Nucleus Silver Cluster Caging a Pair of [EuW10 O36 ]9- Polyoxometalates.

PubMed

Zhang, Shan-Shan; Su, Hai-Feng; Wang, Zhi; Wang, Xing-Po; Chen, Wen-Xian; Zhao, Quan-Qin; Tung, Chen-Ho; Sun, Di; Zheng, Lan-Sun

2018-02-06

The largest known polyoxometalate (POM)-templated silver-alkynyl cluster, [(EuW 10 O 36 ) 2 @Ag 72 (tBuC≡C) 48 Cl 2 ⋅4 BF 4 ] (SD/Ag20), was isolated under solvothermal conditions and structurally characterized. It was confirmed by single-crystal X-ray diffraction (SCXRD) as a {EuW 10 } 2 -in-{Ag 72 } clusters-in-cluster rod-like compound. The high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) shows that such a double anion-templated cluster is assembled from a crucial single anion-templated Ag 42 intermediate in the solution. The crystallization of Ag 42 species (SD/Ag21), followed by SCXRD, gave an important clue about the assembly route of SD/Ag20 in solution: the Ag 42 cluster eliminates six silver atoms laterally, then fuses together at the vacant face to form the final Ag 72 cluster (elimination-fusion mechanism). The characteristic emission of [EuW 10 O 36 ] 9- is well maintained in SD/Ag20. This work not only provides a new method for the synthesis of larger silver clusters as well as the functional integration of the silver cluster and POMs, but also gives deep insights about the high-nuclear silver cluster assembly mechanism. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

BCI-FES system for neuro-rehabilitation of stroke patients

NASA Astrophysics Data System (ADS)

Jure, Fabricio A.; Carrere, Lucía C.; Gentiletti, Gerardo G.; Tabernig, Carolina B.

2016-04-01

Nowadays, strokes are a growing cause of mortality and many people remain with motor sequelae and troubles in the daily activities. To treat this sequelae, alternative rehabilitation techniques are needed. In this article a Brain Computer Interface (BCI) system to control a Functional Electrical Stimulation (FES) system is presented. It can be used as a novel tool in easy setup clinical routines, to improve the rehabilitation process by mean of detecting patient´s motor intention, performing it by FES and finally receiving appropriate feedback The BCI-FES system presented here, consists of three blocks: the first one decodes the patient´s intention and it is composed by the patient, the acquisition hardware and the processing software (Emotiv EPOC®). The second block, based on Arduino’s technology, transforms the information into a valid command signal. The last one excites the patient´s neuromuscular system by means of a FES device. In order to evaluate the cerebral activity sensed by the device, topographic maps were obtained. The BCI-FES system was able to detect the patient´s motor intention and control the FES device. At the time of this publication, the system it’s being employing in a rehabilitation program with patients post stroke.

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex

PubMed Central

Fox, Nicholas G.; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Barondeau, David P.

2015-01-01

Iron–sulfur (Fe–S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe–S assembly complex. PMID:26016518

Enhanced magnetostriction derived from magnetic single domain structures in cluster-assembled SmCo films

NASA Astrophysics Data System (ADS)

Bai, Yulong; Yang, Bo; Guo, Fei; Lu, Qingshan; Zhao, Shifeng

2017-11-01

Cluster-assembled SmCo alloy films were prepared by low energy cluster beam deposition. The structure, magnetic domain, magnetization, and magnetostriction of the films were characterized. It is shown that the as-prepared films are assembled in compact and uniformly distributed spherical cluster nanoparticles, most of which, after vacuum in situ annealing at 700 K, aggregated to form cluster islands. These cluster islands result in transformations from superparamagnetic states to magnetic single domain (MSD) states in the films. Such MSD structures contribute to the enhanced magnetostrictive behaviors with a saturation magnetostrictive coefficient of 160 × 10-6 in comparison to 105 × 10-6 for the as-prepared films. This work demonstrates candidate materials that could be applied in nano-electro-mechanical systems, low power information storage, and weak magnetic detecting devices.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

PubMed Central

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

Effectiveness of gait training using an electromechanical gait trainer, with and without functional electric stimulation, in subacute stroke: a randomized controlled trial.

PubMed

Tong, Raymond K; Ng, Maple F; Li, Leonard S

2006-10-01

To compare the therapeutic effects of conventional gait training (CGT), gait training using an electromechanical gait trainer (EGT), and gait training using an electromechanical gait trainer with functional electric stimulation (EGT-FES) in people with subacute stroke. Nonblinded randomized controlled trial. Rehabilitation hospital for adults. Fifty patients were recruited within 6 weeks after stroke onset; 46 of these completed the 4-week training period. Participants were randomly assigned to 1 of 3 gait intervention groups: CGT, EGT, or EGT-FES. The experimental intervention was a 20-minute session per day, 5 days a week (weekdays) for 4 weeks. In addition, all participants received their 40-minute sessions of regular physical therapy every weekday as part of their treatment by the hospital. Five-meter walking speed test, Elderly Mobility Scale (EMS), Berg Balance Scale, Functional Ambulatory Category (FAC), Motricity Index leg subscale, FIM instrument score, and Barthel Index. The EGT and EGT-FES groups had statistically significantly more improvement than the CGT group in the 5-m walking speed test (CGT vs EGT, P=.011; CGT vs EGT-FES, P=.001), Motricity Index (CGT vs EGT-FES, P=.011), EMS (CGT vs EGT, P=.006; CGT vs EGT-FES, P=.009), and FAC (CGT vs EGT, P=.005; CGT vs EGT-FES, P=.002) after the 4 weeks of training. No statistically significant differences were found between the EGT and EGT-FES groups in all outcome measures. In this sample with subacute stroke, participants who trained on the electromechanical gait trainer with body-weight support, with or without FES, had a faster gait, better mobility, and improvement in functional ambulation than participants who underwent conventional gait training. Future studies with assessor blinding and larger sample sizes are warranted.

Fat embolism syndrome in long bone fracture--clinical experience in a tertiary referral center in Taiwan.

PubMed

Tsai, I-Tzun; Hsu, Chin-Jung; Chen, Ying-Hao; Fong, Yi-Chin; Hsu, Horng-Chaung; Tsai, Chun-Hao

2010-08-01

Fat embolism syndrome (FES) is a potentially fatal complication of long bone fractures. There have been no reports of FES in long bone fractures in this decade in Taiwan. The purpose of this study was to review the FES experiences in a tertiary referral center between January 1997 and February 2008. Between January 1997 and February 2008, 13 patients with long bone fractures with documented FES in our institution were reviewed. FES was diagnosed clinically by at least 2 major criteria or 1 major with at least 4 minor signs of Gurd's criteria. The incidences of FES, less than those reported in the literature, were 0.15% in fracture of the tibia, 0.78% in fracture of the femur and 2.4% in multiple fractures. The mortality rate of FES, similar to other available results, was about 7.7%. All cases were less than 35 years old, except for 1 70-year-old male. Fat embolism occurred within an average of 48.5 hours after long bone fracture. Eleven presented with sudden drop in hemoglobin level, dropping 4.2 g/dL on average. Nine presented with thrombocytopenia, and 10 presented with sudden drop in platelet count, dropping 140,000/dL on average. Two had cerebral sequelae without recovery at the last 48-month follow-up. This 12-year interval retrospective study revealed modern epidemiologic results for FES in long bone fracture. Compared with the available literature in the recent decade, the incidence of FES in long bone fracture in our institution is less and the mortality rate is similar. Copyright 2010 Elsevier. Published by Elsevier B.V. All rights reserved.

Aging of Nanocrystalline Mackinawite (FeS): Mineralogical and Physicochemical Properties

NASA Astrophysics Data System (ADS)

Jeong, H. Y.; Lee, H.

2011-12-01

Due to the extraordinary physical properties and high surface areas, nanocrystalline minerals have been widely investigated for their potential uses in treating contaminated groundwaters and surface waters. Most previous studies in this field have focused on either preparation of nanocrystalline minerals or measurement of their reactivity with environmental contaminants. Nanocrystalline minerals, due to the inherent thermodynamic instability, tend to change the physicochemical and mineralogical properties over time, usually resulting in the decreased reactivity. Thus, to better assess the long-term effectiveness of nanocrystalline minerals in field applications, such "aging" effects should be clearly delineated. In the present work, we have investigated the aging impact on nanocrystalline mackinawite (FeS), the ubiquitous Fe-bearing mineral in anoxic sulfidic sediments. Mackinawite (FeS) is known to be an effective scavenger for metal pollutants and a strong reducing reagent for chromate and chlorinated organic compounds. Our preliminary results indicate that nanocrystalline FeS ages via Ostwald ripening, particle aggregation, or mineralogical transformation. By X-ray diffraction (XRD) analysis, aging of nanocrystalline FeS via Ostwald ripening is found to be dominant at acidic pH. Cryogenic transmission electron microscopy (TEM) shows that particle aggregation is most evident at neutral pH. Transformation of nanosized FeS into a more thermodynamically stable greigite (Fe3S4) is observed in the presence of folic acid at acidic pH. The pH-dependent aging process may be linked with changes in the apparent solubility and surface charge of FeS with pH. The Ostwald ripening or particle aggregation of nanocrystalline FeS leads to the decrease surface area, thus causing the decreased reactivity. Given the less reactivity of greigite, the transformation of nanocrystalline FeS to greigite is also expected to result in the decreased reactivity.

Real-time estimation of FES-induced joint torque with evoked EMG : Application to spinal cord injured patients.

PubMed

Li, Zhan; Guiraud, David; Andreu, David; Benoussaad, Mourad; Fattal, Charles; Hayashibe, Mitsuhiro

2016-06-22

Functional electrical stimulation (FES) is a neuroprosthetic technique for restoring lost motor function of spinal cord injured (SCI) patients and motor-impaired subjects by delivering short electrical pulses to their paralyzed muscles or motor nerves. FES induces action potentials respectively on muscles or nerves so that muscle activity can be characterized by the synchronous recruitment of motor units with its compound electromyography (EMG) signal is called M-wave. The recorded evoked EMG (eEMG) can be employed to predict the resultant joint torque, and modeling of FES-induced joint torque based on eEMG is an essential step to provide necessary prediction of the expected muscle response before achieving accurate joint torque control by FES. Previous works on FES-induced torque tracking issues were mainly based on offline analysis. However, toward personalized clinical rehabilitation applications, real-time FES systems are essentially required considering the subject-specific muscle responses against electrical stimulation. This paper proposes a wireless portable stimulator used for estimating/predicting joint torque based on real time processing of eEMG. Kalman filter and recurrent neural network (RNN) are embedded into the real-time FES system for identification and estimation. Prediction results on 3 able-bodied subjects and 3 SCI patients demonstrate promising performances. As estimators, both Kalman filter and RNN approaches show clinically feasible results on estimation/prediction of joint torque with eEMG signals only, moreover RNN requires less computational requirement. The proposed real-time FES system establishes a platform for estimating and assessing the mechanical output, the electromyographic recordings and associated models. It will contribute to open a new modality for personalized portable neuroprosthetic control toward consolidated personal healthcare for motor-impaired patients.

[Functional electrical stimulation based on a working pattern influences function of lower extremity in subjects with early stroke and effects on diffusion tensor imaging: a randomized controlled trial].

PubMed

Chen, Danfeng; Yan, Tiebin; Li, Guandong; Li, Fangming; Liang, Qitang

2014-10-14

To explore the possible mechanisms for improving lower extremity motor function in patients with early stroke through combining magnetic resonance diffusion tensor imaging (DTI) technology and functional electrical stimulation (FES) based on human walking patterns. From August 2012 to September 2013, a total of 48 eligible patients were stratified according to age, gender, disease course, Brunnstrom staging and types of stroke. And the Minimize software was used to divided them randomly into four-channel FES group (n = 18), dual-channel FES group (n = 15) and comfort stimulation group (n = 15). For all three groups, general medication and standard rehabilitation were provided. Based on normal walking pattern design of FES treatment, four-channel FES groups received the stimulations of quadriceps, hamstring, anterior tibialis and medial gastrocnemius. For the dual-channel FES group, the stimulations of tibialis anterior, peroneus longus and peroneus brevis muscles were applied. In comfort electrical stimulation group, the electrode positions were identical to the stimulation group, but there was no current output during stimulation. Before and after 3-week treatment, three groups received weekly rehabilitation evaluations of Fugl-Meyer assessment (FMA), posture assessment of stroke scale (PASS), Brunel balance assessment (BBA), Berg balance scale (BBS) and modified Barthel index (MBI). Before and after treatment, DTI examination was performed for some patients. Among three groups, general patient profiles and pre-treatment evaluations showed no significant difference. For intra-group comparisons versus pre-treatment, at week 1, 2 and 3, the scores of PASS, BBA, BBS, FMA and MBI had statistically significant differences (P < 0.05); At week 3 post-treatment, when four-channel and double-channel FES groups were compared versus pre-treatment, the scores of ipsilateral FA had statistically significant differences (P < 0.05). At week 1 post-treatment, MBI had statistically significant difference among 3 groups (P = 0.037). As compared with placebo, four-channel group had statistically significant difference [(52 ± 12) vs (38 ± 18), P < 0.05]; At week 2 post-treatment, the scores of PASS and MBI were (29 ± 3, 73 ± 13) in four-channel FES group versus (24 ± 8, 60 ± 17) in dual-channel FES group. And the scores of PASS, BBA, BBS, FMA and MBI were (9 ± 3, 8.3 ± 2.4, 37 ± 7, 22 ± 5, 73 ± 13) in four-channel FES group versus (21 ± 7, 6.2 ± 3.1, 24 ± 16, 15 ± 8, 47 ± 20) in comfort electrical stimulation group. When dual-channel FES and comfort stimulation groups were compared, MBI had significant statistical difference [(60 ± 17) vs (47 ± 20), P < 0.05]. At week 3 post-treatment, four-channel and dual-channel FES groups were compared, there was also statistical significance in FMA [(25 ± 5) vs (20 ± 7), P = 0.055]. The scores of PASS, BBS, FMA and MBI were (31 ± 3, 43 ± 8, 25 ± 5, 81 ± 13) in four-channel FES group versus (25 ± 8, 29 ± 17, 17 ± 9, 54 ± 25) in comfort stimulation group respectively. When dual-channel FES and comfort stimulation groups were compared, the scores of MBI were (71 ± 15) and (54 ± 25) respectively. And the difference was statistically significant (P < 0.05). At week 3 post-treatment, the scores of FA significantly increased [four-channel FES group (0.321 ± 0.172) vs comfort stimulation group (0.217 ± 0.135) (P = 0.020)]. When dual-channel FES group (0.333 ± 0.164) and comfort stimulation group (0.217 ± 0.135) (P = 0.049) were compared, the differences were statistically significant. DTI showed that four-channel FES group increased significantly, but contralateral fiber bundle was not obvious. And the improvements of dual-channel FES and comfort stimulation groups were insignificant. Compared with traditional dual-channel FES, functional electrical stimulation based on human walking patterns is more efficacious. And it helps to restore brain structure and function and promote motor function recovery in patients with early stroke.

Cerebral Fat Embolism: Recognition, Complications, and Prognosis.

PubMed

Godoy, Daniel Agustín; Di Napoli, Mario; Rabinstein, Alejandro A

2017-09-20

Fat embolism syndrome (FES) is a rare syndrome caused by embolization of fat particles into multiple organs including the brain. It typically manifests with petechial rash, deteriorating mental status, and progressive respiratory insufficiency, usually occurring within 24-48 h of trauma with long-bone fractures or an orthopedic surgery. The diagnosis of FES is based on clinical and imaging findings, but requires exclusion of alternative diagnoses. Although there is no specific treatment for FES, prompt recognition is important because it can avoid unnecessary interventions and clarify prognosis. Patients with severe FES can become critically ill, but even comatose patients with respiratory failure may recover favorably. Prophylactic measures, such as early stabilization of fractures and certain intraoperative techniques, may help decrease the incidence and severity of FES.

Interpretation of the photoelectron spectra of FeS(2)(-) by a multiconfiguration computational approach.

PubMed

Clima, Sergiu; Hendrickx, Marc F A

2007-11-01

The ground states of FeS(2) and FeS(2)(-), and several low-lying excited electronic states of FeS(2) that are responsible for the FeS(2)(-) photoelectron spectrum, are calculated. At the B3LYP level an open, quasi-linear [SFeS](-) conformation is found as the most stable structure, which is confirmed at the ab initio CASPT2 computational level. Both the neutral and the anionic unsaturated complexes possess high-spin electronic ground states. For the first time a complete assignment of the photoelectron spectrum of FeS(2)(-) is proposed. The lowest energy band in this spectrum is ascribed to an electron detachment from the two highest-lying 3dpi antibonding orbitals (with respect to the iron-sulfur bonding) of iron. The next-lowest experimental band corresponds to an electron removal from nonbonding, nearly pure sulfur orbitals. The two highest bands in the spectra are assigned as electron detachments from pi and sigma bonding mainly sulfur orbitals.

Exploring hierarchical FeS2/C composite nanotubes arrays as advanced cathode for lithium ion batteries

NASA Astrophysics Data System (ADS)

Pan, G. X.; Cao, F.; Xia, X. H.; Zhang, Y. J.

2016-11-01

Rational construction of advanced FeS2 cathode is one of research hotspots, and of great importance for developing high-performance lithium ion batteries (LIBs). Herein we report a facile hydrolysis-sulfurization method for fabrication of FeS2/C nanotubes arrays with the help of sacrificial Co2(OH)2CO3 nanowires template and glucose carbonization. Self-supported FeS2/C nanotubes consist of interconnected nanoburrs of 5-20 nm, and show hierarchical porous structure. The FeS2/C nanotubes arrays are demonstrated with enhanced cycling life and noticeable high-rate capability with capacities ranging from 735 mAh g-1 at 0.25 C to 482 mAh g-1 at 1.5 C, superior to those FeS2 counterparts in the literature. The composite nanotubes arrays architecture plays positive roles in the electrochemical enhancement due to combined advantages of large electrode-electrolyte contact area, good strain accommodation, improved electrical conductivity, and enhanced structural stability.

Flywheel Energy Storage Technology Workshop

NASA Astrophysics Data System (ADS)

Okain, D.; Howell, D.

Advances in recent years of high strength/lightweight materials, high performance magnetic bearings, and power electronics technology has spurred a renewed interest by the transportation, utility, and manufacturing industries in flywheel energy storage (FES) technologies. FES offers several advantages over conventional electrochemical energy storage, such as high specific energy and specific power, fast charging time, long service life, high turnaround efficiency (energy out/energy in), and no hazardous/toxic materials or chemicals are involved. Potential applications of FES units include power supplies for hybrid and electric vehicles, electric vehicle charging stations, space systems, and pulsed power devices. Also, FES units can be used for utility load leveling, uninterruptable power supplies to protect electronic equipment and electrical machinery, and for intermittent wind or photovoltaic energy sources. The purpose of this workshop is to provide a forum to highlight technologies that offer a high potential to increase the performance of FES systems and to discuss potential solutions to overcome present FES application barriers. This document consists of viewgraphs from 27 presentations.

FES in Europe and Beyond: Current Translational Research

PubMed Central

Coste, Christine Azevedo; Mayr, Winfried; Bijak, Manfred; Musarò, Antonio; Carraro, Ugo

2016-01-01

Capacity of adult neural and muscle tissues to respond to external Electrical Stimulation (ES) is the biological basis for the development and implementation of mobility impairment physiotherapy protocols and of related assistive technologies, e.g, Functional Electrical Stimulation (FES). All body tissues, however, respond to electrical stimulation and, indeed, the most successful application of FES is electrical stimulation of the heart to revert or limit effects of arrhythmias (Pace-makers and Defibrillators). Here, we list and discuss results of FES current research activities, in particular those presented at 2016 Meetings: the PaduaMuscleDays, the Italian Institute of Myology Meeting, the 20th International Functional Electrical Stimulation Society (IFESS) conference held in Montpellier and the Vienna Workshop on FES. Several papers were recently e-published in the European Journal of Translational Myology as reports of meeting presentations. All the events and publications clearly show that FES research in Europe and beyond is alive and promisses translation of results into clinical management of a very large population of persons with deficiencies. PMID:28078074

Sulfur Modifications of the Wobble U34 in tRNAs and their Intracellular Localization in Eukaryotic Cells

PubMed Central

Nakai, Yumi; Nakai, Masato; Yano, Takato

2017-01-01

The wobble uridine (U34) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNALysUUU, tRNAGluUUC, and tRNAGlnUUG, harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of U34, respectively. Both modifications are necessary to construct the proper anticodon loop structure and to enable them to exert their functions in translation. Thio-modification of U34 (s2U34) is found in both cytosolic tRNAs (cy-tRNAs) and mitochondrial tRNAs (mt-tRNAs). Although l-cysteine desulfurase is required in both cases, subsequent sulfur transfer pathways to cy-tRNAs and mt-tRNAs are different due to their distinct intracellular locations. The s2U34 formation in cy-tRNAs involves a sulfur delivery system required for the biosynthesis of iron-sulfur (Fe/S) clusters and certain resultant Fe/S proteins. This review addresses presumed sulfur delivery pathways for the s2U34 formation in distinct intracellular locations, especially that for cy-tRNAs in comparison with that for mt-tRNAs. PMID:28218716

Redox cofactors insertion in prokaryotic molybdoenzymes occurs via a conserved folding mechanism

PubMed Central

Arias-Cartin, Rodrigo; Ceccaldi, Pierre; Schoepp-Cothenet, Barbara; Frick, Klaudia; Blanc, Jean-Michel; Guigliarelli, Bruno; Walburger, Anne; Grimaldi, Stéphane; Friedrich, Thorsten; Receveur-Brechot, Véronique; Magalon, Axel

2016-01-01

A major gap of knowledge in metalloproteins is the identity of the prefolded state of the protein before cofactor insertion. This holds for molybdoenzymes serving multiple purposes for life, especially in energy harvesting. This large group of prokaryotic enzymes allows for coordination of molybdenum or tungsten cofactors (Mo/W-bisPGD) and Fe/S clusters. Here we report the structural data on a cofactor-less enzyme, the nitrate reductase respiratory complex and characterize the conformational changes accompanying Mo/W-bisPGD and Fe/S cofactors insertion. Identified conformational changes are shown to be essential for recognition of the dedicated chaperone involved in cofactors insertion. A solvent-exposed salt bridge is shown to play a key role in enzyme folding after cofactors insertion. Furthermore, this salt bridge is shown to be strictly conserved within this prokaryotic molybdoenzyme family as deduced from a phylogenetic analysis issued from 3D structure-guided multiple sequence alignment. A biochemical analysis with a distantly-related member of the family, respiratory complex I, confirmed the critical importance of the salt bridge for folding. Overall, our results point to a conserved cofactors insertion mechanism within the Mo/W-bisPGD family. PMID:27886223

Sulfur Modifications of the Wobble U34 in tRNAs and their Intracellular Localization in Eukaryotic Cells.

PubMed

Nakai, Yumi; Nakai, Masato; Yano, Takato

2017-02-18

The wobble uridine (U 34 ) of transfer RNAs (tRNAs) for two-box codon recognition, i.e., tRNA Lys UUU , tRNA Glu UUC , and tRNA Gln UUG , harbor a sulfur- (thio-) and a methyl-derivative structure at the second and fifth positions of U 34 , respectively. Both modifications are necessary to construct the proper anticodon loop structure and to enable them to exert their functions in translation. Thio-modification of U 34 (s²U 34 ) is found in both cytosolic tRNAs (cy-tRNAs) and mitochondrial tRNAs (mt-tRNAs). Although l-cysteine desulfurase is required in both cases, subsequent sulfur transfer pathways to cy-tRNAs and mt-tRNAs are different due to their distinct intracellular locations. The s²U 34 formation in cy-tRNAs involves a sulfur delivery system required for the biosynthesis of iron-sulfur (Fe/S) clusters and certain resultant Fe/S proteins. This review addresses presumed sulfur delivery pathways for the s²U 34 formation in distinct intracellular locations, especially that for cy-tRNAs in comparison with that for mt-tRNAs.

Cooperativity in self-limiting equilibrium self-associating systems

NASA Astrophysics Data System (ADS)

Freed, Karl F.

2012-11-01

A wide variety of highly cooperative self-assembly processes in biological and synthetic systems involve the assembly of a large number (m) of units into clusters, with m narrowly peaked about a large size m0 ≫ 1 and with a second peak centered about the m = 1 unassembled monomers. While very specific models have been proposed for the assembly of, for example, viral capsids and core-shell micelles of ß-casein, no available theory describes a thermodynamically general mechanism for this double peaked, highly cooperative equilibrium assembly process. This study provides a general mechanism for these cooperative processes by developing a minimal Flory-Huggins type theory. Beginning from the simplest non-cooperative, free association model in which the equilibrium constant for addition of a monomer to a cluster is independent of cluster size, the new model merely allows more favorable growth for clusters of intermediate sizes. The theory is illustrated by computing the phase diagram for cases of self-assembly on cooling or heating and for the mass distribution of the two phases.

Bi cluster-assembled interconnects produced using SU8 templates

NASA Astrophysics Data System (ADS)

Partridge, J. G.; Matthewson, T.; Brown, S. A.

2007-04-01

Bi clusters with an average diameter of 25 nm have been deposited from an inert gas aggregation source and assembled into thin-film interconnects which are formed between planar electrical contacts and supported on Si substrates passivated with Si3N4 or thermally grown oxide. A layer of SU8 (a negative photoresist based on EPON SU-8 epoxy resin) is patterned using optical or electron-beam lithography, and it defines the position and dimensions of the cluster film. The conduction between the contacts is monitored throughout the deposition/assembly process, and subsequent I(V) characterization is performed in situ. Bi cluster-assembled interconnects have been fabricated with nanoscale widths and with up to 1:1 thickness:width aspect ratios. The conductivity of these interconnects has been increased, post-deposition, using a simple thermal annealing process.

Using Functional Electrical Stimulation Mediated by Iterative Learning Control and Robotics to Improve Arm Movement for People With Multiple Sclerosis.

PubMed

Sampson, Patrica; Freeman, Chris; Coote, Susan; Demain, Sara; Feys, Peter; Meadmore, Katie; Hughes, Ann-Marie

2016-02-01

Few interventions address multiple sclerosis (MS) arm dysfunction but robotics and functional electrical stimulation (FES) appear promising. This paper investigates the feasibility of combining FES with passive robotic support during virtual reality (VR) training tasks to improve upper limb function in people with multiple sclerosis (pwMS). The system assists patients in following a specified trajectory path, employing an advanced model-based paradigm termed iterative learning control (ILC) to adjust the FES to improve accuracy and maximise voluntary effort. Reaching tasks were repeated six times with ILC learning the optimum control action from previous attempts. A convenience sample of five pwMS was recruited from local MS societies, and the intervention comprised 18 one-hour training sessions over 10 weeks. The accuracy of tracking performance without FES and the amount of FES delivered during training were analyzed using regression analysis. Clinical functioning of the arm was documented before and after treatment with standard tests. Statistically significant results following training included: improved accuracy of tracking performance both when assisted and unassisted by FES; reduction in maximum amount of FES needed to assist tracking; and less impairment in the proximal arm that was trained. The system was well tolerated by all participants with no increase in muscle fatigue reported. This study confirms the feasibility of FES combined with passive robot assistance as a potentially effective intervention to improve arm movement and control in pwMS and provides the basis for a follow-up study.

Mirror therapy combined with biofeedback functional electrical stimulation for motor recovery of upper extremities after stroke: a pilot randomized controlled trial.

PubMed

Kim, Jung Hee; Lee, Byoung-Hee

2015-06-01

The objective of this study was to evaluate the effects of mirror therapy in combination with biofeedback functional electrical stimulation (BF-FES) on motor recovery of the upper extremities after stroke. Twenty-nine patients who suffered a stroke > 6 months prior participated in this study and were randomly allocated to three groups. The BF-FES + mirror therapy and FES + mirror therapy groups practiced training for 5 × 30 min sessions over a 4-week period. The control group received a conventional physical therapy program. The following clinical tools were used to assess motor recovery of the upper extremities: electrical muscle tester, electrogoniometer, dual-inclinometer, electrodynamometer, the Box and Block Test (BBT) and Jabsen Taylor Hand Function Test (JHFT), the Functional Independence Measure, the Modified Ashworth Scale, and the Stroke Specific Quality of Life (SSQOL) assessment. The BF-FES + mirror therapy group showed significant improvement in wrist extension as revealed by the Manual Muscle Test and Range of Motion (p < 0.05). The BF-FES + mirror therapy group showed significant improvement in the BBT, JTHT, and SSQOL compared with the FES + mirror therapy group and control group (p < 0.05). We found that BF-FES + mirror therapy induced motor recovery and improved quality of life. These results suggest that mirror therapy, in combination with BF-FES, is feasible and effective for motor recovery of the upper extremities after stroke. Copyright © 2014 John Wiley & Sons, Ltd.

Beyond assembly bias: exploring secondary halo biases for cluster-size haloes

DOE PAGES

Mao, Yao-Yuan; Zentner, Andrew R.; Wechsler, Risa H.

2017-12-01

Secondary halo bias, commonly known as ‘assembly bias’, is the dependence of halo clustering on a halo property other than mass. This prediction of the Λ Cold Dark Matter cosmology is essential to modelling the galaxy distribution to high precision and interpreting clustering measurements. As the name suggests, different manifestations of secondary halo bias have been thought to originate from halo assembly histories. We show conclusively that this is incorrect for cluster-size haloes. We present an up-to-date summary of secondary halo biases of high-mass haloes due to various halo properties including concentration, spin, several proxies of assembly history, and subhalomore » properties. While concentration, spin, and the abundance and radial distribution of subhaloes exhibit significant secondary biases, properties that directly quantify halo assembly history do not. In fact, the entire assembly histories of haloes in pairs are nearly identical to those of isolated haloes. In general, a global correlation between two halo properties does not predict whether or not these two properties exhibit similar secondary biases. For example, assembly history and concentration (or subhalo abundance) are correlated for both paired and isolated haloes, but follow slightly different conditional distributions in these two cases. Lastly, this results in a secondary halo bias due to concentration (or subhalo abundance), despite the lack of assembly bias in the strict sense for cluster-size haloes. Due to this complexity, caution must be exercised in using any one halo property as a proxy to study the secondary bias due to another property.« less

Beyond assembly bias: exploring secondary halo biases for cluster-size haloes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Yao-Yuan; Zentner, Andrew R.; Wechsler, Risa H.

Secondary halo bias, commonly known as ‘assembly bias’, is the dependence of halo clustering on a halo property other than mass. This prediction of the Λ Cold Dark Matter cosmology is essential to modelling the galaxy distribution to high precision and interpreting clustering measurements. As the name suggests, different manifestations of secondary halo bias have been thought to originate from halo assembly histories. We show conclusively that this is incorrect for cluster-size haloes. We present an up-to-date summary of secondary halo biases of high-mass haloes due to various halo properties including concentration, spin, several proxies of assembly history, and subhalomore » properties. While concentration, spin, and the abundance and radial distribution of subhaloes exhibit significant secondary biases, properties that directly quantify halo assembly history do not. In fact, the entire assembly histories of haloes in pairs are nearly identical to those of isolated haloes. In general, a global correlation between two halo properties does not predict whether or not these two properties exhibit similar secondary biases. For example, assembly history and concentration (or subhalo abundance) are correlated for both paired and isolated haloes, but follow slightly different conditional distributions in these two cases. Lastly, this results in a secondary halo bias due to concentration (or subhalo abundance), despite the lack of assembly bias in the strict sense for cluster-size haloes. Due to this complexity, caution must be exercised in using any one halo property as a proxy to study the secondary bias due to another property.« less

Solid-Solution Anion-Enhanced Electrochemical Performances of Metal Sulfides/Selenides for Sodium-Ion Capacitors: The Case of FeS2- xSe x.

PubMed

Long, Yaqiong; Yang, Jing; Gao, Xin; Xu, Xuena; Fan, Weiliu; Yang, Jian; Hou, Shifeng; Qian, Yitai

2018-04-04

Transition-metal sulfides/selenides are explored as advanced electrode materials for nonaqueous sodium-ion capacitors, using FeS 2- x Se x as an example. A solid solution of S/Se in FeS 2- x Se x allows it to combine the high capacity of FeS 2 and the good diffusion kinetics of FeSe 2 together, thereby exhibiting excellent cycle stability (∼220 mA h g -1 after 6000 cycles at 2 A g -1 ) and superior rate capability (∼210 mA h g -1 at 40 A g -1 ) within 0.8-3.0 V. These results are much better than those of FeS 2 and FeSe 2 , confirming the advantages of S/Se solid solution, as supported by EIS spectra, DFT calculations, and electronic conductivity. As FeS 2- x Se x is paired with the activated carbon (AC) as Na-ion capacitors, this device is also better than sodium-ion batteries of FeS 2- x Se x //Na 3 V 2 (PO 4 ) 3 and sodium-ion capacitors of metal oxides//AC, particularly at high rates. These results open a new door for the applications of sulfides/selenides in another device of electrochemical energy storage.

An Exploratory Investigation on the Use of Closed-Loop Electrical Stimulation to Assist Individuals with Stroke to Perform Fine Movements with Their Hemiparetic Arm.

PubMed

Lew, Brian; Alavi, Nezam; Randhawa, Bubblepreet K; Menon, Carlo

2016-01-01

Stroke is the leading cause of upper limb impairments resulting in disability. Modern rehabilitation includes training with robotic exoskeletons and functional electrical stimulation (FES). However, there is a gap in knowledge to define the detailed use of FES in stroke rehabilitation. In this paper, we explore applying closed-loop FES to the upper extremities of healthy volunteers and individuals with a hemiparetic arm resulting from stroke. We used a set of gyroscopes to monitor arm movements and used a non-linear controller, namely, the robust integral of the sign of the error (RISE), to assess the viability of controlling FES in closed loop. Further, we explored the application of closed-loop FES in improving functional tasks performed by individuals with stroke. Four healthy individuals of ages 27-32 years old and five individuals with stroke of ages 61-83 years old participated in this study. We used the Rehastim FES unit (Hasomed Ltd.) with real-time modulation of pulse width and amplitude. Both healthy and stroke individuals were tested in RISE-controlled single and multi-joint upper limb motions following first a sinusoidal trajectory. Individuals with stroke were also asked to perform the following functional tasks: picking up a basket, picking and placing an object on a table, cutting a pizza, pulling back a chair, eating with a spoon, as well as using a stapler and grasping a pen. Healthy individuals were instructed to keep their arm relaxed during the experiment. Most individuals with stroke were able to follow the sinusoid trajectories with their arm joints under the sole excitation of the closed-loop-controlled FES. One individual with stroke, who was unable to perform any of the functional tasks independently, succeeded in completing all the tasks when FES was used. Three other individuals with stroke, who were unable to complete a few tasks independently, completed some of them when FES was used. The remaining stroke participant was able to complete all tasks with and without FES. Our results suggest that individuals with a low Fugl-Meyer score or a higher level of disability may benefit the most with the use of closed-loop-controlled FES.

An Exploratory Investigation on the Use of Closed-Loop Electrical Stimulation to Assist Individuals with Stroke to Perform Fine Movements with Their Hemiparetic Arm

PubMed Central

Lew, Brian; Alavi, Nezam; Randhawa, Bubblepreet K.; Menon, Carlo

2016-01-01

Stroke is the leading cause of upper limb impairments resulting in disability. Modern rehabilitation includes training with robotic exoskeletons and functional electrical stimulation (FES). However, there is a gap in knowledge to define the detailed use of FES in stroke rehabilitation. In this paper, we explore applying closed-loop FES to the upper extremities of healthy volunteers and individuals with a hemiparetic arm resulting from stroke. We used a set of gyroscopes to monitor arm movements and used a non-linear controller, namely, the robust integral of the sign of the error (RISE), to assess the viability of controlling FES in closed loop. Further, we explored the application of closed-loop FES in improving functional tasks performed by individuals with stroke. Four healthy individuals of ages 27–32 years old and five individuals with stroke of ages 61–83 years old participated in this study. We used the Rehastim FES unit (Hasomed Ltd.) with real-time modulation of pulse width and amplitude. Both healthy and stroke individuals were tested in RISE-controlled single and multi-joint upper limb motions following first a sinusoidal trajectory. Individuals with stroke were also asked to perform the following functional tasks: picking up a basket, picking and placing an object on a table, cutting a pizza, pulling back a chair, eating with a spoon, as well as using a stapler and grasping a pen. Healthy individuals were instructed to keep their arm relaxed during the experiment. Most individuals with stroke were able to follow the sinusoid trajectories with their arm joints under the sole excitation of the closed-loop-controlled FES. One individual with stroke, who was unable to perform any of the functional tasks independently, succeeded in completing all the tasks when FES was used. Three other individuals with stroke, who were unable to complete a few tasks independently, completed some of them when FES was used. The remaining stroke participant was able to complete all tasks with and without FES. Our results suggest that individuals with a low Fugl–Meyer score or a higher level of disability may benefit the most with the use of closed-loop-controlled FES. PMID:27014683

Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers

DOEpatents

Alivisatos, A. Paul; Colvin, Vicki L.

1998-01-01

Methods are described for attaching semiconductor nanocrystals to solid inorganic surfaces, using self-assembled bifunctional organic monolayers as bridge compounds. Two different techniques are presented. One relies on the formation of self-assembled monolayers on these surfaces. When exposed to solutions of nanocrystals, these bridge compounds bind the crystals and anchor them to the surface. The second technique attaches nanocrystals already coated with bridge compounds to the surfaces. Analyses indicate the presence of quantum confined clusters on the surfaces at the nanolayer level. These materials allow electron spectroscopies to be completed on condensed phase clusters, and represent a first step towards synthesis of an organized assembly of clusters. These new products are also disclosed.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Yeast Mitoribosome Large Subunit Assembly Proceeds by Hierarchical Incorporation of Protein Clusters and Modules on the Inner Membrane.

PubMed

Zeng, Rui; Smith, Erin; Barrientos, Antoni

2018-03-06

Mitoribosomes are specialized for the synthesis of hydrophobic membrane proteins encoded by mtDNA, all essential for oxidative phosphorylation. Despite their linkage to human mitochondrial diseases and the recent cryoelectron microscopy reconstruction of yeast and mammalian mitoribosomes, how they are assembled remains obscure. Here, we dissected the yeast mitoribosome large subunit (mtLSU) assembly process by systematic genomic deletion of 44 mtLSU proteins (MRPs). Analysis of the strain collection unveiled 37 proteins essential for functional mtLSU assembly, three of which are critical for mtLSU 21S rRNA stability. Hierarchical cluster analysis of mtLSU subassemblies accumulated in mutant strains revealed co-operative assembly of protein sets forming structural clusters and preassembled modules. It also indicated crucial roles for mitochondrion-specific membrane-binding MRPs in anchoring newly transcribed 21S rRNA to the inner membrane, where assembly proceeds. Our results define the yeast mtLSU assembly landscape in vivo and provide a foundation for studies of mitoribosome assembly across evolution. Copyright © 2018 Elsevier Inc. All rights reserved.

Iron Sulfide Attenuates the Methanogenic Toxicity of Elemental Copper and Zinc Oxide Nanoparticles and their Soluble Metal Ion Analogs

PubMed Central

Gonzalez-Estrella, Jorge; Gallagher, Sara; Sierra-Alvarez, Reyes; Field, Jim A.

2016-01-01

Elemental copper (Cu0) and zinc oxide (ZnO) nanoparticle (NP) toxicity to methanogens has been attributed to the release of soluble metal ions. Iron sulfide (FeS) partially controls the soluble concentration of heavy metals and their toxicity in aquatic environments. Heavy metals displace the Fe from FeS forming poorly soluble metal sulfides in the FeS matrix. Therefore, FeS may be expected to attenuate the NP toxicity. This work assessed FeS as an attenuator of the methanogenic toxicity of Cu0 and ZnO NPs and their soluble salt analogs. The toxicity attenuation capacity of fine (25–75 µm) and coarse (500 to 1200 µm) preparations of FeS (FeS-f and FeS-c respectively) was tested in the presence of highly inhibitory concentrations of CuCl2, ZnCl2 Cu0 and ZnO NPs. FeS-f attenuated methanogenic toxicity better than FeS-c. The results revealed that 2.5× less FeS-f than FeS-c was required to recover the methanogenic activity to 50% (activity normalized to uninhibited controls). The results also indicated that a molar FeS-f/Cu0 NP, FeS-f/ZnO NP, FeS-f/ZnCl2, and FeS-f/CuCl2 ratio of 2.14, 2.14, 4.28, and 8.56 respectively, was necessary to recover the methanogenic activity to >75%. Displacement experiments demonstrated that CuCl2 and ZnCl2 partially displaced Fe from FeS. As a whole, the results indicate that not all the sulfide in FeS was readily available to react with the soluble Cu and Zn ions which may explain the need for a large stoichiometric excesses of FeS to highly attenuate Cu and Zn toxicity. Overall, this study provides evidence that FeS attenuates the toxicity caused by Cu0 and ZnO NPs and their soluble ion analogs to methanogens. PMID:26803736

Motor recovery and cortical plasticity after functional electrical stimulation in a rat model of focal stroke.

PubMed

Cecatto, Rebeca Boltes; Maximino, Jessica Ruivo; Chadi, Gerson

2014-09-01

The aim of this study was to investigate the functional responses and plastic cortical changes in a sample of animals with sequelae of cerebral ischemia that were subjected to a model of functional electrical stimulation (FES). Rats received an ischemic cortical lesion (Rose Bengal method) and were randomized and submitted to an FES stimulation (1-2 mA, 30 Hz, 20-40 mins for 14 days) or sham stimulation. The Foot Fault Test was performed before inducing the cortical lesion and also before and after FES. Brain immunochemistry labeling with microtubule-associated protein-2 and neurofilament-200 markers was performed after FES. The authors found a decreased percentage of errors in the Foot Fault Test (P < 0.001) in the stimulated group compared with the sham group after FES. FES has not altered the lesion size. Spontaneous motor parameters returned to basal values in both groups. The qualitative analysis showed an increased amount of radial microtubule-associated protein-2 immunoreactive fibers in the preserved cortex adjacent to stroke site in the stimulated animals. Regarding the measurements of neurofilament-200 immunostaining, there were no differences between the hemispheres or groups in area or intensity. Acute and short period of FES led to motor recovery of ankle joint neurodisability. The extent to which compensatory plasticity occurs after stroke or after FES and the extent to which it contributes to functional recovery are yet unclear. The changes induced by the stimulation may improve the ability of the nervous system to undergo spontaneous recovery, which is of substantial interest for neurorehabilitation strategies.

Remediation of hexavalent chromium spiked soil by using synthesized iron sulfide particles.

PubMed

Li, Yujie; Wang, Wanyu; Zhou, Liqiang; Liu, Yuanyuan; Mirza, Zakaria A; Lin, Xiang

2017-02-01

Carboxymethyl cellulose (CMC) stabilized microscale iron sulfide (FeS) particles were synthesized and applied to remediate hexavalent chromium (Cr(VI)) spiked soil. The effects of parameters including dosage of FeS particles, soil moisture, and natural organic matter (NOM) in soil were investigated with comparison to iron sulfate (FeSO 4 ). The results show that the stabilized FeS particles can reduce Cr(VI) and immobilize Cr in soil quickly and efficiently. The soil moisture ranging from 40% to 70% and NOM in soil had no significant effects on Cr(VI) remediation by FeS particles. When molar ratio of FeS to Cr(VI) was 1.5:1, about 98% of Cr(VI) in soil was reduced by FeS particles in 3 d and Cr(VI) concentration decreased from 1407 mg kg -1 to 16 mg kg -1 . The total Cr and Cr(VI) in Toxicity Characteristic Leaching Procedure (TCLP) leachate were reduced by 98.4% and 99.4%, respectively. In FeS particles-treated soil, the exchangeable Cr fraction was mainly converted to Fe-Mn oxides bound fraction because of the precipitation of Cr(III)-Fe(III) hydroxides. The physiologically based extraction test (PBET) bioaccessibility of Cr was decreased from 58.67% to 6.98%. Compared to FeSO 4 , the high Cr(VI) removal and Cr immobilization efficiency makes prepared FeS particles a great potential in field application of Cr(VI) contaminated soil remediation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Early assembly of the most massive galaxies.

PubMed

Collins, Chris A; Stott, John P; Hilton, Matt; Kay, Scott T; Stanford, S Adam; Davidson, Michael; Hosmer, Mark; Hoyle, Ben; Liddle, Andrew; Lloyd-Davies, Ed; Mann, Robert G; Mehrtens, Nicola; Miller, Christopher J; Nichol, Robert C; Romer, A Kathy; Sahlén, Martin; Viana, Pedro T P; West, Michael J

2009-04-02

The current consensus is that galaxies begin as small density fluctuations in the early Universe and grow by in situ star formation and hierarchical merging. Stars begin to form relatively quickly in sub-galactic-sized building blocks called haloes which are subsequently assembled into galaxies. However, exactly when this assembly takes place is a matter of some debate. Here we report that the stellar masses of brightest cluster galaxies, which are the most luminous objects emitting stellar light, some 9 billion years ago are not significantly different from their stellar masses today. Brightest cluster galaxies are almost fully assembled 4-5 billion years after the Big Bang, having grown to more than 90 per cent of their final stellar mass by this time. Our data conflict with the most recent galaxy formation models based on the largest simulations of dark-matter halo development. These models predict protracted formation of brightest cluster galaxies over a Hubble time, with only 22 per cent of the stellar mass assembled at the epoch probed by our sample. Our findings suggest a new picture in which brightest cluster galaxies experience an early period of rapid growth rather than prolonged hierarchical assembly.

Structure and dynamics of optically directed self-assembly of nanoparticles

PubMed Central

Roy, Debjit; Mondal, Dipankar; Goswami, Debabrata

2016-01-01

Self-assembly of nanoparticles leading to the formation of colloidal clusters often serves as the representative analogue for understanding molecular assembly. Unravelling the in situ structure and dynamics of such clusters in liquid suspensions is highly challenging. Presently colloidal clusters are first isolated from their generating environment and then their structures are probed by light scattering methods. In order to measure the in situ structure and dynamics of colloidal clusters, we have generated them using the high-repetition-rate femtosecond laser pulse optical tweezer. Since the constituent of our dimer, trimer or tetramer clusters are 250 nm radius two-photon resonant fluorophore coated nanospheres under the optical trap, they inherently produce Two-Photon Fluorescence, which undergo intra-nanosphere Fluorescence Energy Transfer. This unique energy transfer signature, in turn, enables us to visualize structures and orientations of these colloidal clusters during the process of their formation and subsequent dynamics in a liquid suspension. We also show that due to shape-birefringence, orientation and structural control of these colloidal clusters are possible as the polarization of the trapping laser is changed from linear to circular. We thus report important progress in sampling the smallest possible aggregates of nanoparticles, dimers, trimers or tetramers, formed early in the self-assembly process. PMID:27006305

Thermal activated ("thermal") battery technology. Part IIIa: FeS 2 cathode material

NASA Astrophysics Data System (ADS)

Masset, Patrick J.; Guidotti, Ronald A.

This article presents an overview of the pyrite FeS 2 used as cathode material in thermally activated ("thermal") batteries. A large emphasis was placed on the physicochemical properties and electrochemical performance of the pyrite FeS 2, including the discharge mechanisms, self-discharge phenomena, and recent developments.

Oxygen-tolerant [NiFe]-hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster.

PubMed

Lukey, Michael J; Roessler, Maxie M; Parkin, Alison; Evans, Rhiannon M; Davies, Rosalind A; Lenz, Oliver; Friedrich, Baerbel; Sargent, Frank; Armstrong, Fraser A

2011-10-26

An important clue to the mechanism for O(2) tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron-sulfur cluster that is proximal to the active site. The O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron-sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O(2).

A Polymerase With Potential: The Fe-S Cluster in Human DNA Primase.

PubMed

Holt, Marilyn E; Salay, Lauren E; Chazin, Walter J

2017-01-01

Replication of DNA in eukaryotes is primarily executed by the combined action of processive DNA polymerases δ and ɛ. These enzymes cannot initiate synthesis of new DNA without the presence of a primer on the template ssDNA. The primers on both the leading and lagging strands are generated by DNA polymerase α-primase (pol-prim). DNA primase is a DNA-dependent RNA polymerase that synthesizes the first ~10 nucleotides and then transfers the substrate to polymerase α to complete primer synthesis. The mechanisms governing the coordination and handoff between primase and polymerase α are largely unknown. Isolated DNA primase contains a [4Fe-4S] 2+ cluster that has been shown to serve as a redox switch modulating DNA binding affinity. This discovery suggests a mechanism for modulating the priming activity of primase and handoff to polymerase α. In this chapter, we briefly discuss the current state of knowledge of primase structure and function, including the role of its iron-sulfur cluster. This is followed by providing the methods for expressing, purifying, and biophysically/structurally characterizing primase and its iron-sulfur cluster-containing domain, p58C. © 2017 Elsevier Inc. All rights reserved.

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

PubMed

Rousset, M; Montet, Y; Guigliarelli, B; Forget, N; Asso, M; Bertrand, P; Fontecilla-Camps, J C; Hatchikian, E C

1998-09-29

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Solubility of K in Fe-S liquid, silicate-K/Fe-S/liq equilibria, and their planetary implications

NASA Technical Reports Server (NTRS)

Gangully, J.; Kennedy, G. C.

1977-01-01

Potassium has been found to have extremely limited absolute solubility in Fe-S liquid in the pressure-temperature range of 18 to 40 kbars, 1050 to 1150 C, and fO2 within the field of metallic iron. It also partitioned into a certain silicate phase highly in preference to Fe-S liquid at 30 kbar and 1100 C. The dependence of the partitioning of K between solid silicate and Fe-S liquid on fO2 and compositions of mineral solid solutions have been analyzed. These experimental data, along with those of others, limit the amount of K that could fractionate in Fe-S liquid layers or a core in the early history of the moon and, thus, act as localized heat sources in its thermal history models; the data also seem to argue against a chondritic abundance of potassium for earth. The question of fractionation of enough K-40 in an Fe-S liquid outer core of earth to provide the necesary thermal energy for the geomagnetic dynamo remains unresolved.

Reliability and Validity of the Short Falls Efficacy Scale International in English, Mandarin, and Bahasa Malaysia in Malaysia.

PubMed

Tan, Maw Pin; Nalathamby, Nemala; Mat, Sumaiyah; Tan, Pey June; Kamaruzzaman, Shahrul Bahyah; Morgan, Karen

2018-01-01

While the prevalence of falls among Malaysian older adults is comparable to other older populations around the world, little is currently known about fear of falling in Malaysia. The Falls Efficacy Scale International (FES-I) and short FES-I scales to measure fear of falling have not yet been validated for use within the Malaysian population, and are currently not available in Bahasa Malaysia (BM). A total of 402 participants aged ≥63 years were recruited. The questionnaire was readministered to 149 participants, 4 to 8 weeks after the first administration to determine test-retest reliability. The original version of the 7-item short FES-I is available in English, while the Mandarin was adapted from the 16-item Mandarin FES-I. The BM version was translated according to protocol by four experts. The internal structure of the FES-I was examined by factor analysis. The 7-item short FES-I showed good internal reliability and test-retest reliability for English, Mandarin, and BM versions for Malaysia.

Interaction of post-stroke voluntary effort and functional neuromuscular electrical stimulation

PubMed Central

Makowski, Nathaniel; Knutson, Jayme; Chae, John; Crago, Patrick

2012-01-01

Functional Electrical Stimulation (FES) may be able to augment functional arm and hand movement after stroke. Post-stroke neuroprostheses that incorporate voluntary effort and FES to produce the desired movement need to consider how the forces generated by voluntary effort and FES combine together, even in the same muscle, in order to provide an appropriate level of stimulation to elicit the desired assistive force. The goal of this study was to determine if the force produced by voluntary effort and FES add together independently of effort, or if the increment in force is dependent on the level of voluntary effort. Isometric force matching tasks were performed under different combinations of voluntary effort and electrical stimulation. Participants reached a steady level of force and while attempting to maintain a constant effort level, FES was applied to augment the force. Results indicate that the increment in force produced by FES decreases as the level of initial voluntary effort increases. Potential mechanisms causing the change in force output are proposed, but the relative contribution of each mechanism is unknown. PMID:23516086

Does aspartate 170 of the D1 polypeptide ligate the manganese cluster in photosystem II? An EPR and ESEEM Study.

PubMed

Debus, Richard J; Aznar, Constantino; Campbell, Kristy A; Gregor, Wolfgang; Diner, Bruce A; Britt, R David

2003-09-16

Aspartate 170 of the D1 polypeptide provides part of the high-affinity binding site for the first Mn(II) ion that is photooxidized during the light-driven assembly of the (Mn)(4) cluster in photosystem II [Campbell, K. A., Force, D. A., Nixon, P. J., Dole, F., Diner, B. A., and Britt, R. D. (2000) J. Am. Chem. Soc. 122, 3754-3761]. However, despite a wealth of data on D1-Asp170 mutants accumulated over the past decade, there is no consensus about whether this residue ligates the assembled (Mn)(4) cluster. To address this issue, we have conducted an EPR and ESEEM (electron spin-echo envelope modulation) study of D1-D170H PSII particles purified from the cyanobacterium Synechocystis sp. PCC 6803. The line shapes of the S(1) and S(2) state multiline EPR signals of D1-D170H PSII particles are unchanged from those of wild-type PSII particles, and the signal amplitudes correlate approximately with the lower O(2) evolving activity of the mutant PSII particles (40-60% compared to that of the wild type). These data provide further evidence that the assembled (Mn)(4) clusters in D1-D170H cells function normally, even though the assembly of the (Mn)(4) cluster is inefficient in this mutant. In the two-pulse frequency domain ESEEM spectrum of the 9.2 GHz S(2) state multiline EPR signal of D1-D170H PSII particles, the histidyl nitrogen modulation observed at 4-5 MHz is unchanged from that of wild-type PSII particles and no significant new modulation is observed. Three scenarios are presented to explain this result. (1) D1-Asp170 ligates the assembled (Mn)(4) cluster, but the hyperfine couplings to the ligating histidyl nitrogen of D1-His170 are too large or anisotropic to be detected by ESEEM analyses conducted at 9.2 GHz. (2) D1-Asp170 ligates the assembled (Mn)(4) cluster, but D1-His170 does not. (3) D1-Asp170 does not ligate the assembled (Mn)(4) cluster.

Cluster-assembled materials based on M12N12 (M = Al, Ga) fullerene-like clusters.

PubMed

Yong, Yongliang; Song, Bin; He, Pimo

2011-09-28

We report the results of density functional theory calculations on cluster-assembled materials based on M(12)N(12) (M = Al, Ga) fullerene-like clusters. Our results show that the M(12)N(12) fullerene-like structure with six isolated four-membered rings (4NRs) and eight six-membered rings (6NRs) has a T(h) symmetry and a large HOMO-LUMO gap, indicating that the M(12)N(12) cluster would be ideal building blocks for the synthesis of cluster-assembled materials. Via the coalescence of M(12)N(12) building blocks, we find that the M(12)N(12) clusters can bind into stable assemblies by either 6NR or 4NR face coalescence, which enables the construction of rhombohedral or cubic nanoporous framework of varying porosity. The rhombohedral-MN phase is energetically more favorable than the cubic-MN phase. The M(12)N(12) fullerene-like structures in both phases are maintained and the M-N bond lengths between M(12)N(12) monomers are slightly larger than that in isolated M(12)N(12) clusters and the bulk wurtzite phases. The band analysis of both phases reveals that they are all wide-gap semiconductors. Because of the nanoporous character of these phases, they could be used for gas storage, heterogeneous catalysis, filtration and so on.

Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs.

PubMed

Majumdar, Chandrima; Nuñez, Nicole N; Raetz, Alan G; Khuu, Cindy; David, Sheila S

2018-01-01

Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron-sulfur (Fe-S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease. © 2018 Elsevier Inc. All rights reserved.

Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

PubMed

Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

2018-05-17

Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Mössbauer spectroscopy of the chloroplast-targeted DnaJ-like proteins CDJ3 and CDJ4

NASA Astrophysics Data System (ADS)

Auerbach, H.; Kalienkova, V.; Schroda, M.; Schünemann, V.

2017-11-01

The heat shock protein 70 (HSP70) in the chloroplast of Chlamydomonas reinhardtii, termed HSP70B, interacts with chloroplast-targeted DnaJ-like proteins (CDJs). In this work we focus on two CDJ co-chaperones (CDJ3 and CDJ4) of HSP70B which contain a redox-active Fe-S cluster (Dorn et al. Biochem. J. 427, 205 [2010]). We have performed Mössbauer spectroscopy on 57Fe enriched CDJ3 an) CDJ4. Our results indicate that both proteins have unusual [4Fe4S] 2+ clusters showing structural inhomogeneity of the two [Fe 2.5+-Fe 2.5+] pairs. The spectra have been analyzed by means of two components with δ-values characteristic for Fe 2.5+ centers, but the differences in Δ E Q indicate variations in their tetrahedral coordination spheres.

Interaction of FeS 2 and Sulfur in Li-S Battery System

DOE PAGES

Sun, Ke; Cama, Christina A.; DeMayo, Rachel A.; ...

2016-09-09

Many transition metal sulfides are electronically conductive, electrochemically active and reversible in reactions with lithium. However, the application of transition metal sulfides as sulfur cathode additives in lithium-sulfur (Li-S) batteries has not been fully explored. In this study, Pyrite (FeS 2) is studied as a capacity contributing conductive additive in sulfur cathode for Li-S batteries. Electrochemically discharging the S-FeS 2 composite electrodes to 1.0 V activates the FeS 2 component, contributing to the improved Li-S cell discharge energy density. However, direct activation of the FeS 2 component in a fresh S-FeS 2 cell results in a significant shuttling effect inmore » the subsequent charging process, preventing further cell cycling. The slight FeS 2 solubility in electrolyte and its activation alone in S-FeS 2 cells are not the root causes of the severe shuttling effect. The observed severe shuttling effect is strongly correlated to the 1st charging of the activated S-FeS 2 electrode that promotes iron dissolution in electrolyte and the deposition of electronically conductive FeS on the anode SEI. Pre-cycling of the S-FeS 2 cell prior to the FeS 2 activation or the use of LiNO 3 electrolyte additive help to prevent the severe shuttling effect and allow the cell to cycle between 2.6 V to 1.0 V with an extra capacity contribution from the FeS2 components. However, a more effective method of anode pre-passivation is still needed to fully protect the lithium surface from FeS deposition and allow the S-FeS 2 electrode to maintain high energy density over extended cycles. A mechanism explaining the observed phenomena based on the experimental data is proposed and discussed« less

Feasibility study of a take-home array-based functional electrical stimulation system with automated setup for current functional electrical stimulation users with foot-drop.

PubMed

Prenton, Sarah; Kenney, Laurence P; Stapleton, Claire; Cooper, Glen; Reeves, Mark L; Heller, Ben W; Sobuh, Mohammad; Barker, Anthony T; Healey, Jamie; Good, Timothy R; Thies, Sibylle B; Howard, David; Williamson, Tracey

2014-10-01

To investigate the feasibility of unsupervised community use of an array-based automated setup functional electrical stimulator for current foot-drop functional electrical stimulation (FES) users. Feasibility study. Gait laboratory and community use. Participants (N=7) with diagnosis of unilateral foot-drop of central neurologic origin (>6mo) who were regular users of a foot-drop FES system (>3mo). Array-based automated setup FES system for foot-drop (ShefStim). Logged usage, logged automated setup times for the array-based automated setup FES system and diary recording of problems experienced, all collected in the community environment. Walking speed, ankle angles at initial contact, foot clearance during swing, and the Quebec User Evaluation of Satisfaction with Assistive Technology version 2.0 (QUEST version 2.0) questionnaire, all collected in the gait laboratory. All participants were able to use the array-based automated setup FES system. Total setup time took longer than participants' own FES systems, and automated setup time was longer than in a previous study of a similar system. Some problems were experienced, but overall, participants were as satisfied with this system as their own FES system. The increase in walking speed (N=7) relative to no stimulation was comparable between both systems, and appropriate ankle angles at initial contact (N=7) and foot clearance during swing (n=5) were greater with the array-based automated setup FES system. This study demonstrates that an array-based automated setup FES system for foot-drop can be successfully used unsupervised. Despite setup's taking longer and some problems, users are satisfied with the system and it would appear as effective, if not better, at addressing the foot-drop impairment. Further product development of this unique system, followed by a larger-scale and longer-term study, is required before firm conclusions about its efficacy can be reached. Copyright © 2014 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Cross-cultural adaptation and measurement properties testing of the Iconographical Falls Efficacy Scale (Icon-FES).

PubMed

Franco, Marcia Rodrigues; Pinto, Rafael Zambelli; Delbaere, Kim; Eto, Bianca Yumie; Faria, Maíra Sgobbi; Aoyagi, Giovana Ayumi; Steffens, Daniel; Pastre, Carlos Marcelo

2018-02-14

The Iconographical Falls Efficacy Scale (Icon-FES) is an innovative tool to assess concern of falling that uses pictures as visual cues to provide more complete environmental contexts. Advantages of Icon-FES over previous scales include the addition of more demanding balance-related activities, ability to assess concern about falling in highly functioning older people, and its normal distribution. To perform a cross-cultural adaptation and to assess the measurement properties of the 30-item and 10-item Icon-FES in a community-dwelling Brazilian older population. The cross-cultural adaptation followed the recommendations of international guidelines. We evaluated the measurement properties (i.e. internal consistency, test-retest reproducibility, standard error of the measurement, minimal detectable change, construct validity, ceiling/floor effect, data distribution and discriminative validity), in 100 community-dwelling people aged ≥60 years. The 30-item and 10-item Icon-FES-Brazil showed good internal consistency (alpha and omega >0.70) and excellent intra-rater reproducibility (ICC 2,1 =0.96 and 0.93, respectively). According to the standard error of the measurement and minimal detectable change, the magnitude of change needed to exceed the measurement error and variability were 7.2 and 3.4 points for the 30-item and 10-item Icon-FES, respectively. We observed an excellent correlation between both versions of the Icon-FES and Falls Efficacy Scale - International (rho=0.83, p<0.001 [30-item version]; 0.76, p<0.001 [10-item version]). Icon-FES versions showed normal distribution, no floor/ceiling effects and were able to discriminate between groups relating to fall risk factors. Icon-FES-Brazil is a semantically and linguistically appropriate tool with acceptable measurement properties to evaluate concern about falling among the community-dwelling older population. Copyright © 2018 Associação Brasileira de Pesquisa e Pós-Graduação em Fisioterapia. Publicado por Elsevier Editora Ltda. All rights reserved.

Falls and confidence related quality of life outcome measures in an older British cohort

PubMed Central

Parry, S; Steen, N; Galloway, S; Kenny, R; Bond, J

2001-01-01

Falls are common in older subjects and result in loss of confidence and independence. The Falls Efficacy Scale (FES) and the Activities-specific Balance Confidence scale (ABC) were developed in North America to quantify these entities, but contain idiom unfamiliar to an older British population. Neither has been validated in the UK. The FES and the ABC were modified for use within British culture and the internal consistency and test-retest reliability of the modified scales (FES-UK and ABC-UK) assessed. A total of 193 consecutive, ambulant, new, and return patients (n=119; 62%) and their friends and relatives ("visitors", n=74; 38%) were tested on both scales, while the last 60 subjects were retested within one week. Internal reliability was excellent for both scales (Cronbach's alpha 0.97 (FES-UK), and 0.98 (ABC-UK)). Test-retest reliability was good for both scales, though superior for the ABC-UK (intraclass correlation coefficient 0.58 (FES-UK), 0.89 (ABC-UK)). There was evidence to suggest that the ABC-UK was better than the FES-UK at distinguishing between older patients and younger patients (|tABC| = 4.4; |tFES| = 2.3); and between fallers and non-fallers (|tABC| = 8.7; |tFES| = 5.0) where the t statistics are based on the comparison of two independent samples. The ABC-UK and FES-UK are both reliable and valid measures for the assessment of falls and balance related confidence in older adults. However, better test-retest reliability and more robust differentiation of subgroups in whom falls related quality of life would be expected to be different make the ABC-UK the current instrument of choice in assessing this entity in older British subjects.


Keywords: quality of life; falls; elderly; health status measurement PMID:11161077

Functional electrical stimulation for chronic heart failure: a meta-analysis.

PubMed

Smart, Neil A; Dieberg, Gudrun; Giallauria, Francesco

2013-07-15

We conducted a meta-analysis of randomized, controlled trials of combined electrical stimulation versus conventional exercise training or placebo control in heart failure patients. A systematic search was conducted of Medline (Ovid) (1950-September 2011), Embase.com (1974-September 2011), Cochrane Central Register of Controlled Trials and CINAHL (1981-September 2011). The search strategy included a mix of MeSH and free text terms for the key concepts heart failure, exercise training and functional electrical stimulation (FES). FES produced inferior improvements in peak VO2 when compared to cycle training: mean difference (MD) -0.32 ml.kg(-1).min(-1) (95% C.I. -0.63 to -0.02 ml.kg(-1).min(-1), p=0.04), however FES elicited superior improvements in peak VO2: MD 2.30 ml.kg(-1).min(-1) (95% C.I. 1.98 to 2.62 ml.kg(-1).min(-1), p<0.00001); and six minute walk distance to sedentary care or sham FES; MD 46.9 m (95% C.I. 22.5 to 71.3m, p=0.0002). There was no difference in change in quality of life between cycling and FES, but FES elicited significantly larger improvements in Minnesota Living with Heart Failure score than placebo or sham treatment; MD 1.15 (95% C.I. 0.69 to 1.61, p<0.00001). Moreover, the total FES intervention hours were strongly correlated with change in peak VO2, (r=0.80, p=0.02). Passive or active exercise is beneficial for patients with moderate to severe heart failure, but active cycling, or other aerobic/resistance activity is preferred in patients with heart failure who are able to exercise, and FES is the preferred modality in those unable to actively exercise. The benefits of FES may however, be smaller than those observed in conventional exercise training. Aggregate hours of electrical stimulation therapy were associated with larger improvements in cardio-respiratory fitness. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Brain-computer interface controlled functional electrical stimulation system for ankle movement.

PubMed

Do, An H; Wang, Po T; King, Christine E; Abiri, Ahmad; Nenadic, Zoran

2011-08-26

Many neurological conditions, such as stroke, spinal cord injury, and traumatic brain injury, can cause chronic gait function impairment due to foot-drop. Current physiotherapy techniques provide only a limited degree of motor function recovery in these individuals, and therefore novel therapies are needed. Brain-computer interface (BCI) is a relatively novel technology with a potential to restore, substitute, or augment lost motor behaviors in patients with neurological injuries. Here, we describe the first successful integration of a noninvasive electroencephalogram (EEG)-based BCI with a noninvasive functional electrical stimulation (FES) system that enables the direct brain control of foot dorsiflexion in able-bodied individuals. A noninvasive EEG-based BCI system was integrated with a noninvasive FES system for foot dorsiflexion. Subjects underwent computer-cued epochs of repetitive foot dorsiflexion and idling while their EEG signals were recorded and stored for offline analysis. The analysis generated a prediction model that allowed EEG data to be analyzed and classified in real time during online BCI operation. The real-time online performance of the integrated BCI-FES system was tested in a group of five able-bodied subjects who used repetitive foot dorsiflexion to elicit BCI-FES mediated dorsiflexion of the contralateral foot. Five able-bodied subjects performed 10 alternations of idling and repetitive foot dorsifiexion to trigger BCI-FES mediated dorsifiexion of the contralateral foot. The epochs of BCI-FES mediated foot dorsifiexion were highly correlated with the epochs of voluntary foot dorsifiexion (correlation coefficient ranged between 0.59 and 0.77) with latencies ranging from 1.4 sec to 3.1 sec. In addition, all subjects achieved a 100% BCI-FES response (no omissions), and one subject had a single false alarm. This study suggests that the integration of a noninvasive BCI with a lower-extremity FES system is feasible. With additional modifications, the proposed BCI-FES system may offer a novel and effective therapy in the neuro-rehabilitation of individuals with lower extremity paralysis due to neurological injuries.

Energy Storage System

NASA Technical Reports Server (NTRS)

1996-01-01

SatCon Technology Corporation developed the drive train for use in the Chrysler Corporation's Patriot Mark II, which includes the Flywheel Energy Storage (FES) system. In Chrysler's experimental hybrid- electric car, the hybrid drive train uses an advanced turboalternator that generates electricity by burning a fuel; a powerful, compact electric motor; and a FES that eliminates the need for conventional batteries. The FES system incorporates technology SatCon developed in more than 30 projects with seven NASA centers, mostly for FES systems for spacecraft attitude control and momentum recovery. SatCon will continue to develop the technology with Westinghouse Electric Corporation.

Fat embolism syndrome

PubMed Central

Kwiatt, Michael E.; Seamon, Mark J.

2013-01-01

Fat embolism syndrome (FES) is an ill-defined clinical entity that arises from the systemic manifestations of fat emboli within the microcirculation. Embolized fat within capillary beds cause direct tissue damage as well as induce a systemic inflammatory response resulting in pulmonary, cutaneous, neurological, and retinal symptoms. This is most commonly seen following orthopedic trauma; however, patients with many clinical conditions including bone marrow transplant, pancreatitis, and following liposuction. No definitive diagnostic criteria or tests have been developed, making the diagnosis of FES difficult. While treatment for FES is largely supportive, early operative fixation of long bone fractures decreases the likelihood of a patient developing FES. PMID:23724388

Fat embolism syndrome.

PubMed

Kwiatt, Michael E; Seamon, Mark J

2013-01-01

Fat embolism syndrome (FES) is an ill-defined clinical entity that arises from the systemic manifestations of fat emboli within the microcirculation. Embolized fat within capillary beds cause direct tissue damage as well as induce a systemic inflammatory response resulting in pulmonary, cutaneous, neurological, and retinal symptoms. This is most commonly seen following orthopedic trauma; however, patients with many clinical conditions including bone marrow transplant, pancreatitis, and following liposuction. No definitive diagnostic criteria or tests have been developed, making the diagnosis of FES difficult. While treatment for FES is largely supportive, early operative fixation of long bone fractures decreases the likelihood of a patient developing FES.

Simulation-based mastery learning for endoscopy using the endoscopy training system: a strategy to improve endoscopic skills and prepare for the fundamentals of endoscopic surgery (FES) manual skills exam.

PubMed

Ritter, E Matthew; Taylor, Zachary A; Wolf, Kathryn R; Franklin, Brenton R; Placek, Sarah B; Korndorffer, James R; Gardner, Aimee K

2018-01-01

The fundamentals of endoscopic surgery (FES) program has considerable validity evidence for its use in measuring the knowledge, skills, and abilities required for competency in endoscopy. Beginning in 2018, the American Board of Surgery will require all candidates to have taken and passed the written and performance exams in the FES program. Recent work has shown that the current ACGME/ABS required case volume may not be enough to ensure trainees pass the FES skills exam. The aim of this study was to investigate the feasibility of a simulation-based mastery-learning curriculum delivered on a novel physical simulation platform to prepare trainees to pass the FES manual skills exam. The newly developed endoscopy training system (ETS) was used as the training platform. Seventeen PGY 1 (10) and PGY 2 (7) general surgery residents completed a pre-training assessment consisting of all 5 FES tasks on the GI Mentor II. Subjects then trained to previously determined expert performance benchmarks on each of 5 ETS tasks. Once training benchmarks were reached for all tasks, a post-training assessment was performed with all 5 FES tasks. Two subjects were lost to follow-up and never returned for training or post-training assessment. One additional subject failed to complete any portion of the curriculum, but did return for post-training assessment. The group had minimal endoscopy experience (median 0, range 0-67) and minimal prior simulation experience. Three trainees (17.6%) achieved a passing score on the pre-training FES assessment. Training consisted of an average of 48 ± 26 repetitions on the ETS platform distributed over 5.1 ± 2 training sessions. Seventy-one percent achieved proficiency on all 5 ETS tasks. There was dramatic improvement demonstrated on the mean post-training FES assessment when compared to pre-training (74.0 ± 8 vs. 50.4 ± 16, p < 0.0001, effect size = 2.4). The number of ETS tasks trained to proficiency correlated moderately with the score on the post-training assessment (r = 0.57, p = 0.028). Fourteen (100%) subjects who trained to proficiency on at least one ETS task passed the post-training FES manual skills exam. This simulation-based mastery learning curriculum using the ETS is feasible for training novices and allows for the acquisition of the technical skills required to pass the FES manual skills exam. This curriculum should be strongly considered by programs wishing to ensure that trainees are prepared for the FES exam.

Semiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers

DOEpatents

Alivisatos, A.P.; Colvin, V.L.

1998-05-12

Methods are described for attaching semiconductor nanocrystals to solid inorganic surfaces, using self-assembled bifunctional organic monolayers as bridge compounds. Two different techniques are presented. One relies on the formation of self-assembled monolayers on these surfaces. When exposed to solutions of nanocrystals, these bridge compounds bind the crystals and anchor them to the surface. The second technique attaches nanocrystals already coated with bridge compounds to the surfaces. Analyses indicate the presence of quantum confined clusters on the surfaces at the nanolayer level. These materials allow electron spectroscopies to be completed on condensed phase clusters, and represent a first step towards synthesis of an organized assembly of clusters. These new products are also disclosed. 10 figs.

The effect of functional electrical stimulation on the physiological cost of gait in people with multiple sclerosis.

PubMed

Paul, L; Rafferty, D; Young, S; Miller, L; Mattison, P; McFadyen, A

2008-08-01

Functional electrical stimulation (FES) is used clinically in the management of drop foot in people suffering from neurological conditions. The aim of the study was to investigate the effects of FES, in terms of speed and physiological cost of gait, in people with multiple sclerosis (pwMS). Twelve pwMS and 12 healthy matched controls walked at their own preferred walking speed (PWS) for 5 min around a 10 m elliptical course. Subjects with MS completed the protocol with and without using their FES. In addition, control subjects completed the protocol twice more walking at the same PWS of the pwMS to which they were matched. Wearing FES lead to a significant improvement in walking speed (0.49 ms(-1) and 0.43 ms(-1) with and without their FES respectively; P<0.001) and a significant reduction in the physiological cost of gait (0.41 mL min(-1) kg(-1) m(-1) and 0.46 mL min(-1) kg(-1) m(-1) with and without FES respectively; P=0.017) in pwMS. The speed of walking, oxygen uptake, and physiological cost were significantly different between pwMS and controls both at preferred and matched speeds. Although pwMS exhibit a higher physiological cost of walking, FES offers an orthotic benefit to pwMS and should be considered as a possible treatment option.

Functional electrical stimulation enhancement of upper extremity functional recovery during stroke rehabilitation: a pilot study.

PubMed

Alon, Gad; Levitt, Alan F; McCarthy, Patricia A

2007-01-01

To test if functional electrical stimulation (FES) can enhance the recovery of upper extremity function during early stroke rehabilitation. Open-label block-randomized trial, begun during inpatient rehabilitation and continued at the patients' home. Patients were assigned to either FES combined with task-specific upper extremity rehabilitation (n = 7) or a control group that received task-specific therapy alone (n = 8) over 12 weeks. Outcome measures . Hand function (Box & Blocks, B & B; Jebsen-Taylor light object lift, J-T) and motor control (modified Fugl-Meyer, mF-M) were video-recorded for both upper extremities at baseline, 4, 8, and 12 weeks. B&B mean score at 12 weeks favored (P = .049) the FES group (42.3 +/- 16.6 blocks) over the control group (26.3 +/- 11.0 blocks). The FES group J-T task was 6.7 +/- 2.9 seconds and faster (P = .049) than the 11.8 +/- 5.4 seconds of the control group. Mean mF-M score of the FES group at 12 weeks was 49.3 +/- 5.1 points out of 54, compared to the control group that scored 40.6 +/- 8.2 points (P = .042). All patients regained hand function. Upper extremity task-oriented training that begins soon after stroke that incorporates FES may improve upper extremity functional use in patients with mild/moderate paresis more than task-oriented training without FES.

Effect of Self-Assembly of Fullerene Nano-Particles on Lipid Membrane

PubMed Central

Zhang, Saiqun; Mu, Yuguang; Zhang, John Z. H.; Xu, Weixin

2013-01-01

Carbon nanoparticles can penetrate the cell membrane and cause cytotoxicity. The diffusion feature and translocation free energy of fullerene through lipid membranes is well reported. However, the knowledge on self-assembly of fullerenes and resulting effects on lipid membrane is poorly addressed. In this work, the self-assembly of fullerene nanoparticles and the resulting influence on the dioleoylphosphtidylcholine (DOPC) model membrane were studied by using all-atom molecular dynamics simulations with explicit solvents. Our simulation results confirm that gathered small fullerene cluster can invade lipid membrane. Simulations show two pathways: 1) assembly process is completely finished before penetration; 2) assembly process coincides with penetration. Simulation results also demonstrate that in the membrane interior, fullerene clusters tend to stay at the position which is 1.0 nm away from the membrane center. In addition, the diverse microscopic stacking mode (i.e., equilateral triangle, tetrahedral pentahedral, trigonal bipyramid and octahedron) of these small fullerene clusters are well characterized. Thus our simulations provide a detailed high-resolution characterization of the microscopic structures of the small fullerene clusters. Further, we found the gathered small fullerene clusters have significant adverse disturbances to the local structure of the membrane, but no great influence on the global integrity of the lipid membrane, which suggests the prerequisite of high-content fullerene for cytotoxicity. PMID:24204827

The plasma membrane proteome of maize roots grown under low and high iron conditions.

PubMed

Hopff, David; Wienkoop, Stefanie; Lüthje, Sabine

2013-10-08

Iron (Fe) homeostasis is essential for life and has been intensively investigated for dicots, while our knowledge for species in the Poaceae is fragmentary. This study presents the first proteome analysis (LC-MS/MS) of plasma membranes isolated from roots of 18-day old maize (Zea mays L.). Plants were grown under low and high Fe conditions in hydroponic culture. In total, 227 proteins were identified in control plants, whereas 204 proteins were identified in Fe deficient plants and 251 proteins in plants grown under high Fe conditions. Proteins were sorted by functional classes, and most of the identified proteins were classified as signaling proteins. A significant number of PM-bound redox proteins could be identified including quinone reductases, heme and copper-containing proteins. Most of these components were constitutive, and others could hint at an involvement of redox signaling and redox homeostasis by change in abundance. Energy metabolism and translation seem to be crucial in Fe homeostasis. The response to Fe deficiency includes proteins involved in development, whereas membrane remodeling and assembly and/or repair of Fe-S clusters is discussed for Fe toxicity. The general stress response appears to involve proteins related to oxidative stress, growth regulation, an increased rigidity and synthesis of cell walls and adaption of nutrient uptake and/or translocation. This article is part of a Special Issue entitled: Plant Proteomics in Europe. Copyright © 2013 Elsevier B.V. All rights reserved.

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells

DOE PAGES

Dumitrache, Alexandru; Klingeman, Dawn M.; Natzke, Jace; ...

2017-02-27

Clostridium thermocellum forms biofilms adherent to lignocellulosic feedstock in a typical continuous cell-monolayer to efficiently break down and uptake cellulose hydrolysates. The sessile cells of biofilms may revert to non-adherent planktonic cells through generation of offspring cells or microenvironment constraints such as limited surface area. These interdependent cell populations co-exist and have different contributions to culture activity and growth. Here, we developed a novel bioreactor design to rapidly harvest sessile and planktonic cell populations for omics studies. In RNA-seq analyses, within 3299 protein coding genes, 59% (or 1958 genes) were differentially expressed with a minimum two-fold change between the twomore » cell populations isolated simultaneously at high culture activity. Furthermore, sessile cells had definitive greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division; planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. Our knowledge of these cellular adaptations will aid the engineering of industrially relevant strains for consolidated bioprocessing of solid lignocellulosic biomass« less

Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dumitrache, Alexandru; Klingeman, Dawn M.; Natzke, Jace

Clostridium thermocellum forms biofilms adherent to lignocellulosic feedstock in a typical continuous cell-monolayer to efficiently break down and uptake cellulose hydrolysates. The sessile cells of biofilms may revert to non-adherent planktonic cells through generation of offspring cells or microenvironment constraints such as limited surface area. These interdependent cell populations co-exist and have different contributions to culture activity and growth. Here, we developed a novel bioreactor design to rapidly harvest sessile and planktonic cell populations for omics studies. In RNA-seq analyses, within 3299 protein coding genes, 59% (or 1958 genes) were differentially expressed with a minimum two-fold change between the twomore » cell populations isolated simultaneously at high culture activity. Furthermore, sessile cells had definitive greater expression of genes involved in catabolism of carbohydrates by glycolysis and pyruvate fermentation, ATP generation by proton gradient, the anabolism of proteins and lipids and cellular functions critical for cell division; planktonic cells had notably higher gene expression for flagellar motility and chemotaxis, cellulosomal cellulases and anchoring scaffoldins, and a range of stress induced homeostasis mechanisms such as oxidative stress protection by antioxidants and flavoprotein co-factors, methionine repair, Fe-S cluster assembly and repair in redox proteins, cell growth control through tRNA thiolation, recovery of damaged DNA by nucleotide excision repair and removal of terminal proteins by proteases. Our knowledge of these cellular adaptations will aid the engineering of industrially relevant strains for consolidated bioprocessing of solid lignocellulosic biomass« less

Reducing The Cost of Transport and Increasing Walking Distance After Stroke: A Randomized Controlled Trial on Fast Locomotor Training Combined With Functional Electrical Stimulation

PubMed Central

Awad, Louis N.; Reisman, Darcy S.; Pohlig, Ryan T.; Binder-Macleod, Stuart A.

2015-01-01

Background Neurorehabilitation efforts have been limited in their ability to restore walking function after stroke. Recent work has demonstrated proof-of-concept for a Functional Electrical Stimulation (FES)-based combination therapy designed to improve poststroke walking by targeting deficits in paretic propulsion. Objectives To determine the effects on the energy cost of walking (EC) and long-distance walking ability of locomotor training that combines fast walking with FES to the paretic ankle musculature (FastFES). Methods Fifty participants >6 months poststroke were randomized to 12 weeks of gait training at self-selected speeds (SS), fast speeds (Fast), or FastFES. Participants’ 6-Minute Walk Test (6MWT) distance and EC at comfortable (EC-CWS) and fast (EC-Fast) walking speeds were measured pretraining, posttraining, and at a 3-month follow-up. A reduction in EC-CWS, independent of changes in speed, was the primary outcome. Also evaluated were group differences in the number of 6MWT responders and moderation by baseline speed. Results When compared with SS and Fast, FastFES produced larger reductions in EC (p’s ≤0.03). FastFES produced reductions of 24% and 19% in EC-CWS and EC-Fast (p’s <0.001), whereas neither Fast nor SS influenced EC. Between-group 6MWT differences were not observed; however, 73% of FastFES and 68% of Fast participants were responders, in contrast to 35% of SS participants. Conclusions Combining fast locomotor training with FES is an effective approach to reducing the high EC of persons poststroke. Surprisingly, differences in 6MWT gains were not observed between groups. Closer inspection of the 6MWT and EC relationship and elucidation of how reduced EC may influence walking-related disability is warranted. PMID:26621366

Do corticosteroids reduce the risk of fat embolism syndrome in patients with long-bone fractures? A meta-analysis.

PubMed

Bederman, S Samuel; Bhandari, Mohit; McKee, Michael D; Schemitsch, Emil H

2009-10-01

Fat embolism syndrome (FES) is a potentially lethal condition most commonly seen in polytrauma patients with multiple long-bone fractures. Treatment has centred around supportive care and early fracture fixation. Several small clinical trials have suggested corticosteroids benefit patients with FES, but this treatment remains controversial. Our objective was to determine the effect of corticosteroids in preventing FES in patients with long-bone fractures. We conducted a meta-analysis of published studies of patients with long-bone fractures who were randomly assigned to groups receiving corticosteroids or standard treatment for the prevention of FES (1966-2006). Data were extracted on quality, population, intervention and outcomes. Our primary outcome was the development of FES. We used random-effects models to pool results across studies, assessing for study heterogeneity. Of the 104 studies identified, 7 met our eligibility criteria. Overall, the quality of the trials was poor. Our pooled analysis of 389 patients found that corticosteroids reduced the risk of FES by 78% (95% confidence interval [CI] 43%-92%) and that only 8 patients needed to be treated (95% CI 5-13 patients) to prevent 1 case of FES. Similarly, corticosteroids significantly reduced the risk of hypoxia. We found no differences in the rates of mortality or infection. Rates of avascular necrosis were not reported in any of these studies. Evidence suggests that corticosteroids may be beneficial in preventing FES and hypoxia but not mortality in patients with long-bone fractures. The risk of infection is not increased with the use of corticosteroids. However, methodological limitations of these trials necessitate a large confirmatory randomized trial.

Role of interleukin-6 as an early marker of fat embolism syndrome: a clinical study.

PubMed

Prakash, Shiva; Sen, Ramesh Kumar; Tripathy, Sujit Kumar; Sen, Indu Mohini; Sharma, R R; Sharma, Sadhna

2013-07-01

A few animal studies have shown that IL-6 can serve as an early marker of fat embolism syndrome. The degree to which this is true in human trauma victims is unknown. In this clinical study, we sought to determine (1) whether elevated serum IL-6 levels at 6, 12, and 24 hours in patients with skeletal trauma were associated with the development of fat embolism syndrome (FES) within 72 hours after injury, and (2) at what time after trauma peak IL-6 levels are observed. Forty-eight patients between 16 and 40 years old who presented to our tertiary trauma center within 6 hours of injury with long bone and/or pelvic fractures were included in this study. Serum IL-6 levels were measured at 6, 12, and 24 hours after injury. The patients were observed clinically and monitored for 72 hours for development of FES symptoms. Gurd's criteria were used to diagnose FES. Elevated serum IL-6 levels 12 hours after trauma correlated with an increased likelihood of having FES develop; no significant relationship was observed between IL-6 levels at 6 or 24 hours and the development of FES. Patients with FES had a mean IL-6 level of 131 pg/mL, whereas those without FES had a mean IL-6 level of 72 pg/mL. Peak IL-6 levels were observed at 12 hours. An elevated serum IL-6 level may be useful as an early marker of FES in patients with isolated skeletal trauma. Level II, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

Do corticosteroids reduce the risk of fat embolism syndrome in patients with long-bone fractures? A meta-analysis

PubMed Central

Bederman, S. Samuel; Bhandari, Mohit; McKee, Michael D.; Schemitsch, Emil H.

2009-01-01

Background Fat embolism syndrome (FES) is a potentially lethal condition most commonly seen in polytrauma patients with multiple long-bone fractures. Treatment has centred around supportive care and early fracture fixation. Several small clinical trials have suggested corticosteroids benefit patients with FES, but this treatment remains controversial. Our objective was to determine the effect of corticosteroids in preventing FES in patients with long-bone fractures. Methods We conducted a meta-analysis of published studies of patients with long-bone fractures who were randomly assigned to groups receiving corticosteroids or standard treatment for the prevention of FES (1966–2006). Data were extracted on quality, population, intervention and outcomes. Our primary outcome was the development of FES. We used random-effects models to pool results across studies, assessing for study heterogeneity. Results Of the 104 studies identified, 7 met our eligibility criteria. Overall, the quality of the trials was poor. Our pooled analysis of 389 patients found that corticosteroids reduced the risk of FES by 78% (95% confidence interval [CI] 43%–92%) and that only 8 patients needed to be treated (95% CI 5–13 patients) to prevent 1 case of FES. Similarly, corticosteroids significantly reduced the risk of hypoxia. We found no differences in the rates of mortality or infection. Rates of avascular necrosis were not reported in any of these studies. Conclusion Evidence suggests that corticosteroids may be beneficial in preventing FES and hypoxia but not mortality in patients with long-bone fractures. The risk of infection is not increased with the use of cortisosteroids. However, methodological limitations of these trials necessitate a large confirmatory randomized trial. PMID:19865573

Long-Term Follow-up to a Randomized Controlled Trial Comparing Peroneal Nerve Functional Electrical Stimulation to an Ankle Foot Orthosis for Patients With Chronic Stroke.

PubMed

Bethoux, Francois; Rogers, Helen L; Nolan, Karen J; Abrams, Gary M; Annaswamy, Thiru; Brandstater, Murray; Browne, Barbara; Burnfield, Judith M; Feng, Wuwei; Freed, Mitchell J; Geis, Carolyn; Greenberg, Jason; Gudesblatt, Mark; Ikramuddin, Farha; Jayaraman, Arun; Kautz, Steven A; Lutsep, Helmi L; Madhavan, Sangeetha; Meilahn, Jill; Pease, William S; Rao, Noel; Seetharama, Subramani; Sethi, Pramod; Turk, Margaret A; Wallis, Roi Ann; Kufta, Conrad

2015-01-01

Evidence supports peroneal nerve functional electrical stimulation (FES) as an effective alternative to ankle foot orthoses (AFO) for treatment of foot drop poststroke, but few long-term, randomized controlled comparisons exist. Compare changes in gait quality and function between FES and AFOs in individuals with foot drop poststroke over a 12-month period. Follow-up analysis of an unblinded randomized controlled trial (ClinicalTrials.gov #NCT01087957) conducted at 30 rehabilitation centers comparing FES to AFOs over 6 months. Subjects continued to wear their randomized device for another 6 months to final 12-month assessments. Subjects used study devices for all home and community ambulation. Multiply imputed intention-to-treat analyses were utilized; primary endpoints were tested for noninferiority and secondary endpoints for superiority. Primary endpoints: 10 Meter Walk Test (10MWT) and device-related serious adverse event rate. Secondary endpoints: 6-Minute Walk Test (6MWT), GaitRite Functional Ambulation Profile, and Modified Emory Functional Ambulation Profile (mEFAP). A total of 495 subjects were randomized, and 384 completed the 12-month follow-up. FES proved noninferior to AFOs for all primary endpoints. Both FES and AFO groups showed statistically and clinically significant improvement for 10MWT compared with initial measurement. No statistically significant between-group differences were found for primary or secondary endpoints. The FES group demonstrated statistically significant improvements for 6MWT and mEFAP Stair-time subscore. At 12 months, both FES and AFOs continue to demonstrate equivalent gains in gait speed. Results suggest that long-term FES use may lead to additional improvements in walking endurance and functional ambulation; further research is needed to confirm these findings. © The Author(s) 2015.

Resolution of holograms produced by the fluid experiment system and the holography ground system

NASA Technical Reports Server (NTRS)

Brooks, Howard L.

1987-01-01

The Fluid Experiment System (FES) was developed to study low temperature crystal growth of triglycine sulfate from solution in a low gravity environment onboard Spacelab. The first flight of FES was in 1985. FES uses an optical system to take holograms of the growing crystal to be analyzed after the mission in the Holography Ground System (HGS) located in the Test Laboratory at Marshall Space Flight Center. Microscopic observation of the images formed by the reconstructed holograms is critical to determining crystal growth rate and particle velocity. FES and HGS were designed for a resolution of better than 20 micrometers, but initial observation of the flight holograms show a limit of 80 micrometers. The resolution of the FES holograms is investigated, as well as the role of beam intensity ratio and exposure time on the resolution of HGS produced holograms.

Pore-scale spectral induced polarization (SIP) signaturesassociated with FeS biomineral transformations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slater, Lee; Ntarlagiannis, Dimitrios; Personna, Yves R.

2007-10-01

The authors measured Spectral Induced Polarization (SIP) signatures in sand columns during (1) FeS biomineralization produced by sulfate reducing bacteria (D. vulgaris) under anaerboci conditions, and (2) subsequent biomineral dissolution upon return to an aerobic state. The low-frequency (0.1-10 Hz peak) relaxations produced during biomineralization can be modeled with a Cole-Cole formulation, from which the evolution of the polarization magnitude and relaxation length scale can be estimated. They find that the modeled time constant is consistent with the polarizable elements being biomineral encrused pores. Evolution of the model parameters is consistent with FeS surface area increases and pore-size reduction duringmore » biomineral growth, and subsequent biomineral dissolution (FeS surface area decreases and pore expansion) upon return to the aerobic state. They conclude that SIP signatures are diagnostic of pore-scale geometrical changes associated with FeS biomineralization by sulfate reducing bacteria.« less

Use of Early Inhaled Nitric Oxide Therapy in Fat Embolism Syndrome to Prevent Right Heart Failure

PubMed Central

Koyfman, Leonid; Kutz, Ruslan; Frenkel, Amit; Gruenbaum, Shaun E.; Zlotnik, Alexander; Klein, Moti

2014-01-01

Fat embolism syndrome (FES) is a life-threatening condition in which multiorgan dysfunction manifests 48–72 hours after long bone or pelvis fractures. Right ventricular (RV) failure, especially in the setting of pulmonary hypertension, is a frequent feature of FES. We report our experience treating 2 young, previously healthy trauma patients who developed severe hypoxemia in the setting of FES. Neither patient had evidence of RV dysfunction on echocardiogram. The patients were treated with inhaled nitric oxide (NO), and their oxygenation significantly improved over the subsequent few days. Neither patient developed any cardiovascular compromise. Patients with FES that have severe hypoxemia and evidence of adult respiratory distress syndrome (ARDS) are likely at risk for developing RV failure. We recommend that these patients with FES and severe refractory hypoxemia should be treated with inhaled NO therapy prior to the onset of RV dysfunction. PMID:25180103

Clinical effectiveness analysis of dextran 40 plus dexamethasone on the prevention of fat embolism syndrome.

PubMed

Liu, Xi-Ming; Huang, Jin-Cheng; Wang, Guo-Dong; Lan, Sheng-Hui; Wang, Hua-Song; Pan, Chang-Wu; Zhang, Ji-Ping; Cai, Xian-Hua

2014-01-01

This study aims to investigate the clinical efficacy of Dextran 40 plus dexamethasone on the prevention of fat embolism syndrome (FES) in high-risk patients with long bone shaft fractures. According to the different preventive medication, a total of 1837 cases of long bone shaft fracture patients with injury severity score (ISS) > 16 were divided into four groups: dextran plus dexamethasone group, dextran group, dexamethasone group and control group. The morbidity and mortality of FES in each group were analyzed with pairwise comparison analysis. There were totally 17 cases of FES and 1 case died. The morbidity of FES was 0.33% in dextran plus dexamethasone group and significantly lowers than that of other groups (P < 0.05). There was no significant difference among other groups (P > 0.05). Conclusion from our data is dextran 40 plus dexamethasone can effectively prevent long bone shaft fractures occurring in high-risk patients with FES.

Feedback Error Learning Controller for Functional Electrical Stimulation Assistance in a Hybrid Robotic System for Reaching Rehabilitation

PubMed Central

Resquín, Francisco; Gonzalez-Vargas, Jose; Ibáñez, Jaime; Brunetti, Fernando; Pons, José Luis

2016-01-01

Hybrid robotic systems represent a novel research field, where functional electrical stimulation (FES) is combined with a robotic device for rehabilitation of motor impairment. Under this approach, the design of robust FES controllers still remains an open challenge. In this work, we aimed at developing a learning FES controller to assist in the performance of reaching movements in a simple hybrid robotic system setting. We implemented a Feedback Error Learning (FEL) control strategy consisting of a feedback PID controller and a feedforward controller based on a neural network. A passive exoskeleton complemented the FES controller by compensating the effects of gravity. We carried out experiments with healthy subjects to validate the performance of the system. Results show that the FEL control strategy is able to adjust the FES intensity to track the desired trajectory accurately without the need of a previous mathematical model. PMID:27990245

Synthesis of nanostructured marcasite FeS2 for energy storage applications

NASA Astrophysics Data System (ADS)

Kaur, Gurpreet; Sharma, Pooja D.; Thakur, Anup; Kumar, Manjeet; Bala, Rajni; Kumar, Akshay

2018-05-01

The synthesis of marcasite FeS2 is of great interest as this area is seldom studied due to its sophisticated synthesis methods. In fulfillment of growing energy demands, there is need of cost effective alternates for energy storage devices. Nanostructured marcasite iron disulfide (FeS2) is a promising candidate as anode material for energy storage devices. FeS2 exist in many phases out of which marcasite and pyrite are best suitable for energy storage applications. Purity of the phase is a big challenge for its application oriented use. Pure marcasite (FeS2) has been synthesized by low cost, environmentally friendly hydrothermal route. The synthesized material has been characterized by X-ray Diffraction (XRD). Cyclic voltammetry results show the significant electrochemical performance of marcasite. This work purposes a vision to develop marcasite based electrode material for energy storage devices.

Activation Thermodynamics and H/D Kinetic Isotope Effect of the H ox to H red H + Transition in [FeFe] Hydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratzloff, Michael W.; Wilker, Molly B.; Mulder, David W.

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here in this paper we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox→H redH + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ~2.5-foldmore » kinetic isotope effect. Overall these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox→H redH + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.« less

Activation Thermodynamics and H/D Kinetic Isotope Effect of the H ox to H red H + Transition in [FeFe] Hydrogenase

DOE PAGES

Ratzloff, Michael W.; Wilker, Molly B.; Mulder, David W.; ...

2017-08-29

Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here in this paper we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The H ox→H redH + reduction step measured by Fourier-transform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol -1 and a ~2.5-foldmore » kinetic isotope effect. Overall these results support electron injection from CdSe into CaI involving F-clusters, and that the H ox→H redH + step of catalytic proton reduction in CaI proceeds by a proton-dependent process.« less

Influence of cluster-assembly parameters on the field emission properties of nanostructured carbon films

NASA Astrophysics Data System (ADS)

Ducati, C.; Barborini, E.; Piseri, P.; Milani, P.; Robertson, J.

2002-11-01

Supersonic cluster beam deposition has been used to produce films with different nanostructures by controlling the deposition parameters such as the film thickness, substrate temperature and cluster mass distribution. The field emission properties of cluster-assembled carbon films have been characterized and correlated to the evolution of the film nanostructure. Threshold fields ranging between 4 and 10 V/mum and saturation current densities as high as 0.7 mA have been measured for samples heated during deposition. A series of voltage ramps, i.e., a conditioning process, was found to initiate more stable and reproducible emission. It was found that the presence of graphitic particles (onions, nanotube embryos) in the films substantially enhances the field emission performance. Films patterned on a micrometer scale have been conditioned spot by spot by a ball-tip anode, showing that a relatively high emission site density can be achieved from the cluster-assembled material.

Haspin kinase regulates microtubule-organizing center clustering and stability through Aurora kinase C in mouse oocytes.

PubMed

Balboula, Ahmed Z; Nguyen, Alexandra L; Gentilello, Amanda S; Quartuccio, Suzanne M; Drutovic, David; Solc, Petr; Schindler, Karen

2016-10-01

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes. © 2016. Published by The Company of Biologists Ltd.

Role of protein-glutathione contacts in defining glutaredoxin-3 [2Fe-2S] cluster chirality, ligand exchange and transfer chemistry.

PubMed

Sen, Sambuddha; Cowan, J A

2017-10-01

Monothiol glutaredoxins (Grx) serve as intermediate cluster carriers in iron-sulfur cluster trafficking. The [2Fe-2S]-bound holo forms of Grx proteins display cysteinyl coordination from exogenous glutathione (GSH), in addition to contact from protein-derived Cys. Herein, we report mechanistic studies that investigate the role of exogenous glutathione in defining cluster chirality, ligand exchange, and the cluster transfer chemistry of Saccharomyces cerevisiae Grx3. Systematic perturbations were introduced to the glutathione-binding site by substitution of conserved charged amino acids that form crucial electrostatic contacts with the glutathione molecule. Native Grx3 could also be reconstituted in the absence of glutathione, with either DTT, BME or free L-cysteine as the source of the exogenous Fe-S ligand contact, while retaining full functional reactivity. The delivery of the [2Fe-2S] cluster to Grx3 from cluster donor proteins such as Isa, Nfu, and a [2Fe-2S](GS) 4 complex, revealed that electrostatic contacts are of key importance for positioning the exogenous glutathione that in turn influences the chiral environment of the cluster. All Grx3 derivatives were reconstituted by standard chemical reconstitution protocols and found to transfer cluster to apo ferredoxin 1 (Fdx1) at rates comparable to native protein, even when using DTT, BME or free L-cysteine as a thiol source in place of GSH during reconstitution. Kinetic analysis of cluster transfer from holo derivatives to apo Fdx1 has led to a mechanistic model for cluster transfer chemistry of native holo Grx3, and identification of the likely rate-limiting step for the reaction.

Assessment of Supportive, Conflicted, and Controlling Dimensions of Family Functioning: A Principal Components Analysis of Family Environment Scale Subscales in a College Sample.

ERIC Educational Resources Information Center

Kronenberger, William G.; Thompson, Robert J., Jr.; Morrow, Catherine

1997-01-01

A principal components analysis of the Family Environment Scale (FES) (R. Moos and B. Moos, 1994) was performed using 113 undergraduates. Research supported 3 broad components encompassing the 10 FES subscales. These results supported previous research and the generalization of the FES to college samples. (SLD)

Alveolar hemorrhage in a case of fat embolism syndrome: A case report with short systemic review.

PubMed

Dash, Sananta Kumar; Bansal, Avdesh; Wankhade, Bhushan Sudhakar; Sharma, Rakesh

2013-04-01

Fat embolism and fat embolism syndrome (FES) are well-known complications of long bone fracture and surgery involving manipulation of skeletal elements. Many non-traumatic causes of FES have been suggested but they constitute only a small portion. FES presents with classical symptoms of petechiae, hypoxemia, central nervous system symptoms along with other features such as tachycardia and pyrexia. Diagnosis of FES relies on clinical judgment rather than objective findings such as emboli present in the retinal vessels on fundoscopy, fat globules present in urine and sputum, a sudden inexplicable drop in hematocrit or platelet values, increasing erythrocyte sedimentation rate.

Pulmonary Embolization of Fat and Bone Marrow in Cynomolgus Macaques (Macaca fascicularis)

PubMed Central

Fong, Derek L.; Murnane, Robert D.; Hotchkiss, Charlotte E.; Green, Damian J.; Hukkanen, Renee R.

2011-01-01

Fat embolization (FE), the introduction of bone marrow elements into circulation, is a known complication of bone fractures. Although FE has been described in other animal models, this study represents the first reported cases of FE and bone marrow embolism in nonhuman primates. Histopathologic findings from cynomolgus macaques (Macaca fascicularis) indicated that in all 5 cases, fat and bone marrow embolization occurred subsequent to multiple bone marrow biopsies. In the most severe case, extensive embolization was associated pulmonary damage consistent with acute respiratory distress syndrome. Fat embolism syndrome (FES) is an infrequent clinical outcome of FE and is triggered by systemic biochemical and mechanical responses to fat in circulation. Although clinical criteria diagnostic of FES were not investigated at the time of death, this severe case may represent the fulminant form of FES, which occurs within 12 h after trauma. Bone marrow biopsy as an etiology of FES has been reported only once in humans. In addition, the association of embolization with bone marrow biopsies suggests that nonhuman primates may be a useful animal model of FE. FE and FES represent important research confounders and FES should be considered as a differential diagnosis for clinical complications subsequent to skeletal trauma. PMID:21819686

Pulmonary embolization of fat and bone marrow in cynomolgus Macaques (Macaca fascicularis).

PubMed

Fong, Derek L; Murnane, Robert D; Hotchkiss, Charlotte E; Green, Damian J; Hukkanen, Renee R

2011-02-01

Fat embolization (FE), the introduction of bone marrow elements into circulation, is a known complication of bone fractures. Although FE has been described in other animal models, this study represents the first reported cases of FE and bone marrow embolism in nonhuman primates. Histopathologic findings from cynomolgus macaques (Macaca fascicularis) indicated that in all 5 cases, fat and bone marrow embolization occurred subsequent to multiple bone marrow biopsies. In the most severe case, extensive embolization was associated pulmonary damage consistent with acute respiratory distress syndrome. Fat embolism syndrome (FES) is an infrequent clinical outcome of FE and is triggered by systemic biochemical and mechanical responses to fat in circulation. Although clinical criteria diagnostic of FES were not investigated at the time of death, this severe case may represent the fulminant form of FES, which occurs within 12 h after trauma. Bone marrow biopsy as an etiology of FES has been reported only once in humans. In addition, the association of embolization with bone marrow biopsies suggests that nonhuman primates may be a useful animal model of FE. FE and FES represent important research confounders and FES should be considered as a differential diagnosis for clinical complications subsequent to skeletal trauma.

Reduced Graphene Oxide-Wrapped FeS2 Composite as Anode for High-Performance Sodium-Ion Batteries

NASA Astrophysics Data System (ADS)

Wang, Qinghong; Guo, Can; Zhu, Yuxuan; He, Jiapeng; Wang, Hongqiang

2018-06-01

Iron disulfide is considered to be a potential anode material for sodium-ion batteries due to its high theoretical capacity. However, its applications are seriously limited by the weak conductivity and large volume change, which results in low reversible capacity and poor cycling stability. Herein, reduced graphene oxide-wrapped FeS2 (FeS2/rGO) composite was fabricated to achieve excellent electrochemical performance via a facile two-step method. The introduction of rGO effectively improved the conductivity, BET surface area, and structural stability of the FeS2 active material, thus endowing it with high specific capacity, good rate capability, as well as excellent cycling stability. Electrochemical measurements show that the FeS2/rGO composite had a high initial discharge capacity of 1263.2 mAh g-1 at 100 mA g-1 and a high discharge capacity of 344 mAh g-1 at 10 A g-1, demonstrating superior rate performance. After 100 cycles at 100 mA g-1, the discharge capacity remained at 609.5 mAh g-1, indicating the excellent cycling stability of the FeS2/rGO electrode.

Design and Test of a Closed-Loop FES System for Supporting Function of the Hemiparetic Hand Based on Automatic Detection using the Microsoft Kinect sensor.

PubMed

Simonsen, Daniel; Spaich, Erika G; Hansen, John; Andersen, Ole K

2016-10-26

This paper describes the design of a FES system automatically controlled in a closed loop using a Microsoft Kinect sensor, for assisting both cylindrical grasping and hand opening. The feasibility of the system was evaluated in real-time in stroke patients with hand function deficits. A hand function exercise was designed in which the subjects performed an arm and hand exercise in sitting position. The subject had to grasp one of two differently sized cylindrical objects and move it forward or backwards in the sagittal plane. This exercise was performed with each cylinder with and without FES support. Results showed that the stroke patients were able to perform up to 29% more successful grasps when they were assisted by FES. Moreover, the hand grasp-and-hold and hold-and-release durations were shorter for the smaller of the two cylinders. FES was appropriately timed in more than 95% of all trials indicating successful closed loop FES control. Future studies should incorporate options for assisting forward reaching in order to target a larger group of stroke patients.

sEMG Signal Acquisition Strategy towards Hand FES Control.

PubMed

Toledo-Peral, Cinthya Lourdes; Gutiérrez-Martínez, Josefina; Mercado-Gutiérrez, Jorge Airy; Martín-Vignon-Whaley, Ana Isabel; Vera-Hernández, Arturo; Leija-Salas, Lorenzo

2018-01-01

Due to damage of the nervous system, patients experience impediments in their daily life: severe fatigue, tremor or impaired hand dexterity, hemiparesis, or hemiplegia. Surface electromyography (sEMG) signal analysis is used to identify motion; however, standardization of electrode placement and classification of sEMG patterns are major challenges. This paper describes a technique used to acquire sEMG signals for five hand motion patterns from six able-bodied subjects using an array of recording and stimulation electrodes placed on the forearm and its effects over functional electrical stimulation (FES) and volitional sEMG combinations, in order to eventually control a sEMG-driven FES neuroprosthesis for upper limb rehabilitation. A two-part protocol was performed. First, personalized templates to place eight sEMG bipolar channels were designed; with these data, a universal template, called forearm electrode set (FELT), was built. Second, volitional and evoked movements were recorded during FES application. 95% classification accuracy was achieved using two sessions per movement. With the FELT, it was possible to perform FES and sEMG recordings simultaneously. Also, it was possible to extract the volitional and evoked sEMG from the raw signal, which is highly important for closed-loop FES control.

Superconductivity and magnetism in iron sulfides intercalated by metal hydroxides† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05268a Click here for additional data file.

PubMed Central

Zhou, Xiuquan; Eckberg, Christopher; Wilfong, Brandon; Liou, Sz-Chian; Vivanco, Hector K.; Paglione, Johnpierre

2017-01-01

Inspired by naturally occurring sulfide minerals, we present a new family of iron-based superconductors. A metastable form of FeS known as the mineral mackinawite forms two-dimensional sheets that can be readily intercalated by various cationic guest species. Under hydrothermal conditions using alkali metal hydroxides, we prepare three different cation and metal hydroxide-intercalated FeS phases including (Li1–xFexOH)FeS, [(Na1–xFex)(OH)2]FeS, and KxFe2–yS2. Upon successful intercalation of the FeS layer, the superconducting critical temperature T c of mackinawite is enhanced from 5 K to 8 K for the (Li1–xFexOH)δ+ intercalate. Layered heterostructures of [(Na1–xFex)(OH)2]FeS resemble the natural mineral tochilinite, which contains an iron square lattice interleaved with a hexagonal hydroxide lattice. Whilst heterostructured [(Na1–xFex)(OH)2]FeS displays long-range magnetic ordering near 15 K, KxFe2–yS2 displays short range antiferromagnetism. PMID:28580110

XAFS of short-lived reduction products of structural and functional models of the [Fe Fe] hydrogenase H-cluster

NASA Astrophysics Data System (ADS)

Bondin, Mark I.; Borg, Stacey J.; Cheah, Mun-Hon; Best, Stephen P.

2006-11-01

Thiolate-bridged diiron compounds that are related to the active site of the [Fe-Fe] hydrogenase enzyme have been shown to act as electrocatalysts for reduction of protons. The use of XAFS for clarification of the structures of intermediates formed following reduction of related diiron carbonyl compounds is described. These measurements allow the determination of Fe-Fe and Fe-S bond lengths with good reliability and when used in conjunction with the standard bonding models this provides a means of validating the structures proposed for longer-lived ( t>20 s at -50 °C) reaction intermediates.

Testing the Large-scale Environments of Cool-core and Non-cool-core Clusters with Clustering Bias

NASA Astrophysics Data System (ADS)

Medezinski, Elinor; Battaglia, Nicholas; Coupon, Jean; Cen, Renyue; Gaspari, Massimo; Strauss, Michael A.; Spergel, David N.

2017-02-01

There are well-observed differences between cool-core (CC) and non-cool-core (NCC) clusters, but the origin of this distinction is still largely unknown. Competing theories can be divided into internal (inside-out), in which internal physical processes transform or maintain the NCC phase, and external (outside-in), in which the cluster type is determined by its initial conditions, which in turn leads to different formation histories (I.e., assembly bias). We propose a new method that uses the relative assembly bias of CC to NCC clusters, as determined via the two-point cluster-galaxy cross-correlation function (CCF), to test whether formation history plays a role in determining their nature. We apply our method to 48 ACCEPT clusters, which have well resolved central entropies, and cross-correlate with the SDSS-III/BOSS LOWZ galaxy catalog. We find that the relative bias of NCC over CC clusters is b = 1.42 ± 0.35 (1.6σ different from unity). Our measurement is limited by the small number of clusters with core entropy information within the BOSS footprint, 14 CC and 34 NCC clusters. Future compilations of X-ray cluster samples, combined with deep all-sky redshift surveys, will be able to better constrain the relative assembly bias of CC and NCC clusters and determine the origin of the bimodality.

Testing the Large-scale Environments of Cool-core and Non-cool-core Clusters with Clustering Bias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medezinski, Elinor; Battaglia, Nicholas; Cen, Renyue

2017-02-10

There are well-observed differences between cool-core (CC) and non-cool-core (NCC) clusters, but the origin of this distinction is still largely unknown. Competing theories can be divided into internal (inside-out), in which internal physical processes transform or maintain the NCC phase, and external (outside-in), in which the cluster type is determined by its initial conditions, which in turn leads to different formation histories (i.e., assembly bias). We propose a new method that uses the relative assembly bias of CC to NCC clusters, as determined via the two-point cluster-galaxy cross-correlation function (CCF), to test whether formation history plays a role in determiningmore » their nature. We apply our method to 48 ACCEPT clusters, which have well resolved central entropies, and cross-correlate with the SDSS-III/BOSS LOWZ galaxy catalog. We find that the relative bias of NCC over CC clusters is b = 1.42 ± 0.35 (1.6 σ different from unity). Our measurement is limited by the small number of clusters with core entropy information within the BOSS footprint, 14 CC and 34 NCC clusters. Future compilations of X-ray cluster samples, combined with deep all-sky redshift surveys, will be able to better constrain the relative assembly bias of CC and NCC clusters and determine the origin of the bimodality.« less

Fuel-Mediated Transient Clustering of Colloidal Building Blocks.

PubMed

van Ravensteijn, Bas G P; Hendriksen, Wouter E; Eelkema, Rienk; van Esch, Jan H; Kegel, Willem K

2017-07-26

Fuel-driven assembly operates under the continuous influx of energy and results in superstructures that exist out of equilibrium. Such dissipative processes provide a route toward structures and transient behavior unreachable by conventional equilibrium self-assembly. Although perfected in biological systems like microtubules, this class of assembly is only sparsely used in synthetic or colloidal analogues. Here, we present a novel colloidal system that shows transient clustering driven by a chemical fuel. Addition of fuel causes an increase in hydrophobicity of the building blocks by actively removing surface charges, thereby driving their aggregation. Depletion of fuel causes reappearance of the charged moieties and leads to disassembly of the formed clusters. This reassures that the system returns to its initial, equilibrium state. By taking advantage of the cyclic nature of our system, we show that clustering can be induced several times by simple injection of new fuel. The fuel-mediated assembly of colloidal building blocks presented here opens new avenues to the complex landscape of nonequilibrium colloidal structures, guided by biological design principles.

Association between fast food purchasing and the local food environment.

PubMed

Thornton, Lukar E; Kavanagh, A M

2012-12-03

In this study, an instrument was created to measure the healthy and unhealthy characteristics of food environments and investigate associations between the whole of the food environment and fast food consumption. In consultation with other academic researchers in this field, food stores were categorised to either healthy or unhealthy and weighted (between +10 and -10) by their likely contribution to healthy/unhealthy eating practices. A healthy and unhealthy food environment score (FES) was created using these weightings. Using a cross-sectional study design, multilevel multinomial regression was used to estimate the effects of the whole food environment on the fast food purchasing habits of 2547 individuals. Respondents in areas with the highest tertile of the healthy FES had a lower likelihood of purchasing fast food both infrequently and frequently compared with respondents who never purchased, however only infrequent purchasing remained significant when simultaneously modelled with the unhealthy FES (odds ratio (OR) 0.52; 95% confidence interval (CI) 0.32-0.83). Although a lower likelihood of frequent fast food purchasing was also associated with living in the highest tertile of the unhealthy FES, no association remained once the healthy FES was included in the models. In our binary models, respondents living in areas with a higher unhealthy FES than healthy FES were more likely to purchase fast food infrequently (OR 1.35; 95% CI 1.00-1.82) however no association was found for frequent purchasing. Our study provides some evidence to suggest that healthier food environments may discourage fast food purchasing.

Cardiorespiratory demand of acute voluntary cycling with functional electrical stimulation in individuals with multiple sclerosis with severe mobility impairment.

PubMed

Edwards, Thomas; Motl, Robert W; Pilutti, Lara A

2018-01-01

Exercise training is one strategy for improving cardiorespiratory fitness (CRF) in multiple sclerosis (MS); however, few modalities are accessible for those with severe mobility impairment. Functional electrical stimulation (FES) cycling is an adapted exercise modality with the potential for improving CRF in people with severe MS. The objective of this study was to characterize the cardiorespiratory response of acute voluntary cycling with FES in people with MS with severe mobility impairment, and to compare this response to passive leg cycling. Eleven participants with MS that required assistance for ambulation completed a single bout of voluntary cycling with FES or passive leg cycling. Oxygen consumption, heart rate (HR), work rate (WR), and ratings of perceived exertion (RPE) were recorded throughout the session. For the FES group, mean exercising oxygen consumption was 8.7 ± 1.8 mL/(kg·min) -1 , or 63.5% of peak oxygen consumption. Mean HR was 102 ± 9.7 bpm, approximately 76.4% of peak HR. Mean WR was 27.0 ± 9.2 W, or 57.3% of peak WR, and median RPE was 13.5 (interquartile range = 5.5). Active cycling with FES was significantly (p < 0.05) more intense than passive leg cycling based on oxygen consumption, HR, WR, and RPE during exercise. In conclusion, voluntary cycling with FES elicited an acute response that corresponded with moderate-to vigorous-intensity activity, suggesting that active cycling with FES can elicit a sufficient stimulus for improving CRF.

Cross-cultural adaptation and measurement properties of the Arabic version of the Fall Efficacy Scale International.

PubMed

Alghadir, Ahmad H; Al-Momani, Murad; Marchetti, Gregory F; Whitney, Susan L

2015-07-01

To translate the Falls Efficacy Scale International (FES-I) into Arabic according to the World Health Organization`s (WHO) criteria and to evaluate the concurrent validity of the FES-I in persons living with balance and vestibular disorders. This cross-sectional descriptive study included 43 persons with balance and vestibular disorders presenting to an outpatient dizziness center at King Abdulaziz University Hospital, Riyadh, Kingdom of Saudi Arabia between June 2012 and May 2013. All participants completed the Arabic version of the FES-I and the Dizziness Handicap Inventory (DHI) during their assessment with the clinical audiologist. In addition, subjects completed the Dynamic Gait Index 4-item (DGI-4) gait test. An additional 55 control participants also completed the Arabic FES-I, the DGI-4, and the Arabic DHI. Forty-three participants with vestibular disorders (36 females, 7 males) with a mean age of 32 years (standard deviation (SD) 10 years, range 18-56 years) and 55 control participants (27 females, 28 males) with a mean age of 33, (SD-12), and age range of 18-78 participated. The correlation between the Arabic FES-I and the Arabic DHI was 0.75 in patients and 0.77 in control participants. The correlation between the Arabic FES-I and the DGI-4 was r=-0.30 (p=0.003). The Arabic FES-I has established concurrent validity and may be helpful for measuring an individual`s concern of falling in people with vestibular and balance disorders.

Adsorption and Desulfurization Mechanism of Thiophene on Layered FeS(001), (011), and (111) Surfaces: A Dispersion-Corrected Density Functional Theory Study

PubMed Central

2017-01-01

Layered transition-metal chalcogenides have emerged as a fascinating new class of materials for catalysis. Here, we present periodic density functional theory (DFT) calculations of the adsorption of thiophene and the direct desulfurization reaction pathways on the (001), (011), and (111) surfaces of layered FeS. The fundamental aspects of the thiophene adsorption, including the initial adsorption geometries, adsorption energies, structural parameters, and electronic properties, are presented. From the calculated adsorption energies, we show that the flat adsorption geometries, wherein the thiophene molecule forms multiple π-bonds with the FeS surfaces, are energetically more favorable than the upright adsorption geometries, with the strength of adsorption decreasing in the order FeS(111) > FeS(011) > FeS(001). The adsorption of the thiophene onto the reactive (011) and (111) surfaces is shown to be characterized by charge transfer from the interacting Fe d-band to the π-system of the thiophene molecule, which causes changes of the intramolecular structure including loss of aromaticity and elongation of the C–S bonds. The thermodynamic and kinetic analysis of the elementary steps involved in the direct desulfurization of thiophene on the reactive FeS surfaces is also presented. Direct desulfurization of thiophene occurs preferentially on the (111) surface, as reflected by the overall exothermic reaction energy calculated for the process (ER = −0.15 eV), with an activation energy of 1.58 eV. PMID:29348782

De novo assembly of the transcriptome of the non-model plant Streptocarpus rexii employing a novel heuristic to recover locus-specific transcript clusters.

PubMed

Chiara, Matteo; Horner, David S; Spada, Alberto

2013-01-01

De novo transcriptome characterization from Next Generation Sequencing data has become an important approach in the study of non-model plants. Despite notable advances in the assembly of short reads, the clustering of transcripts into unigene-like (locus-specific) clusters remains a somewhat neglected subject. Indeed, closely related paralogous transcripts are often merged into single clusters by current approaches. Here, a novel heuristic method for locus-specific clustering is compared to that implemented in the de novo assembler Oases, using the same initial transcript collections, derived from Arabidopsis thaliana and the developmental model Streptocarpus rexii. We show that the proposed approach improves cluster specificity in the A. thaliana dataset for which the reference genome is available. Furthermore, for the S. rexii data our filtered transcript collection matches a larger number of distinct annotated loci in reference genomes than the Oases set, while containing a reduced overall number of loci. A detailed discussion of advantages and limitations of our approach in processing de novo transcriptome reconstructions is presented. The proposed method should be widely applicable to other organisms, irrespective of the transcript assembly method employed. The S. rexii transcriptome is available as a sophisticated and augmented publicly available online database.

Clustering effects in ionic polymers: Molecular dynamics simulations.

PubMed

Agrawal, Anupriya; Perahia, Dvora; Grest, Gary S

2015-08-01

Ionic clusters control the structure, dynamics, and transport in soft matter. Incorporating a small fraction of ionizable groups in polymers substantially reduces the mobility of the macromolecules in melts. These ionic groups often associate into random clusters in melts, where the distribution and morphology of the clusters impact the transport in these materials. Here, using molecular dynamic simulations we demonstrate a clear correlation between cluster size and morphology with the polymer mobility in melts of sulfonated polystyrene. We show that in low dielectric media ladderlike clusters that are lower in energy compared with spherical assemblies are formed. Reducing the electrostatic interactions by enhancing the dielectric constant leads to morphological transformation from ladderlike clusters to globular assemblies. Decrease in electrostatic interaction significantly enhances the mobility of the polymer.

FeS/S/FeS(2) redox system and its oxidoreductase-like chemistry in the iron-sulfur world.

PubMed

Wang, Wei; Yang, Bin; Qu, Youpeng; Liu, Xiaoyang; Su, Wenhui

2011-06-01

The iron-sulfur world (ISW) theory is an intriguing prediction regarding the origin of life on early Earth. It hypothesizes that life arose as a geochemical process from inorganic starting materials on the surface of sulfide minerals in the vicinity of deep-sea hot springs. During the last two decades, many experimental studies have been carried out on this topic, and some interesting results have been achieved. Among them, however, the processes of carbon/nitrogen fixation and biomolecular assembly on the mineral surface have received an inordinate amount of attention. To the present, an abiotic model for the oxidation-reduction of intermediates participating in metabolic pathways has been ignored. We examined the oxidation-reduction effect of a prebiotic FeS/S/FeS(2) redox system on the interconversion between several pairs of α-hydroxy acids and α-keto acids (i.e., lactate/pyruvate, malate/oxaloacetate, and glycolate/glyoxylate). We found that, in the absence of FeS, elemental sulfur (S) oxidized α-hydroxy acids to form corresponding keto acids only at a temperature higher than its melting point (113°C); in the presence of FeS, such reactions occurred more efficiently through a coupled reaction mechanism, even at a temperature below the phase transition point of S. On the other hand, FeS was shown to have the capacity to reversibly reduce the keto acids. Such an oxidoreductase-like chemistry of the FeS/S/FeS(2) redox system suggests that it can determine the redox homeostasis of metabolic intermediates in the early evolutionary phase of life. The results provide a possible pathway for the development of primordial redox biochemistry in the iron-sulfur world. Key Words: Iron-sulfur world-FeS/S/FeS(2) redox system-Oxidoreductase-like chemistry. Astrobiology 11, 471-476.

Exploration of Hand Grasp Patterns Elicitable Through Non-Invasive Proximal Nerve Stimulation.

PubMed

Shin, Henry; Watkins, Zach; Hu, Xiaogang

2017-11-29

Various neurological conditions, such as stroke or spinal cord injury, result in an impaired control of the hand. One method of restoring this impairment is through functional electrical stimulation (FES). However, traditional FES techniques often lead to quick fatigue and unnatural ballistic movements. In this study, we sought to explore the capabilities of a non-invasive proximal nerve stimulation technique in eliciting various hand grasp patterns. The ulnar and median nerves proximal to the elbow joint were activated transcutanously using a programmable stimulator, and the resultant finger flexion joint angles were recorded using a motion capture system. The individual finger motions averaged across the three joints were analyzed using a cluster analysis, in order to classify the different hand grasp patterns. With low current intensity (<5 mA and 100 µs pulse width) stimulation, our results show that all of our subjects demonstrated a variety of consistent hand grasp patterns including single finger movement and coordinated multi-finger movements. This study provides initial evidence on the feasibility of a proximal nerve stimulation technique in controlling a variety of finger movements and grasp patterns. Our approach could also be developed into a rehabilitative/assistive tool that can result in flexible movements of the fingers.

Binding of dinitrogen to an iron-sulfur-carbon site

NASA Astrophysics Data System (ADS)

Čorić, Ilija; Mercado, Brandon Q.; Bill, Eckhard; Vinyard, David J.; Holland, Patrick L.

2015-10-01

Nitrogenases are the enzymes by which certain microorganisms convert atmospheric dinitrogen (N2) to ammonia, thereby providing essential nitrogen atoms for higher organisms. The most common nitrogenases reduce atmospheric N2 at the FeMo cofactor, a sulfur-rich iron-molybdenum cluster (FeMoco). The central iron sites that are coordinated to sulfur and carbon atoms in FeMoco have been proposed to be the substrate binding sites, on the basis of kinetic and spectroscopic studies. In the resting state, the central iron sites each have bonds to three sulfur atoms and one carbon atom. Addition of electrons to the resting state causes the FeMoco to react with N2, but the geometry and bonding environment of N2-bound species remain unknown. Here we describe a synthetic complex with a sulfur-rich coordination sphere that, upon reduction, breaks an Fe-S bond and binds N2. The product is the first synthetic Fe-N2 complex in which iron has bonds to sulfur and carbon atoms, providing a model for N2 coordination in the FeMoco. Our results demonstrate that breaking an Fe-S bond is a chemically reasonable route to N2 binding in the FeMoco, and show structural and spectroscopic details for weakened N2 on a sulfur-rich iron site.

On the efficiency of FES cycling: a framework and systematic review.

PubMed

Hunt, K J; Fang, J; Saengsuwan, J; Grob, M; Laubacher, M

2012-01-01

Research and development in the art of cycling using functional electrical stimulation (FES) of the paralysed leg muscles has been going on for around thirty years. A range of physiological benefits has been observed in clinical studies but an outstanding problem with FES-cycling is that efficiency and power output are very low. The present work had the following aims: (i) to provide a tutorial introduction to a novel framework and methods of estimation of metabolic efficiency using example data sets, and to propose benchmark measures for evaluating FES-cycling performance; (ii) to systematically review the literature pertaining specifically to the metabolic efficiency of FES-cycling, to analyse the observations and possible explanations for the low efficiency, and to pose hypotheses for future studies which aim to improve performance. We recommend the following as benchmark measures for assessment of the performance of FES-cycling: (i) total work efficiency, delta efficiency and stimulation cost; (ii) we recommend, further, that these benchmark measures be complemented by mechanical measures of maximum power output, sustainable steady-state power output and endurance. Performance assessments should be carried out at a well-defined operating point, i.e. under conditions of well controlled work rate and cadence, because these variables have a strong effect on energy expenditure. Future work should focus on the two main factors which affect FES-cycling performance, namely: (i) unfavourable biomechanics, i.e. crude recruitment of muscle groups, non-optimal timing of muscle activation, and lack of synergistic and antagonistic joint control; (ii) non-physiological recruitment of muscle fibres, i.e. mixed recruitment of fibres of different type and deterministic constant-frequency stimulation. We hypothesise that the following areas may bring better FES-cycling performance: (i) study of alternative stimulation strategies for muscle activation including irregular stimulation patterns (e.g. doublets, triplets, stochastic patterns) and variable frequency stimulation trains, where it appears that increasing frequency over time may be profitable; (ii) study of better timing parameters for the stimulated muscle groups, and addition of more muscle groups: this path may be approached using EMG studies and constrained numerical optimisation employing dynamic models; (iii) development of optimal stimulation protocols for muscle reconditioning and FES-cycle training.

NMR studies of conformational states of proteins involved in biosynthesis of iron-sulfur clusters

NASA Astrophysics Data System (ADS)

Dai, Ziqi

Iron-sulfur (Fe-S) clusters are the most ancient and ubiquitous cofactors that exist throughout evolution. The most important biosynthetic system of the cluster in both prokaryotes and eukaryotes is the ISC system. Defects in this system can be lethal and have been associated with a number of human diseases. Previous works show that a number of proteins are involved in the [Fe-S] biosynthetic processes and the structural flexibility may play an important role. For example, it was shown that apo-IscU, the scaffold protein, from Escherichia coli populates two functionally important conformational states, one dynamically disordered (D-state) and the other more structured (S-state) (Kim et al., 2009; Kim et al., 2012c). To further investigate the characteristics and transition of the conformational states of proteins involved in this system, I performed extensive NMR studies. Here, I present the findings based on my studies of two important players of the ISC system, IscU and HscB. In this research, I find that a peptidyl-prolyl cis/trans isomerization might account for the slow step in the S-D interconversion of IscU. More specifically, P14 and P101 are trans in the S-state, but become cis in the D-state. In addition, I discover that IscU is very responsive to pH changes, and I postulate that this response is correlated to conserved histidine residues, H10 and H105. Moreover, my thermodynamic analyses reveal that the S-D equilibrium of IscU is also very sensitive to change in temperature, pressure, and amino acid sequence compared to other proteins. In the study, I also discovered a novel state of IscU, the unfolded U-state. I suspect that this state may serve as an intermediate of interconversion between IscU S-/D-states. Finally, I extended the effort to HscB, and find that it may possess more conformational flexibility than expected earlier. I postulate that this flexibility may be the cause of the line-broadening observed during interaction of HscB with IscU (Fuzery et al., 2008; Kim et al., 2009) and HscA.

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

PubMed Central

Rousset, Marc; Montet, Yael; Guigliarelli, Bruno; Forget, Nicole; Asso, Marcel; Bertrand, Patrick; Fontecilla-Camps, Juan C.; Hatchikian, E. Claude

1998-01-01

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed. PMID:9751716

Acceleration effects observed in optical data taken in Spacelab 3 FES

NASA Technical Reports Server (NTRS)

Trolinger, James; Lal, Ravindra; Ruff, Rudy

1990-01-01

Optical instrumentation in the Fluids Experiment System (FES) is briefly described. Samples of the data produced by the schlieren and holography systems during the Spacelab 3 flight are then presented with some of the holographic interferometry data being presented for the first time. Acceleration effects that can be observed in these data are discussed and the potential for using them as a basis for measurement is explored. This includes the tracking of deliberately introduced tracer particles and density gradients in the FES, the analysis of the existing concentration gradients, and a new fiber optic G-meter concept. Finally, some of the plans for acceleration measurement in the upcoming International Microgravity-1/FES are described.

CYP76M7 Is an ent-Cassadiene C11α-Hydroxylase Defining a Second Multifunctional Diterpenoid Biosynthetic Gene Cluster in Rice[W][OA

PubMed Central

Swaminathan, Sivakumar; Morrone, Dana; Wang, Qiang; Fulton, D. Bruce; Peters, Reuben J.

2009-01-01

Biosynthetic gene clusters are common in microbial organisms, but rare in plants, raising questions regarding the evolutionary forces that drive their assembly in multicellular eukaryotes. Here, we characterize the biochemical function of a rice (Oryza sativa) cytochrome P450 monooxygenase, CYP76M7, which seems to act in the production of antifungal phytocassanes and defines a second diterpenoid biosynthetic gene cluster in rice. This cluster is uniquely multifunctional, containing enzymatic genes involved in the production of two distinct sets of phytoalexins, the antifungal phytocassanes and antibacterial oryzalides/oryzadiones, with the corresponding genes being subject to distinct transcriptional regulation. The lack of uniform coregulation of the genes within this multifunctional cluster suggests that this was not a primary driving force in its assembly. However, the cluster is dedicated to specialized metabolism, as all genes in the cluster are involved in phytoalexin metabolism. We hypothesize that this dedication to specialized metabolism led to the assembly of the corresponding biosynthetic gene cluster. Consistent with this hypothesis, molecular phylogenetic comparison demonstrates that the two rice diterpenoid biosynthetic gene clusters have undergone independent elaboration to their present-day forms, indicating continued evolutionary pressure for coclustering of enzymatic genes encoding components of related biosynthetic pathways. PMID:19825834

Metagenome assembly through clustering of next-generation sequencing data using protein sequences.

PubMed

Sim, Mikang; Kim, Jaebum

2015-02-01

The study of environmental microbial communities, called metagenomics, has gained a lot of attention because of the recent advances in next-generation sequencing (NGS) technologies. Microbes play a critical role in changing their environments, and the mode of their effect can be solved by investigating metagenomes. However, the difficulty of metagenomes, such as the combination of multiple microbes and different species abundance, makes metagenome assembly tasks more challenging. In this paper, we developed a new metagenome assembly method by utilizing protein sequences, in addition to the NGS read sequences. Our method (i) builds read clusters by using mapping information against available protein sequences, and (ii) creates contig sequences by finding consensus sequences through probabilistic choices from the read clusters. By using simulated NGS read sequences from real microbial genome sequences, we evaluated our method in comparison with four existing assembly programs. We found that our method could generate relatively long and accurate metagenome assemblies, indicating that the idea of using protein sequences, as a guide for the assembly, is promising. Copyright © 2015 Elsevier B.V. All rights reserved.

Out of touch with reality? Social perception in first-episode schizophrenia

PubMed Central

Salone, Anatolia; Ferri, Francesca; De Berardis, Domenico; Romani, Gian Luca; Ferro, Filippo M.; Gallese, Vittorio

2013-01-01

Social dysfunction has been recognized as an elementary feature of schizophrenia, but it remains a crucial issue whether social deficits in schizophrenia concern the inter-subjective domain or primarily have their roots in disturbances of self-experience. Social perception comprises vicarious processes grounding an experiential inter-relationship with others as well as self-regulation processes allowing to maintain a coherent sense of self. The present study investigated whether the functional neural basis underlying these processes is altered in first-episode schizophrenia (FES). Twenty-four FES patients and 22 healthy control participants underwent functional magnetic resonance imaging during a social perception task requiring them to watch videos depicting other individuals' inanimate and animate/social tactile stimulations, and a tactile localizer condition. Activation in ventral premotor cortex for observed bodily tactile stimulations was reduced in the FES group and negatively correlated with self-experience disturbances. Moreover, FES patients showed aberrant differential activation in posterior insula for first-person tactile experiences and observed affective tactile stimulations. These findings suggest that social perception in FES at a pre-reflective level is characterized by disturbances of self-experience, including impaired multisensory representations and self-other distinction. However, the results also show that social perception in FES involves more complex alterations of neural activation at multiple processing levels. PMID:22275166

Cycling induced by electrical stimulation improves muscle activation and symmetry during pedaling in hemiparetic patients.

PubMed

Ambrosini, Emilia; Ferrante, Simona; Ferrigno, Giancarlo; Molteni, Franco; Pedrocchi, Alessandra

2012-05-01

A randomized controlled trial, involving 35 post-acute hemiparetic patients, demonstrated that a four-week treatment of cycling induced by functional electrical stimulation (FES-cycling) promotes motor recovery. Analyzing additional data acquired during that study, the present work investigated whether these improvements were associated to changes in muscle strength and motor coordination. Participants were randomized to receive FES-cycling or placebo FES-cycling. Clinical outcome measures were: the Motricity Index (MI), the gait speed, the electromyography activation of the rectus femoris and biceps femoris, and the mechanical work produced by each leg during voluntary pedaling. To provide a comparison with normal values, healthy adults also carried out the pedaling test. Patients were evaluated before, after training, and at follow-up visits. A significant treatment effect in favor of FES-treated patients was found in terms of MI scores and unbalance in mechanical works, while differences in gait speed were not significant (ANCOVA). Significant improvements in the activation of the paretic muscles were highlighted in the FES group, while no significant change was found in the placebo group (Friedman test). Our findings suggested that improvements in motor functions induced by FES-cycling training were associated with a more symmetrical involvement of the two legs and an improved motor coordination.

Metrological characterization of a cycle-ergometer to optimize the cycling induced by functional electrical stimulation on patients with stroke.

PubMed

Comolli, Lorenzo; Ferrante, Simona; Pedrocchi, Alessandra; Bocciolone, Marco; Ferrigno, Giancarlo; Molteni, Franco

2010-05-01

Functional electrical stimulation (FES) is a well established method in the rehabilitation of stroke patients. Indeed, a bilateral movement such as cycling induced by FES would be crucial for these patients who had an unilateral motor impairment and had to recover an equivalent use of limbs. The aim of this study was to develop a low-cost meteorologically qualified cycle-ergometer, optimized for patients with stroke. A commercial ergometer was instrumented with resistive strain gauges and was able to provide the torque produced at the right and left crank, independently. The developed system was integrated with a stimulator, obtaining a novel FES cycling device able to control in real-time the movement unbalance. A dynamic calibration of the sensors was performed and a total torque uncertainty was computed. The system was tested on a healthy subject and on a stroke patient. Results demonstrated that the proposed sensors could be successfully used during FES cycling sessions where the maximum torque produced is about 9Nm, an order of magnitude less than the torque produced during voluntary cycling. This FES cycling system will assist in future investigations on stroke rehabilitation by means of FES and in new exercise regimes designed specifically for patients with unilateral impairments.

Cooperative Control for A Hybrid Rehabilitation System Combining Functional Electrical Stimulation and Robotic Exoskeleton

PubMed Central

Zhang, Dingguo; Ren, Yong; Gui, Kai; Jia, Jie; Xu, Wendong

2017-01-01

Functional electrical stimulation (FES) and robotic exoskeletons are two important technologies widely used for physical rehabilitation of paraplegic patients. We developed a hybrid rehabilitation system (FEXO Knee) that combined FES and an exoskeleton for swinging movement control of human knee joints. This study proposed a novel cooperative control strategy, which could realize arbitrary distribution of torque generated by FES and exoskeleton, and guarantee harmonic movements. The cooperative control adopted feedfoward control for FES and feedback control for exoskeleton. A parameter regulator was designed to update key parameters in real time to coordinate FES controller and exoskeleton controller. Two muscle groups (quadriceps and hamstrings) were stimulated to generate active torque for knee joint in synchronization with torque compensation from exoskeleton. The knee joint angle and the interactive torque between exoskeleton and shank were used as feedback signals for the control system. Central pattern generator (CPG) was adopted that acted as a phase predictor to deal with phase confliction of motor patterns, and realized synchronization between the two different bodies (shank and exoskeleton). Experimental evaluation of the hybrid FES-exoskeleton system was conducted on five healthy subjects and four paraplegic patients. Experimental results and statistical analysis showed good control performance of the cooperative control on torque distribution, trajectory tracking, and phase synchronization. PMID:29311798

Cooperative Control for A Hybrid Rehabilitation System Combining Functional Electrical Stimulation and Robotic Exoskeleton.

PubMed

Zhang, Dingguo; Ren, Yong; Gui, Kai; Jia, Jie; Xu, Wendong

2017-01-01

Functional electrical stimulation (FES) and robotic exoskeletons are two important technologies widely used for physical rehabilitation of paraplegic patients. We developed a hybrid rehabilitation system (FEXO Knee) that combined FES and an exoskeleton for swinging movement control of human knee joints. This study proposed a novel cooperative control strategy, which could realize arbitrary distribution of torque generated by FES and exoskeleton, and guarantee harmonic movements. The cooperative control adopted feedfoward control for FES and feedback control for exoskeleton. A parameter regulator was designed to update key parameters in real time to coordinate FES controller and exoskeleton controller. Two muscle groups (quadriceps and hamstrings) were stimulated to generate active torque for knee joint in synchronization with torque compensation from exoskeleton. The knee joint angle and the interactive torque between exoskeleton and shank were used as feedback signals for the control system. Central pattern generator (CPG) was adopted that acted as a phase predictor to deal with phase confliction of motor patterns, and realized synchronization between the two different bodies (shank and exoskeleton). Experimental evaluation of the hybrid FES-exoskeleton system was conducted on five healthy subjects and four paraplegic patients. Experimental results and statistical analysis showed good control performance of the cooperative control on torque distribution, trajectory tracking, and phase synchronization.

Molecular characterization of human xanthine oxidoreductase: the enzyme is grossly deficient in molybdenum and substantially deficient in iron-sulphur centres

PubMed Central

2005-01-01

XOR (xanthine oxidoreductase) purified from human milk was shown to contain 0.04 atom of Mo and 0.09 molecule of molybdopterin/subunit. On the basis of UV/visible and CD spectra, the human enzyme was approx. 30% deficient in iron-sulphur centres. Mo(V) EPR showed the presence of a weak rapid signal corresponding to the enzyme of low xanthine oxidase activity and a slow signal indicating a significant content of desulpho-form. Resulphuration experiments, together with calculations based on enzymic activity and Mo content, led to an estimate of 50–60% desulpho-form. Fe/S EPR showed, in addition to the well-known Fe/S I and Fe/S II species, the presence of a third Fe/S signal, named Fe/S III, which appears to replace partially Fe/S I. Comparison is made with similarly prepared bovine milk XOR, which has approx. 15-fold higher enzymic activity and Mo content. Taken along with evidence of low Mo content in the milk of other mammals, these findings add further support to the idea that XOR protein plays a physiological role in milk (e.g. in secretion) equal in importance to its catalytic function as an enzyme. PMID:15679468

Effectiveness of functional electrical stimulation on walking speed, functional walking category, and clinically meaningful changes for people with multiple sclerosis.

PubMed

Street, Tamsyn; Taylor, Paul; Swain, Ian

2015-04-01

To determine the effectiveness of functional electrical stimulation (FES) on drop foot in patients with multiple sclerosis (MS), using data from standard clinical practice. Case series with a consecutive sample of FES users collected between 2008 and 2013. Specialist FES center at a district general hospital. Patients with MS who have drop foot (N=187) (117 women, 70 men; mean age, 55y [range, 27-80y]; mean duration since diagnosis, 11.7y [range, 1-56y]). A total of 166 patients were still using FES after 20 weeks, with 153 patients completing the follow-up measures. FES of the common peroneal nerve (178 unilateral, 9 bilateral FES users). Clinically meaningful changes (ie, >.05m/s and >0.1m/s) and functional walking category derived from 10-m walking speed. An increase in walking speed was found to be highly significant (P<.001), both initially where a minimum clinically meaningful change was observed (.07m/s) and after 20 weeks with a substantial clinically meaningful change (.11m/s). After 20 weeks, treatment responders displayed a 27% average improvement in their walking speed. No significant training effect was found. Overall functional walking category was maintained or improved in 95% of treatment responders. FES of the dorsiflexors is a well-accepted intervention that enables clinically meaningful changes in walking speed, leading to a preserved or an increased functional walking category. Copyright © 2015 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.