Sulfur Protein Research Articles

Evolution of Chlamydomonas reinhardtii ferredoxins and their interactions with [FeFe]-hydrogenases.

Ferredoxins are soluble iron sulphur proteins which function as electron donors in a number of metabolic pathways in a broad range of organisms. In photosynthetic organisms, PETF, or ferredoxin 1 (FDX1), is the most studied ferredoxin due to its essential role in photosynthesis, where it transfers electrons from photosystem I to ferredoxin-NADP+ oxidoreductase. However, PETF can also transfer electrons to a large number of other proteins. One important PETF electron acceptor found in green microalgae is the biologically and biotechnologically important [FeFe]-hydrogenase HYDA, which catalyses the production of molecular hydrogen (H2) from protons and electrons. The interaction between PETF and HYDA is of considerable interest, as PETF is the primary electron donor to HYDA and electron supply is one of the main limiting factors for H2 production on a commercial scale. Although there is no three dimensional structure of the PETF-HYDA complex available, protein variants, nuclear magnetic resonance titration studies, molecular dynamics and modelling have provided considerable insight into the residues essential for forming and maintaining the interaction. In this review, we discuss the most recent findings with regard to ferredoxin-HYDA interactions and the evolution of the various Chlamydomonas reinhardtii ferredoxin isoforms. Finally, we provide an outlook on new PETF-based biotechnological approaches for improved H2 production efficiencies.

Investigating the role(s) of SufT and the domain of unknown function 59 (DUF59) in the maturation of iron-sulfur proteins.

Comprehending biology at the molecular and systems levels is predicated upon understanding the functions of proteins. Proteins are typically composed of one or more functional moieties termed domains. Members of Bacteria, Eukarya, and Archaea utilize proteins containing a domain of unknown function (DUF) 59. Proteins requiring iron-sulfur (FeS) clusters containing cofactors are necessary for nearly all organisms making the assembly of functional FeS proteins essential. Recently, studies in eukaryotic and bacterial organisms have shown that proteins containing a DUF59, or those composed solely of DUF59, function in FeS protein maturation and/or intracellular Fe homeostasis. Herein, we review the current literature, discuss potential roles for DUF59, and address future studies that will help advance the field.

Paramagnetic Properties of a Crystalline Iron-Sulfur Protein by Magic-Angle Spinning NMR Spectroscopy.

We present the first solid-state NMR study of an iron-sulfur protein. The combined use of very fast (60 kHz) magic-angle spinning and tailored radiofrequency irradiation schemes allows the detection and the assignment of most of the 1H and 13C resonances of the oxidized high-potential iron-sulfur protein I from Ectothiorhodospira halophila (EhHiPIP I), including those in residues coordinating the Fe4S4 cluster. For these residues, contact shifts as large as 100 and 400 ppm for 1H and 13C resonances, respectively, were observed, which represent the most shifted solid-state NMR signals ever measured in metalloproteins. Interestingly, by targeting EhHiPIP I in a crystalline environment, we were able to capture distinct paramagnetic signatures from the two conformations present in the asymmetric unit. The magnetic properties of the system were verified by following the temperature dependence of the contact-shifted cysteine resonances.

Differences in Gene Expression Profiles between Early and Late Isolates in Monospecies Achromobacter Biofilm.

Bacteria of genus Achromobacter are emerging pathogens in cystic fibrosis (CF) capable of biofilm formation and development of antimicrobial resistance. Evolutionary adaptions in the transition from primary to chronic infection were assessed by transcriptomic analysis of successive isolates of Achromobacter xylosoxidans from a single CF patient. Several efflux pump systems targeting antimicrobial agents were upregulated during the course of the disease, whereas all genes related to motility were downregulated. Genes annotated to subsystems of sulfur metabolism, protein metabolism and potassium metabolism exhibited the strongest upregulation. K+ channel genes were hyperexpressed, and a putative sulfite oxidase was more than 1500 times upregulated. The transcriptome patterns indicated a pivotal role of sulfur metabolism and electrical signalling in Achromobacter biofilms during late stage CF lung disease.

The Q-Cycle Mechanism of the bc1 Complex: A Biologist's Perspective on Atomistic Studies.

The Q-cycle mechanism of the bc1 complex is now well enough understood to allow application of advanced computational approaches to the study of atomistic processes. In addition to the main features of the mechanism, these include control and gating of the bifurcated reaction at the Qo-site, through which generation of damaging reactive oxygen species is minimized. We report a new molecular dynamics model of the Rhodobacter sphaeroides bc1 complex implemented in a native membrane, and constructed so as to eliminate blemishes apparent in earlier Rhodobacter models. Unconstrained MD simulations after equilibration with ubiquinol and ubiquinone respectively at Qo- and Qi-sites show that substrate binding configurations at both sites are different in important details from earlier models. We also demonstrate a new Qo-site intermediate, formed in the sub-ms time range, in which semiquinone remains complexed with the reduced iron sulfur protein. We discuss this, and a spring-loaded mechanism for modulating interactions of the iron sulfur protein with occupants of the Qo-site, in the context of control and gating roles. Such atomistic features of the mechanism can usefully be explored through simulation, but we stress the importance of constraints from physical chemistry and biology, both in setting up a simulation and in interpreting results.

Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein.

Mitochondrial cysteine desulfurase is an essential component of the machinery for iron–sulfur cluster biosynthesis. It has been known that human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the cysteine desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron–sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron–sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.

Evolutionary conservation and in vitro reconstitution of microsporidian iron\u2013sulfur cluster biosynthesis

Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron–sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe–2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron–sulfur protein assembly (CIA) pathway includes the essential Cfd1–Nbp35 scaffold complex that assembles a [4Fe–4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of Fe/S-related proteins and pathways, and their strong conservation among highly reduced parasites, provides compelling evidence for the ancient chimeric ancestry of eukaryotes.

De Novo Design of Iron-Sulfur Proteins.

Iron-sulfur proteins are one of the most abundant and functionally pliable redox proteins found in all living organisms. Because of their crucial role in mediating electron transfer processes, minimalist model systems have been developed as a proxy to study natural Fe-S redox proteins and to dissect rules to enable tuning of their redox and electron transfer activities. This goal has been pursued through computational design, mutagenesis in the first and second coordination sphere, metal substitution, cofactor replacement, and the use of unnatural amino acids to stabilize a given cluster. In this chapter, we discuss the most recent design strategies to introduce various Fe-S clusters into natural and artificial protein scaffolds. Practical approaches for the cluster reconstitution, hydrogen production, and electrochemical characterization are mentioned.

Phenolic compounds responding to zinc and/or cadmium treatments in Gynura pseudochina (L.) DC. extracts and biomass

Gynura pseudochina (L.) DC. is a Zn/Cd hyperaccumulative plant. In an in vivo system under controlled plant age, this research reveals that phenolic compounds and lignification play beneficial roles in protecting G. pseudochina from exposure to an excess of Zn and/or Cd, and Zn reduces Cd toxicity under the dual treatments. The total phenolic content (TPC), total flavonoid content (TFC), and half-maximal inhibitory concentration (IC50) values correspond to the metal dose-response curves. Liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (LC-ESI-QTOF-MS/MS) is used to characterize phenolic compounds and their glycosides, which could play roles in antioxidant activities and in the esterification of the cell wall, especially derivatives of p-coumaric and caffeic acid. Confocal laser scanning microscopy (CLSM) and micro X-ray fluorescence (μ-XRF) imaging revealed that the accumulation of Zn and Cd in the cell wall involves flavonoid compounds. Low extractable pools of Cd and Zn in the leaf extracts indicate that these elements are tightly bound to the plant biomass structures. The bulk X-ray absorption near edge structure (XANES) spectra indicate that Zn2+ and Cd2+ dominate with O and S ligands, which could be provided by cell walls, phenolic compounds, and sulphur protein. Consequently, the benefit of these results is to support the growth of G. pseudochina for phytoremediation in a Zn- and/or Cd-contaminated site.

Proteomic analysis provides insights into the molecular bases of hydrogen gas-induced cadmium resistance in Medicago sativa

Recently, molecular hydrogen (H2) has emerged as a bio-regulator both in animals and plants. Normally, functions of endogenous generated H2 could be mimicked by exogenously applied hydrogen-rich water (HRW) or hydrogen-rich saline (particularly in animals). Although alfalfa seedlings showed more cadmium (Cd) resistance after the administration with HRW, corresponding molecular mechanism is still elusive. To address this gap, iTRAQ-based quantitative proteomics was used. The results showed that a total of 2377 proteins were identified with <1% FDR, and 1254 protein abundance perturbations were confidently assessed. Total of 248 significant differential proteins were identified in Cd- and/or HRW-treated samples. Furthermore, 92 proteins from the 248 proteins were selected for further bioinformatics analysis. Interestingly, results indicated that they were classified into seven categories: defense and response to stress, sulfur compound metabolic process, amino acid and protein metabolic process, carbohydrate and energy metabolic process, secondary metabolic process, oxidation-reduction process, and metal ion homeostasis. In addition, the protein expression patterns were consistent with the results of decreased lipid peroxidation, increased non-protein thiols abundance, as well as iron and zinc content. These suggest that HRW alleviates Cd toxicity mainly by decreasing oxidative damage, enhancing sulfur compound metabolic process, and maintaining nutrient element homeostasis. Biological significanceContamination of soils by Cd has become a potential concern to crops. Medicago sativa is a widely used forage around the world. Recently, hydrogen gas (H2) was suggested as a candidate of signal molecule, and found to effectively attenuate Cd-induced damage in alfalfa seedlings. However, the underlying molecular mechanism still needs to be further elucidated. In the present work, an iTRAQ-based quantitative proteomics was firstly carried out, and the results revealed the main molecular targets and metabolic processes associated with Cd resistance conferred by H2. This study may expand our understanding of hydrogen gas-medicated heavy metal tolerance in plants.

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

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.

Friedreich ataxia-induced pluripotent stem cell-derived neurons show a cellular phenotype that is corrected by a benzamide HDAC inhibitor.

We employed induced pluripotent stem cell (iPSC)-derived neurons obtained from Friedreich ataxia (FRDA) patients and healthy subjects, FRDA neurons and CT neurons, respectively, to unveil phenotypic alterations related to frataxin (FXN) deficiency and investigate if they can be reversed by treatments that upregulate FXN. FRDA and control iPSCs were equally capable of differentiating into a neuronal or astrocytic phenotype. FRDA neurons showed lower levels of iron–sulfur (Fe–S) and lipoic acid-containing proteins, higher labile iron pool (LIP), higher expression of mitochondrial superoxide dismutase (SOD2), increased reactive oxygen species (ROS) and lower reduced glutathione (GSH) levels, and enhanced sensitivity to oxidants compared with CT neurons, indicating deficient Fe–S cluster biogenesis, altered iron metabolism, and oxidative stress. Treatment with the benzamide HDAC inhibitor 109 significantly upregulated FXN expression and increased Fe–S and lipoic acid-containing protein levels, downregulated SOD2 levels, normalized LIP and ROS levels, and almost fully protected FRDA neurons from oxidative stress-mediated cell death. Our findings suggest that correction of FXN deficiency may not only stop disease progression, but also lead to clinical improvement by rescuing still surviving, but dysfunctional neurons.

Functional Identification of Putrescine C- and N-Hydroxylases.

The small polyamine putrescine (1,4-diaminobutane) is ubiquitously and abundantly found in all three domains of life. It is a precursor, through N-aminopropylation or N-aminobutylation, for biosynthesis of the longer polyamines spermidine, sym-homospermidine, spermine, and thermospermine and longer and branched chain polyamines. Putrescine is also biochemically modified for purposes of metabolic regulation and catabolism, e.g. N-acetylation and N-glutamylation, and for incorporation into specialized metabolites, e.g. N-methylation, N-citrylation, N-palmitoylation, N-hydroxylation, and N-hydroxycinnamoylation. Only one example is known where putrescine is modified on a methylene carbon: the formation of 2-hydroxyputrescine by an unknown C-hydroxylase. Here, we report the functional identification of a previously undescribed putrescine 2-hydroxylase, a Rieske-type nonheme iron sulfur protein from the β-proteobacteria Bordetella bronchiseptica and Ralstonia solanacearum. Identification of the putrescine 2-hydroxylase will facilitate investigation of the physiological functions of 2-hydroxyputrescine. One known role of 2-hydroxyputrescine has direct biomedical relevance: its role in the biosynthesis of the cyclic hydroxamate siderophore alcaligin, a potential virulence factor of the causative agent of whooping cough, Bordetella pertussis. We also report the functional identification of a putrescine N-hydroxylase from the γ-proteobacterium Shewanella oneidensis, which is homologous to FAD- and NADPH-dependent ornithine and lysine N-monooxygenases involved in siderophore biosynthesis. Heterologous expression of the putrescine N-hydroxylase in E. coli produced free N-hydroxyputrescine, never detected previously in a biological system. Furthermore, the putrescine C- and N-hydroxylases identified here could contribute new functionality to polyamine structural scaffolds, including C-H bond functionalization in synthetic biology strategies.

The Arabidopsis Iron–Sulfur Protein GRXS17 is a Target of the Ubiquitin E3 Ligases RGLG3 and RGLG4

The stability of signaling proteins in eukaryotes is often controlled by post-translational modifiers. For polyubiquitination, specificity is assured by E3 ubiquitin ligases. Although plant genomes encode hundreds of E3 ligases, only few targets are known, even in the model Arabidopsis thaliana. Here, we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 using a substrate trapping approach involving tandem affinity purification of RING-dead versions. Simultaneously, we used a ubiquitin-conjugating enzym (UBC) panel screen to pinpoint UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 and mediating auto-ubiquitination of RGLG3 and ubiquitination of GRXS17 in vitro. Accordingly, GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta. The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. Our results suggest that the RGLG family is intimately linked to the essential element iron.

The conserved protein Dre2 uses essential [2Fe–2S] and [4Fe–4S] clusters for its function in cytosolic iron–sulfur protein assembly

The cytosolic iron-sulfur (Fe-S) protein assembly (CIA) machinery comprises 11 essential components and matures Fe-S proteins involved in translation and genome maintenance. Maturation is initiated by the electron transfer chain NADPH-diflavin reductase Tah18-Fe-S protein Dre2 that facilitates the de novo assembly of a [4Fe-4S] cluster on the scaffold complex Cfd1-Nbp35. Tah18-Dre2 also play a critical role in the assembly of the diferric tyrosyl radical cofactor of ribonucleotide reductase. Dre2 contains eight conserved cysteine residues as potential co-ordinating ligands for Fe-S clusters but their functional importance and the typeof bound clusters is unclear. In the present study, we use a combination of mutagenesis, cell biological and biochemical as well as UV-visible, EPR and Mössbauer spectroscopic approaches to show that the yeast Dre2 cysteine residues Cys(252), Cys(263), Cys(266) and Cys(268) (motif I) bind a [2Fe-2S] cluster, whereas cysteine residues Cys(311), Cys(314), Cys(322) and Cys(325) (motif II) co-ordinate a [4Fe-4S] cluster. All of these residues with the exception of Cys(252) are essential for cell viability, cytosolic Fe-S protein activity and invivo (55)Fe-S cluster incorporation. The N-terminal methyltransferase-like domain of Dre2 is important for proper Fe-S cluster assembly at motifs I and II, which occurs in an interdependent fashion. Our findings further resolve why recombinant Dre2 from Arabidopsis, Trypanosoma or humans has previously been isolated with a single [2Fe-2S] instead of native [2Fe-2S] plus [4Fe-4S] clusters. In the presence of oxygen, the motif I-bound [2Fe-2S] cluster is labile and the motif II-bound [4Fe-4S] cluster is readily converted into a [2Fe-2S] cluster.

MetalPredator: a web server to predict iron-sulfur cluster binding proteomes.

The prediction of the iron-sulfur proteome is highly desirable for biomedical and biological research but a freely available tool to predict iron-sulfur proteins has not been developed yet. We developed a web server to predict iron-sulfur proteins from protein sequence(s). This tool, called MetalPredator, is able to process complete proteomes rapidly with high recall and precision. The web server is freely available at: http://metalweb.cerm.unifi.it/tools/metalpredator/ andreini@cerm.unifi.it Supplementary data are available at Bioinformatics online.

A Chloroplast-Localized Rubredoxin Family Protein Gene from Puccinellia tenuiflora (PutRUB) Increases NaCl and NaHCO₃ Tolerance by Decreasing H₂O₂ Accumulation.

Rubredoxin is one of the simplest iron–sulfur (Fe–S) proteins. It is found primarily in strict anaerobic bacteria and acts as a mediator of electron transfer participation in different biochemical reactions. The PutRUB gene encoding a chloroplast-localized rubredoxin family protein was screened from a yeast full-length cDNA library of Puccinellia tenuiflora under NaCl and NaHCO3 stress. We found that PutRUB expression was induced by abiotic stresses such as NaCl, NaHCO3, CuCl2 and H2O2. These findings suggested that PutRUB might be involved in plant responses to adversity. In order to study the function of this gene, we analyzed the phenotypic and physiological characteristics of PutRUB transgenic plants treated with NaCl and NaHCO3. The results showed that PutRUB overexpression inhibited H2O2 accumulation, and enhanced transgenic plant adaptability to NaCl and NaHCO3 stresses. This indicated PutRUB might be involved in maintaining normal electron transfer to reduce reactive oxygen species (ROS) accumulation.

Charge-density analysis of an iron-sulfur protein at an ultra-high resolution of 0.48 Å.

The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0-1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins. Here we report a charge-density analysis of the high-potential iron-sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron-sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

Metalloproteins: Mapping elusive electron density.

Iron–sulfur metalloproteins are critical for electron transfer in bacterial metabolism, but most crystal structures are insufficient for their in-depth study. Now, acquisition of an iron–sulfur protein structure at ultra-high resolution enables detailed visualization of its electron distribution.

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

BackgroundBacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron–sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene.ResultsBacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP+-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300 % compared with the wild-type strain.ConclusionsIspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0476-9) contains supplementary material, which is available to authorized users.