RESUMO
Metamonads are a diverse group of heterotrophic microbial eukaryotes adapted to living in hypoxic environments. All metamonads but one harbour metabolically altered 'mitochondrion-related organelles' (MROs) with reduced functions, however the degree of reduction varies. Here, we generate high-quality draft genomes, transcriptomes, and predicted proteomes for five recently discovered free-living metamonads. Phylogenomic analyses placed these organisms in a group we name the 'BaSk' (Barthelonids+Skoliomonads) clade, a deeply branching sister group to the Fornicata, a phylum that includes parasitic and free-living flagellates. Bioinformatic analyses of gene models shows that these organisms are predicted to have extremely reduced MRO proteomes in comparison to other free-living metamonads. Loss of the mitochondrial iron-sulfur cluster assembly system in some organisms in this group appears to be linked to the acquisition in their common ancestral lineage of a SUF-like minimal system Fe/S cluster pathway by lateral gene transfer. One of the isolates, Skoliomonas litria, appears to have lost all other known MRO pathways. No proteins were confidently assigned to the predicted MRO proteome of this organism suggesting that the organelle has been lost. The extreme mitochondrial reduction observed within this free-living anaerobic protistan clade demonstrates that mitochondrial functions may be completely lost even in free-living organisms.
Assuntos
Mitocôndrias , Filogenia , Proteoma , Mitocôndrias/metabolismo , Mitocôndrias/genética , Proteoma/metabolismo , Proteoma/genética , Transcriptoma , Eucariotos/genética , Eucariotos/metabolismo , Eucariotos/classificação , Transferência Genética Horizontal , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genéticaRESUMO
ConspectusCentral to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.
Assuntos
Heme , Heme/química , Heme/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Ferro/química , Ferro/metabolismo , Enxofre/química , Células Artificiais/química , Células Artificiais/metabolismoRESUMO
The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl-1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl-1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2.
Assuntos
Hidrogênio , Hidrogenase , Luz , Oxigênio , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Hidrogenase/metabolismo , Hidrogenase/química , Hidrogênio/metabolismo , Oxigênio/metabolismo , Oxigênio/química , Clostridium beijerinckii/metabolismo , Clostridium beijerinckii/genética , Oxirredução , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , FotossínteseRESUMO
Post-transcriptional modification of nucleosides in transfer RNAs (tRNAs) is an important process for accurate and efficient translation of the genetic information during protein synthesis in all domains of life. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A), within tRNAs. Whereas the mechanism that has prevailed for decades involved persulfide chemistry, more and more tRNA thiolation enzymes have now been shown to contain a [4Fe-4S] cluster. This review summarizes the information over the last ten years concerning the biochemical, spectroscopic and structural characterization of [4Fe-4S]-dependent non-redox tRNA thiolation enzymes.
Assuntos
Proteínas Ferro-Enxofre , RNA de Transferência , RNA de Transferência/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/química , Oxirredução , Processamento Pós-Transcricional do RNA , Humanos , Tiouridina/análogos & derivados , Tiouridina/metabolismo , Tiouridina/químicaRESUMO
The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (â¢NO) and superoxide (O2â¢-) produced by phagocytes contributes to its success as a human pathogen. Recombination of â¢NO and O2â¢- generates peroxynitrite (ONOO-), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward â¢NO and O2â¢- is well established, how Mtb responds to ONOO- remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both â¢NO and O2â¢-, which should combine to produce ONOO-. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO- levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO-. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe-4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe-4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe-S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO- in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO- and targeting Fe-S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).
Assuntos
Metabolismo Energético , Homeostase , Proteínas Ferro-Enxofre , Mycobacterium tuberculosis , Oxirredução , Ácido Peroxinitroso , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Humanos , Óxido Nítrico/metabolismo , Estresse Oxidativo , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/efeitos dos fármacos , Superóxidos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Tuberculose/microbiologia , Tuberculose/metabolismoRESUMO
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila ortholog of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.
Parkinson's disease affects millions of people worldwide, causing progressively worse symptoms like stiffness, tremors and difficulty moving. These issues result from the death of neurons in the brain that produce the neurotransmitter dopamine. While most cases have no known cause, 10 to 15 per cent are due to inherited gene mutations. This includes mutations in the genes that code for the proteins PINK1 and Parkin which are essential for maintaining healthy mitochondria, the powerhouse of the cell. Mutations in this quality control system affect a protein called CISD1, which sits within the outer surface of the mitochondria. CISD1 contains a cluster of iron and sulfur ions, and is involved in regulating iron levels and mitochondrial energy production. However, its role in inherited cases of Parkinson's disease, particularly those related to mutations in PINK1 and Parkin, is poorly understood. To understand the impact of CISD1, Bitar et al. studied genetically modified fruit flies and dopamine-producing neurons from Parkinson's patients with PINK1 mutations. This revealed that losing PINK1 activity led to higher levels of CISD1 proteins which lacked the iron-sulfur cluster due to a bond forming between two CISD1 molecules. Reducing levels of the CISD1-equivalent protein in the flies helped to alleviate most of the symptoms caused by PINK1 and Parkin gene mutations, such as difficulties climbing and impaired wing posture. These findings suggest that iron-depleted CISD1 contributes to the symptoms associated with Parkinson's disease, underscoring its potential as a drug target. Drugs that target CISD1 already exist, which could ease the way for further research. Recent studies have shown that cases of Parkinson's related to mutations in PINK-1 share features with some non-inherited instances of the disease, suggesting that this approach could potentially benefit many patients.
Assuntos
Proteínas de Drosophila , Proteínas Ferro-Enxofre , Mitocôndrias , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Humanos , Mitocôndrias/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fenótipo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mutação com Perda de Função , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
To the best of our knowledge, enzymes that catalyse intramolecular Diels-Alder ([4+2] cycloaddition) reactions are frequently reported in natural product biosynthesis; however, no native enzymes utilising Lewis acid catalysis have been reported. Verticilactam is a representative member of polycyclic macrolactams, presumably produced by spontaneous cycloaddition. We report that the intramolecular [4+2] cycloadditions can be significantly accelerated by ferredoxins (Fds), a class of small iron-sulphur (Fe-S) proteins. Through iron atom substitution by Lewis acidic gallium (Ga) iron and computational calculations, we confirm that the ubiquitous Fe-S cluster efficiently functions as Lewis acid to accelerate the tandem [4+2] cycloaddition and Michael addition reactions by lowering free energy barriers. Our work highlights Nature's ingenious strategy to generate complex molecule structures using the ubiquitous Fe-S protein. Furthermore, our study sheds light on the future design of Fd as a versatile Lewis acid catalyst for [4+2] cycloaddition reactions.
Assuntos
Produtos Biológicos , Reação de Cicloadição , Proteínas Ferro-Enxofre , Ácidos de Lewis , Produtos Biológicos/metabolismo , Produtos Biológicos/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Ácidos de Lewis/química , Ácidos de Lewis/metabolismo , Catálise , Ferro/química , Ferro/metabolismo , Lactamas/metabolismo , Lactamas/química , BiocatáliseRESUMO
Iron-sulfur (Fe-S) clusters are essential redox-active metallocofactors participating in electron transfer, radical chemistry, primary metabolism, and gene regulation. Successful trafficking and incorporation of Fe-S clusters into target proteins are critical to proper cellular function. While biophysical studies of isolated Fe-S proteins provide insight into the structure and function of these inorganic cofactors, few strategies currently exist to directly interrogate Fe-S cluster binding within a cellular environment. Here, we describe a chemoproteomic platform to report on Fe-S cluster incorporation and occupancy directly within a native proteome, enabling the characterization of Fe-S biogenesis pathways and the identification of undiscovered Fe-S proteins.
Assuntos
Proteínas Ferro-Enxofre , Proteômica , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteômica/métodos , Ligação Proteica , Proteoma , Ferro/metabolismo , Enxofre/metabolismo , OxirreduçãoRESUMO
[FeFe]-hydrogenase is nature's most efficient proton reducing and H2-oxidizing enzyme. However, biotechnological applications are hampered by the O2 sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe2(adt)(CO)6-x(CN)x]x- and [Fe2(pdt)(CO)6-x(CN)x]x- (x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe2(adt)(CO)6. Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O2 and ROS like superoxide and H2O2 as the oxygen sensitivity increases with the exchange of ligands from CO to CN-. The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O2 is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.
Assuntos
Domínio Catalítico , Hidrogenase , Proteínas Ferro-Enxofre , Oxigênio , Hidrogenase/química , Hidrogenase/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Teoria da Densidade FuncionalRESUMO
In the current study, we aimed to investigate whether disulfiram (DSF) exerts a neuroprotective role in cerebral ischemiareperfusion (CI-RI) injury by modulating ferredoxin 1 (FDX1) to regulate copper ion (Cu) levels and inhibiting inflammatory responses. To simulate CI-RI, a transient middle cerebral artery occlusion (tMCAO) model in C57/BL6 mice was employed. Mice were administered with or without DSF before and after tMCAO. Changes in infarct volume after tMCAO were observed using TTC staining. Nissl staining and hematoxylin-eosin (he) staining were used to observe the morphological changes of nerve cells at the microscopic level. The inhibitory effect of DSF on initial inflammation was verified by TUNEL assay, apoptosis-related protein detection and iron concentration detection. FDX1 is the main regulatory protein of copper death, and the occurrence of copper death will lead to the increase of HSP70 stress and inflammatory response. Cuproptosis-related proteins and downstream inflammatory factors were detected by western blotting, immunofluorescence staining, and immunohistochemistry. The content of copper ions was detected using a specific kit, while electron microscopy was employed to examine mitochondrial changes. We found that DSF reduced the cerebral infarction volume, regulated the expression of cuproptosis-related proteins, and modulated copper content through down regulation of FDX1 expression. Moreover, DSF inhibited the HSP70/TLR-4/NLRP3 signaling pathway. Collectively, DSF could regulate Cu homeostasis by inhibiting FDX1, acting on the HSP70/TLR4/NLRP3 pathway to alleviate CI/RI. Accordingly, DSF could mitigate inflammatory responses and safeguard mitochondrial integrity, yielding novel therapeutic targets and mechanisms for the clinical management of ischemia-reperfusion injury.
Assuntos
Cobre , Dissulfiram , Homeostase , Inflamação , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão , Animais , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Dissulfiram/farmacologia , Camundongos , Cobre/metabolismo , Homeostase/efeitos dos fármacos , Masculino , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Regulação para Baixo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Modelos Animais de Doenças , Proteínas Ferro-Enxofre/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Apoptose/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptor 4 Toll-Like/metabolismoRESUMO
BACKGROUND: Glutaric aciduria type II (GA2) is a rare genetic disorder inherited in an autosomal recessive manner. Double dosage mutations in GA2 corresponding genes, ETFDH, ETFA, and ETFB, lead to defects in the catabolism of fatty acids, and amino acids lead to broad-spectrum phenotypes, including muscle weakness, developmental delay, and seizures. product of these three genes have crucial role in transferring electrons to the electron transport chain (ETC), but are not directly involve in ETC complexes. METHODS: Here, by using exome sequencing, the cause of periodic cryptic gastrointestinal complications in a 19-year-old girl was resolved after years of diagnostic odyssey. Protein modeling for the novel variant served as another line of validation for it. RESULTS: Exome Sequencing (ES) identified two variants in ETFDH: ETFDH:c.926T>G and ETFDH:c.1141G>C. These variants are likely contributing to the crisis in this case. To the best of our knowledge at the time of writing this manuscript, variant ETFDH:c.926T>G is reported here for the first time. Clinical manifestations of the case and pathological analysis are in consistent with molecular findings. Protein modeling provided another line of evidence proving the pathogenicity of the novel variant. ETFDH:c.926T>G is reported here for the first time in relation to the causation GA2. CONCLUSION: Given the milder symptoms in this case, a review of GA2 cases caused by compound heterozygous mutations was conducted, highlighting the range of symptoms observed in these patients, from mild fatigue to more severe outcomes. The results underscore the importance of comprehensive genetic analysis in elucidating the spectrum of clinical presentations in GA2 and guiding personalized treatment strategies.
Assuntos
Flavoproteínas Transferidoras de Elétrons , Heterozigoto , Proteínas Ferro-Enxofre , Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Humanos , Feminino , Flavoproteínas Transferidoras de Elétrons/genética , Proteínas Ferro-Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Adulto Jovem , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/patologia , Encefalopatias Metabólicas/diagnóstico , Mutação , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Deficiência Múltipla de Acil Coenzima A Desidrogenase/patologiaRESUMO
Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.
Assuntos
Proteínas Ferro-Enxofre , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Masculino , Feminino , Doenças Neuromusculares/genética , Doenças Neuromusculares/enzimologia , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Criança , Núcleo Celular/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Citoplasma/metabolismo , Citoplasma/enzimologia , MetalochaperonasRESUMO
The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.
Assuntos
Azotobacter vinelandii , Proteínas de Bactérias , Modelos Moleculares , Azotobacter vinelandii/química , Cristalografia por Raios X , Proteínas de Bactérias/química , Conformação Proteica , Domínios Proteicos , Ferredoxinas/química , Multimerização Proteica , Proteínas Ferro-Enxofre/químicaRESUMO
In an effort to develop the biomimetic chemistry of [FeFe]hydrogenases for catalytic hydrogen evolution reaction (HER) in aqueous environment, we herein report the integrations of diiron dithiolate complexes into carbon nanotubes (CNTs) through three different strategies and compare the electrochemical HER performances of the as-resulted 2Fe2S/CNT hybrids in neutral aqueous medium. That is, three new diiron dithiolate complexes [{(µ-SCH2)2N(C6H4CH2C(O)R)}Fe2(CO)6] (R = N-oxylphthalimide (1), NHCH2pyrene (2), and NHCH2Ph (3)) were prepared and could be further grafted covalently to CNTs via an amide bond (this 2Fe2S/CNT hybrid is labeled as H1) as well as immobilized noncovalently to CNTs via π-π stacking interaction (H2) or via simple physisorption (H3). Meanwhile, the molecular structures of 1-3 are determined by elemental analysis and spectroscopic as well as crystallographic techniques, whereas the structures and morphologies of H1-H3 are characterized by various spectroscopies and scanning electronic microscopy. Further, the electrocatalytic HER activity trend of H1 > H2 ≈ H3 is observed in 0.1 M phosphate buffer solution (pH = 7) through different electrochemical measurements, whereas the degradation processes of H1-H3 lead to their electrocatalytic deactivation in the long-term electrolysis as proposed by post operando analysis. Thus, this work is significant to extend the potential application of carbon electrode materials engineered with diiron molecular complexes as heterogeneous HER electrocatalysts for water splitting to hydrogen.
Assuntos
Hidrogênio , Hidrogenase , Proteínas Ferro-Enxofre , Nanotubos de Carbono , Nanotubos de Carbono/química , Hidrogenase/química , Hidrogenase/metabolismo , Hidrogênio/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Catálise , Água/química , Complexos de Coordenação/químicaRESUMO
Maintaining the integrity of the genome is fundamental to living organisms. To this end, nature developed several mechanisms to find and promptly repair DNA lesions. Among them, base excision repair (BER) enzymes evolved to efficiently carry out this task. Notably, the mechanisms allowing these proteins to search for, detect, and fix DNA damage on a biologically relevant time scale still remain partially unclear. By taking MutY, a BER enzyme implied in the repair of the 8-oxoguanine-adenine mismatches, as a model system, we shed some light on the repair mechanism through a theoretical-computational approach. First, we estimated the effect of the oxidation state of the MutY iron-sulfur cluster on the protein-DNA binding. Then, the redox thermodynamics of both the protein cluster and DNA nucleobases are calculated. Finally, the charge migration kinetics along the double strand bound to the enzyme has been evaluated. The rationalization of our results indicates that the search for DNA lesions is essentially dictated by the redox chemistry of the species involved, i.e., the iron-sulfur redox cofactor and the DNA bound to the enzyme.
Assuntos
DNA Glicosilases , Reparo do DNA , Oxirredução , DNA Glicosilases/metabolismo , DNA Glicosilases/química , Reparo do DNA/fisiologia , DNA/metabolismo , DNA/química , Cinética , Dano ao DNA , Termodinâmica , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genéticaRESUMO
Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5â Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42â Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48â h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for â¼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to â¼1.5â Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.
Assuntos
Microscopia Crioeletrônica , Microscopia Crioeletrônica/métodos , Modelos Moleculares , Conformação Proteica , Bactérias/enzimologia , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Complexos MultienzimáticosRESUMO
About 50 proteins expressed in plastids of photosynthetic eukaryotes ligate ironsulfur (Fe-S) clusters and ensure vital functions in photosynthesis, sulfur and nitrogen assimilation, but also in the synthesis of pigments, vitamins and hormones. The synthesis of these Fe-S clusters, which are co- or post-translationally incorporated into these proteins, relies on several proteins belonging to the so-called sulfur mobilization (SUF) machinery. An Fe-S cluster is first de novo synthesized on a scaffold protein complex before additional late-acting maturation factors act in the specific transfer, possible conversion and insertion of this cluster into target recipient proteins. In this review, we will summarize what is known about the molecular mechanisms responsible for both the synthesis and transfer steps, focusing in particular on the structural aspects that allow the formation of the required protein complexes.
Assuntos
Proteínas Ferro-Enxofre , Plastídeos , Enxofre , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/química , Plastídeos/metabolismo , Plastídeos/genética , Enxofre/metabolismo , Fotossíntese , Ferro/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genéticaRESUMO
FANCJ/BRIP1, initially identified as DOG-1 (Deletions Of G-rich DNA) in Caenorhabditis elegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe-4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron-sulfur (Fe-S) cluster in DOG-1 and its relationship with the cytosolic iron-sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe-S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1's stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe-S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC's role in Fe-S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Dano ao DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi , Proteínas Ferro-Enxofre , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Citoplasma/metabolismo , Reparo do DNA , Mutação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA HelicasesRESUMO
OBJECTIVE: To explore the clinical characteristics and genetic variants in three children with late-onset Multiple acyl-Coenzyme A dehydrogenase deficiency (MADD type â ¢). METHODS: Clinical data of three children diagnosed with late-onset MADD at the Children's Hospital Affiliated to Zhengzhou University between March 2020 and March 2022 were retrospectively analyzed. All children were subjected to whole exome sequencing (WES), and candidate variants were verified by Sanger sequencing. All children had received improved metabolic therapy and followed up for 1 ~ 3 years. RESULTS: The children had included 2 males and 1 female, and aged from 2 months to 11 years and 7 months. Child 1 had intermittent vomiting, child 2 had weakness in lower limbs, while child 3 had no symptom except abnormal neonatal screening. Tandem mass spectrometry of the three children showed elevation of multiple acylcarnitines with short, medium and long chains. Children 1 and 2 showed increased glutaric acid and multiple dicarboxylic acids by urine Gas chromatography-mass spectrometry (GC-MS) analysis. All children were found to harbor compound heterozygous variants of the ETFDH gene, including a paternal c.1211T>C (p.M404T) and a maternal c.488-22T>G variant in child 1, a paternal c.1717C>T (p.Q573X) and a maternal c.250G>A (p.A84T) variant in child 2, and a paternal c.1285+1G>A and maternal c.629A>G (p.S210N) variant in child 3. As for the treatment, high-dose vitamin B2, levocarnitine and coenzyme Q10 were given to improve the metabolism, in addition with a low fat, hypoproteinic and high carbohydrate diet. All children showed a stable condition with normal growth and development during the follow-up. CONCLUSION: The compound heterozygous variants of the ETFDH gene probably underlay the muscle weakness, remittent vomiting, elevated short, medium, and long chain acylcarnitine, as well as elevated glutaric acid and various dicarboxylic acids in the three children with type â ¢ MADD.
Assuntos
Flavoproteínas Transferidoras de Elétrons , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Humanos , Masculino , Feminino , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Lactente , Criança , Pré-Escolar , Flavoproteínas Transferidoras de Elétrons/genética , Mutação , Estudos Retrospectivos , Carnitina/análogos & derivados , Carnitina/sangue , Proteínas Ferro-Enxofre/genética , Sequenciamento do Exoma , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Variação GenéticaRESUMO
The methylerythritol phosphate (MEP) pathway is responsible for biosynthesis of the precursors of isoprenoid compounds in eubacteria and plastids. It is a metabolic alternative to the well-known mevalonate pathway for isoprenoid production found in archaea and eukaryotes. Recently, a role for the MEP pathway in oxidative stress detection, signalling, and response has been identified. This role is executed in part through the unusual cyclic intermediate, methylerythritol cyclodiphosphate (MEcDP). We postulate that this response is triggered through the oxygen sensitivity of the MEP pathway's terminal iron-sulfur (Fe-S) cluster enzymes. MEcDP is the substrate of IspG, the first Fe-S cluster enzyme in the pathway; it accumulates under oxidative stress conditions and acts as a signalling molecule. It may also act as an antioxidant. Furthermore, evidence is emerging for a broader and highly nuanced role of the MEP pathway in oxidative stress responses, implemented through a complex system of differential regulation and sensitivity at numerous nodes in the pathway. Here, we explore the evidence for such a role (including the contribution of the Fe-S cluster enzymes and different pathway metabolites, especially MEcDP), the evolutionary implications, and the many questions remaining about the behaviour of the MEP pathway in the presence of oxidative stress.