RESUMEN

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

Asunto(s)

Proteínas Hierro-Azufre , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Hierro/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo

RESUMEN

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M-1 s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.

Asunto(s)

Proteínas de Unión a Hierro , Proteínas Hierro-Azufre , Humanos , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Oxidación-Reducción , Superóxidos/metabolismo , Aconitato Hidratasa/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Frataxina

RESUMEN

Fe-S clusters are versatile and essential cofactors that participate in multiple and fundamental biological processes. In Escherichia coli, the biogenesis of these cofactors requires either the housekeeping Isc pathway, or the stress-induced Suf pathway which plays a general role under conditions of oxidative stress or iron limitation. In the present work, the Fe-S cluster assembly Isc and Suf systems of acidophilic Bacteria and Archaea, which thrive in highly oxidative environments, were studied. This analysis revealed that acidophilic microorganisms have a complete set of genes encoding for a single system (either Suf or Isc). In acidophilic Proteobacteria and Nitrospirae, a complete set of isc genes (iscRSUAX-hscBA-fdx), but not genes coding for the Suf system, was detected. The activity of the Isc system was studied in Leptospirillum sp. CF-1 (Nitrospirae). RT-PCR experiments showed that eight candidate genes were co-transcribed and conform the isc operon in this strain. Additionally, RT-qPCR assays showed that the expression of the iscS gene was significantly up-regulated in cells exposed to oxidative stress imposed by 260 mM Fe2(SO4)3 for 1 h or iron starvation for 3 h. The activity of cysteine desulfurase (IscS) in CF-1 cell extracts was also up-regulated under such conditions. Thus, the Isc system from Leptospirillum sp. CF-1 seems to play an active role in stressful environments. These results contribute to a better understanding of the distribution and role of Fe-S cluster protein biogenesis systems in organisms that thrive in extreme environmental conditions.

Asunto(s)

Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Extractos Celulares , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Operón , Azufre/metabolismo

RESUMEN

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

Asunto(s)

Aminoácidos/metabolismo , Arabidopsis/genética , Carbono/metabolismo , Flavoproteínas Transportadoras de Electrones/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flavoproteínas Transportadoras de Electrones/genética , Germinación , Proteínas Hierro-Azufre/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Semillas/enzimología , Semillas/genética , Semillas/crecimiento & desarrollo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo

RESUMEN

Several biological activities depend on iron-sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol-1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich's Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.

Asunto(s)

Dictyostelium/metabolismo , Ataxia de Friedreich/genética , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Secuencia de Aminoácidos/genética , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Biología Computacional , Cristalografía , Dictyostelium/genética , Humanos , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Cinética , Simulación de Dinámica Molecular , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes , Alineación de Secuencia , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Frataxina

RESUMEN

Three different types of electron-transferring metallo-ATPases are able to couple ATP hydrolysis to the reduction of low-potential metal sites, thereby energizing an electron. Besides the Fe-protein known from nitrogenase and homologous enzymes, two other kinds of ATPase with different scaffolds and cofactors are used to achieve a unidirectional, energetic, uphill electron transfer to either reduce inactive Co-corrinoid-containing proteins (RACE-type activators) or a second iron-sulfur cluster-containing enzyme of a unique radical enzymes family (archerases). We have found a new cofactor in the latter enzyme family, that is, a double-cubane cluster with two [4Fe4S] subclusters bridged by a sulfido ligand. An enzyme containing this cofactor catalyzes the ATP-dependent reduction of small molecules, including acetylene. Thus, enzymes containing the double-cubane cofactor are analogous in function and share some structural features with nitrogenases.

Asunto(s)

Proteínas Hierro-Azufre/metabolismo , Nitrogenasa/química , Acetileno/química , Acetileno/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Biocatálisis , Proteínas Hierro-Azufre/química , Modelos Moleculares , Nitrogenasa/metabolismo , Oxidación-Reducción

RESUMEN

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm-1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hierro-Azufre/metabolismo , Sitios de Unión/fisiología , Daño del ADN/fisiología , ADN Glicosilasas/metabolismo , Reparación del ADN/fisiología , Escherichia coli/metabolismo , Oxidación-Reducción , Espectroscopía Infrarroja por Transformada de Fourier/métodos

RESUMEN

KEY MESSAGE: The ISC Fe-S cluster biosynthetic pathway would play a key role in the regulation of iron and sulfur homeostasis in plants. The Arabidopsis thaliana mitochondrial cysteine desulfurase AtNFS1 has an essential role in cellular ISC Fe-S cluster assembly, and this pathway is one of the main sinks for iron (Fe) and sulfur (S) in the plant. In different plant species it has been reported a close relationship between Fe and S metabolisms; however, the regulation of both nutrient homeostasis is not fully understood. In this study, we have characterized AtNFS1 overexpressing and knockdown mutant Arabidopsis plants. Plants showed alterations in the ISC Fe-S biosynthetic pathway genes and in the activity of Fe-S enzymes. Genes involved in Fe and S uptakes, assimilation, and regulation were up-regulated in overexpressing plants and down-regulated in knockdown plants. Furthermore, the plant nutritional status in different tissues was in accordance with those gene activities: overexpressing lines accumulated increased amounts of Fe and S and mutant plant had lower contents of S. In summary, our results suggest that the ISC Fe-S cluster biosynthetic pathway plays a crucial role in the homeostasis of Fe and S in plants, and that it may be important in their regulation.

Asunto(s)

Hierro/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Azufre/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética

RESUMEN

Molybdenum-dependent nitrogenases catalyze the transformation of dinitrogen into ammonia under ambient conditions. The active site (FeMo cofactor) is the structurally and electronically complex weak-field metal cluster [MoFe7S9C] built of Fe4S3 and MoFe3S3C portions connected by three sulfur bridges and containing an interstitial carbon atom centered in an Fe6 trigonal prism. Chemical synthesis of this cluster is a major challenge in biomimetic inorganic chemistry. One synthetic approach of core ligand metathesis has been developed based on the design and synthesis of unprecedented incomplete ([(Tp*)WFe2S3Q3]-) and complete ([(Tp*)WFe3S3Q4]2-) cubane-type clusters containing bridging halide (Q = halide). These clusters are achieved by template-assisted assembly in the presence of sodium benzophenone ketyl reductant; products are controlled by reaction stoichiometry. Incomplete cubane clusters are subject to a variety of metathesis reactions resulting in substitution of a µ2-bridging ligand with other bridges such as N3-, MeO-, and EtS- Reactions of complete cubanes with Me3SiN3 and S8 undergo a redox metathesis process and lead to core ligand displacement and formation of [(Tp*)WFe3S3(µ3-Q)Cl3]- (Q = Me3SiN2-, S2-). This work affords entry to a wide variety of heteroleptic clusters derivable from incomplete and complete cubanes; examples are provided. Among these is the cluster [(Tp*)WFe3S3(µ3-NSiMe3)Cl3]-, one of the very few instances of a synthetic Fe-S cluster containing a light atom (C, N, O) in the core, which constitutes a close mimic of the [MoFe3S3C] fragment in FeMo cofactor. Superposition of them and comparison of metric information disclose a clear structural relationship [Tp* = tris(3,5-dimethyl-1-pyrazolyl)hydroborate(1-)].

Asunto(s)

Complejos de Coordinación/química , Molibdeno/química , Molibdoferredoxina/química , Azufre/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Ligandos , Modelos Moleculares , Estructura Molecular , Nitrogenasa/química , Nitrogenasa/metabolismo , Oxidación-Reducción

RESUMEN

Chemically demanding reductive conversions in biology, such as the reduction of dinitrogen to ammonia or the Birch-type reduction of aromatic compounds, depend on Fe/S-cluster-containing ATPases. These reductions are typically catalyzed by two-component systems, in which an Fe/S-cluster-containing ATPase energizes an electron to reduce a metal site on the acceptor protein that drives the reductive reaction. Here, we show a two-component system featuring a double-cubane [Fe8S9]-cluster [{Fe4S4(SCys)3}2(µ2-S)]. The double-cubane-cluster-containing enzyme is capable of reducing small molecules, such as acetylene (C2H2), azide (N3-), and hydrazine (N2H4). We thus present a class of metalloenzymes akin in fold, metal clusters, and reactivity to nitrogenases.

Asunto(s)

Adenosina Trifosfato/metabolismo , Proteínas Hierro-Azufre/metabolismo , Acetileno/metabolismo , Clonación Molecular , Firmicutes/metabolismo , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación Proteica

RESUMEN

Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•-) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.

Asunto(s)

Ácido Peroxinitroso/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Dióxido de Carbono/química , Coenzimas/metabolismo , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Hemo/química , Hemo/metabolismo , Proteínas Hierro-Azufre/metabolismo , Cinética , Peroxidasas/metabolismo , Ácido Peroxinitroso/química , Proteínas/química

RESUMEN

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

Asunto(s)

Proteínas Bacterianas , Proteínas Hierro-Azufre , Streptomyces , Factores de Transcripción , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Motivos de Nucleótidos , Unión Proteica , Homología de Secuencia de Aminoácido , Streptomyces/genética , Streptomyces/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The lack of available transcriptome data for plants of no economic or agronomic importance limits the identification of miRNAs in many species. Considering the possible similarity of the transcriptome between related species, the present study used expressed sequence tags (ESTs) of Suaeda salsa and Suaeda glauca to identify conserved miRNAs, which were validated in a halophyte, Suaeda maritima, with the aim of identifying salt-responsive miRNAs from naturally salt-tolerant plants, information on which is limited. In this study, computational analysis predicted three miRNA sequences by mapping non-redundant miRNA sequences from miRBase 16.0 on 1534 ESTs of S. salsa and S. glauca. The expression of one could be validated in S. maritima, and was named sma-miR1867. This miRNA was downregulated in response to NaCl treatment. It was predicted to target ferredoxin-thioredoxin reductase (FTR), cell division control protein 6 (CDC6), and ubiquitin-protein ligase (UPL) in S. salsa and/or S. glauca. However, only UPL could be amplified in S. maritima, and RT-qPCR showed that it was upregulated in response to NaCl treatment. These results indicate that, in halophytes, FTR and CDC6 may promote carbon metabolism and cell division, respectively, in the presence of salt, while UPL may regulate the abundance of proteins that are important for salt tolerance in halophytes. Thus, sma-miR1867 could be an essential component of salt resistance in halophytes.

Asunto(s)

Chenopodiaceae/genética , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Proteínas de Plantas/genética , ARN de Planta/genética , Transcriptoma , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chenopodiaceae/efectos de los fármacos , Chenopodiaceae/metabolismo , Biología Computacional , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , MicroARNs/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , ARN de Planta/metabolismo , Tolerancia a la Sal/genética , Plantas Tolerantes a la Sal , Alineación de Secuencia , Cloruro de Sodio/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

Frataxin plays a key role in eukaryotic cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (Fe-S) cluster biosynthesis. However, its precise role has yet to be elucidated. In this work, we studied the subcellular localization of Arabidopsis frataxin, AtFH, using confocal microscopy, and found a novel dual localization for this protein. We demonstrate that plant frataxin is targeted to both the mitochondria and the chloroplast, where it may play a role in Fe-S cluster metabolism as suggested by functional studies on nitrite reductase (NIR) and ferredoxin (Fd), two Fe-S containing chloroplast proteins, in AtFH deficient plants. Our results indicate that frataxin deficiency alters the normal functioning of chloroplasts by affecting the levels of Fe, chlorophyll, and the photosynthetic electron transport chain in this organelle.

Asunto(s)

Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/análisis , Cloroplastos/química , Ferredoxinas/genética , Ferredoxinas/metabolismo , Eliminación de Gen , Proteínas de Unión a Hierro/análisis , Proteínas de Unión a Hierro/genética , Microscopía Confocal , Mitocondrias/química , Proteínas Mitocondriales/fisiología , Nitrito Reductasas/genética , Nitrito Reductasas/metabolismo , Plantas Modificadas Genéticamente , Protoplastos/metabolismo , Protoplastos/ultraestructura , ARN Mensajero/genética , ARN de Planta/genética , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Synthesis of the iron-containing prosthetic groups-heme and iron-sulfur clusters-occurs in mitochondria. The mitochondrion is also an important producer of reactive oxygen species (ROS), which are derived from electrons leaking from the electron transport chain. The coexistence of both ROS and iron in the secluded space of the mitochondrion makes this organelle particularly prone to oxidative damage. Here, we review the elements that configure mitochondrial iron homeostasis and discuss the principles of iron-mediated ROS generation in mitochondria. We also review the evidence for mitochondrial dysfunction and iron accumulation in Alzheimer's disease, Huntington Disease, Friedreich's ataxia, and in particular Parkinson's disease. We postulate that a positive feedback loop of mitochondrial dysfunction, iron accumulation, and ROS production accounts for the process of cell death in various neurodegenerative diseases in which these features are present.

Asunto(s)

Homeostasis , Hierro/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Animales , Muerte Celular , Hemo/metabolismo , Humanos , Hierro/toxicidad , Proteínas Hierro-Azufre/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad

RESUMEN

Antimicrobial drug resistance in pathogens is an increasing human health problem. The rapid loss of effectiveness in antibiotics treatments and the accumulation of multi-resistant microbial strains are increasing worldwide threats. Moreover, several infectious diseases have been neglected for years and new antimicrobial treatments are lacking. In other cases, complexity of infectious organisms has exceeded the efforts to find new drugs to control them. Thus, strategies for the proper development of specific drugs are critically needed. Redox metabolism has already been proved to be a useful target for drug development. During the last years a significant number of electron carriers, enzymes, proteins and protein complexes have been studied and some of them were found to be essential for survival of several microbial pathogens. This review will focus on three major redox metabolic pathways which may provide promising strategies to fight against pathogens: the non-mevalonate pathway for isoprenoids biosynthesis, the iron metabolism and the iron-sulfur proteins.The common attractive link of all these processes is the plant-type ferredoxin-NADP+ reductase, an enzyme that participates in numerous electron transfer reactions and has no homologous enzyme in humans. Research in these redox pathways will open new perspectives for the rational design of drugs against infectious diseases.

Asunto(s)

Antiinfecciosos/farmacología , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Redes y Vías Metabólicas/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Animales , Antiinfecciosos/química , Antiinfecciosos/uso terapéutico , Enfermedades Transmisibles/tratamiento farmacológico , Enfermedades Transmisibles/enzimología , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/parasitología , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Oxidación-Reducción , Terpenos/metabolismo

RESUMEN

Nitric oxide (NO(•); nitrogen monoxide) is known to be a critical regulator of cell and tissue function through mechanisms that utilize its unique physicochemical properties as a small and uncharged free radical with limited reactivity. Here, the basic chemistry and biochemistry of NO(•) are summarized through the description of its chemical reactivity, biological sources, physiological and pathophysiological levels, and cellular transport. The complexity of the interactions of NO(•) with biotargets, which vary from irreversible second-order reactions to reversible formation of nonreactive and reactive nitrosyl complexes, is noted. Emphasis is placed on the kinetics and physiological consequences of the reactions of NO(•) with its better characterized biotargets. These targets are soluble guanylate cyclase (sCG), oxyhemoglobin/hemoglobin (HbO(2)/Hb) and cytochrome c oxidase (CcOx), all of which are ferrous heme proteins that react with NO(•) with second-order rate constants approaching the diffusion limit (k(on) approximately 10(7) to 10(8) M(-1) s(-1)). Likewise, the biotarget responsible for the most described pathophysiological actions of NO(•) is the superoxide anion radical (O(2)(•-)), which reacts with NO(•) in a diffusion-controlled process (k approximately 10(10) M(-1) s(-1)). The reactions of NO(•) with proteins containing iron-sulfur clusters ([FeS]) remain little studied and the reported rate constants of the first steps of these reactions are considerable (k approximately 10(5) M(-1) s(-1)). Not surprisingly, the interactions of proteins containing iron-sulfur clusters with NO(•) remain ambiguous and have been associated with both physiological and pathophysiological effects. Overall, it is emphasized that any claimed biological action of NO(•) should be connected with its interaction with kinetically relevant biotargets. Although reactivity toward biotargets is only one of the factors contributing to cellular and tissue responses mediated by short-lived species, such as NO(•) and other oxygen-derived species, it is a critical factor. Therefore, taking reactivity into account is important to advancing our knowledge on redox signaling mechanisms.

Asunto(s)

Óxido Nítrico/química , Óxido Nítrico/metabolismo , Animales , Transporte Biológico , Hemoproteínas/metabolismo , Humanos , Proteínas Hierro-Azufre/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Frataxin is a nuclear-encoded mitochondrial protein highly conserved in prokaryotes and eukaryotes. Its deficiency was initially described as the phenotype of Friedreich's ataxia, an autosomal recessive disease in humans. Although several functions have been described for frataxin, that is, involvement in Fe-S cluster and heme synthesis, energy conversion and oxidative phosphorylation, iron handling and response to oxidative damage, its precise function remains unclear. Although there is a general consensus on the participation of frataxin in the maintenance of cellular iron homeostasis and in iron metabolism, this protein may have other specific functions in different tissues and organisms.

Asunto(s)

Proteínas de Unión a Hierro/fisiología , Secuencia de Aminoácidos , Animales , Hemo/metabolismo , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Frataxina

RESUMEN

Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups that play essential roles in all living organisms. In vivo [Fe-S] cluster biogenesis requires enzymes involved in iron and sulfur mobilization, assembly of clusters, and delivery to their final acceptor. In these systems, a cysteine desulfurase is responsible for the release of sulfide ions, which are incorporated into a scaffold protein for subsequent [Fe-S] cluster assembly. Although three machineries have been shown to be present in Proteobacteria for [Fe-S] cluster biogenesis (NIF, ISC, and SUF), only the SUF machinery has been found in Firmicutes. We have recently described the structural similarities and differences between Enterococcus faecalis and Escherichia coli SufU proteins, which prompted the proposal that SufU is the scaffold protein of the E. faecalis sufCDSUB system. The present work aims at elucidating the biological roles of E. faecalis SufS and SufU proteins in [Fe-S] cluster assembly. We show that SufS has cysteine desulfurase activity and cysteine-365 plays an essential role in catalysis. SufS requires SufU as activator to [4Fe-4S] cluster assembly, as its ortholog, IscU, in which the conserved cysteine-153 acts as a proximal sulfur acceptor for transpersulfurization reaction.

Asunto(s)

Liasas de Carbono-Azufre/metabolismo , Cisteína/metabolismo , Enterococcus faecalis/enzimología , Proteínas Hierro-Azufre/fisiología , Azufre/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/aislamiento & purificación , Clonación Molecular , Cisteína/química , Enterococcus faecalis/química , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Activación Enzimática , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Unión Proteica , Especificidad por Sustrato , Azufre/química

RESUMEN

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.

Asunto(s)

Complejo I de Transporte de Electrón/antagonistas & inhibidores , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/enzimología , Aconitato Hidratasa/metabolismo , Línea Celular Tumoral , Citosol/enzimología , Humanos , Rotenona/farmacología