The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli.

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E. coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E. coli Fur also binds a [2Fe-2S] cluster in wildtype E. coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box in vitro, and are inactive to complement the function of Fur in vivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E. coli cells.

Ferrodifferentiation regulates neurodevelopment via ROS generation.

Iron is important for life, and iron deficiency impairs development, but whether the iron level regulates neural differentiation remains elusive. In this study, with iron-regulatory proteins (IRPs) knockout embryonic stem cells (ESCs) that showed severe iron deficiency, we found that the Pax6- and Sox2-positive neuronal precursor cells and Tuj1 fibers in IRP1-/-IRP2-/- ESCs were significantly decreased after inducing neural differentiation. Consistently, in vivo study showed that the knockdown of IRP1 in IRP2-/- fetal mice remarkably affected the differentiation of neuronal precursors and the migration of neurons. These findings suggest that low intracellular iron status significantly inhibits neurodifferentiation. When supplementing IRP1-/-IRP2-/- ESCs with iron, these ESCs could differentiate normally. Further investigations revealed that the underlying mechanism was associated with an increase in reactive oxygen species (ROS) production caused by the substantially low level of iron and the down-regulation of iron-sulfur cluster protein ISCU, which, in turn, affected the proliferation and differentiation of stem cells. Thus, the appropriate amount of iron is crucial for maintaining normal neural differentiation that is termed ferrodifferentiation.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth.

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

ETF dehydrogenase advances in molecular genetics and impact on treatment.

Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.

Staphylococcus aureus lacking a functional MntABC manganese import system has increased resistance to copper.

S. aureus USA300 isolates utilize the copBL and copAZ gene products to prevent Cu intoxication. We created and examined a ΔcopAZ ΔcopBL mutant strain (cop-). The cop- strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in the cop- background and isolated strains with Tn insertions in the mntABC operon that permitted growth in the presence of Cu. The mutations were in mntA and they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing a mntA::Tn accumulated less Cu than the parent strain. Mn(II) supplementation improved growth when cop- was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor of mntABC transcription. A ΔmntR strain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction of mntA::Tn. Over-expression of mntABC increased cellular Cu load and sensitivity to Cu. The presence of a mntA::Tn mutation protected iron-sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.

Methane, arsenic, selenium and the origins of the DMSO reductase family.

Mononuclear molybdoenzymes of the dimethyl sulfoxide reductase (DMSOR) family catalyze a number of reactions essential to the carbon, nitrogen, sulfur, arsenic, and selenium biogeochemical cycles. These enzymes are also ancient, with many lineages likely predating the divergence of the last universal common ancestor into the Bacteria and Archaea domains. We have constructed rooted phylogenies for over 1,550 representatives of the DMSOR family using maximum likelihood methods to investigate the evolution of the arsenic biogeochemical cycle. The phylogenetic analysis provides compelling evidence that formylmethanofuran dehydrogenase B subunits, which catalyze the reduction of CO2 to formate during hydrogenotrophic methanogenesis, constitutes the most ancient lineage. Our analysis also provides robust support for selenocysteine as the ancestral ligand for the Mo/W atom. Finally, we demonstrate that anaerobic arsenite oxidase and respiratory arsenate reductase catalytic subunits represent a more ancient lineage of DMSORs compared to aerobic arsenite oxidase catalytic subunits, which evolved from the assimilatory nitrate reductase lineage. This provides substantial support for an active arsenic biogeochemical cycle on the anoxic Archean Earth. Our work emphasizes that the use of chalcophilic elements as substrates as well as the Mo/W ligand in DMSORs has indelibly shaped the diversification of these enzymes through deep time.

A NAC-type transcription factor confers aluminium resistance by regulating cell wall-associated receptor kinase 1 and cell wall pectin.

Transcriptional regulation is important for plants to respond to toxic effects of aluminium (Al). However, our current knowledge to these events is confined to a few transcription factors. Here, we functionally characterized a rice bean (Vigna umbellata) NAC-type transcription factor, VuNAR1, in terms of Al stress response. We demonstrated that rice bean VuNAR1 is a nuclear-localized transcriptional activator, whose expression was specifically upregulated by Al in roots but not in shoot. VuNAR1 overexpressing Arabidopsis plants exhibit improved Al resistance via Al exclusion. However, VuNAR1-mediated Al exclusion is independent of the function of known Al-resistant genes. Comparative transcriptomic analysis revealed that VuNAR1 specifically regulates the expression of genes associated with protein phosphorylation and cell wall modification in Arabidopsis. Transient expression assay demonstrated the direct transcriptional activation of cell wall-associated receptor kinase 1 (WAK1) by VuNAR1. Moreover, yeast one-hybrid assays and MEME motif searches identified a new VuNAR1-specific binding motif in the promoter of WAK1. Compared with wild-type Arabidopsis plants, VuNAR1 overexpressing plants have higher WAK1 expression and less pectin content. Taken together, our results suggest that VuNAR1 regulates Al resistance by regulating cell wall pectin metabolism via directly binding to the promoter of WAK1 and induce its expression.

Adolescent Hyperuricemia with Lipid Storage Myopathy: A Clinical Study.

BACKGROUND In this study, we investigated the clinical and pathological features of patients with lipid storage myopathy (LSM) complicated with hyperuricemia, to improve clinicians' understanding of metabolic multi-muscular disorder with metabolic disorders, and to reduce the risk of missed diagnosis of LSM. MATERIAL AND METHODS From January 2005 to December 2017, 8 patients underwent muscle biopsy and diagnosed by muscle pathology and genetic testing in our hospital. All 8 patients were in compliance with LSM diagnosis. We collected data on the patient's clinical performance, adjuvant examination, treatment, and outcomes to provide a comprehensive report and description of LSM patients with hyperuricemia. RESULTS All patients were diagnosed as having ETFDH gene mutations. The main clinical manifestations of patients were chronic limb and trunk weakness, limb numbness, and muscle pain. The serum creatine kinase (CK) values in all patients were higher than normal values. Electromyography showed 3 cases of simple myogenic damage and 3 cases of neurogenic injury. Hematuria metabolic screening showed that 2 patients had elevated glutaric aciduria, and 1 patient had elevated fatty acyl carnitine in the blood. All patients were given riboflavin treatment, and the clinical symptoms were significantly improved, and 3 patients returned to normal uric acid levels after treatment. Pathological staining showed an abnormal deposition of lipid droplets in muscle fibers. CONCLUSIONS If an adolescent hyperuricemia patient has abnormal limb weakness, exercise intolerance, and elevated serum CK values, clinicians need to be highly alert to the possibility of LSM. Early diagnosis and treatment of LSM should improve the clinical symptoms and quality of life and reduce complications.

Improved subtyping affords better discrimination of Trichomonas gallinae strains and suggests hybrid lineages.

Trichomonas gallinae is a protozoan pathogen that causes avian trichomonosis typically associated with columbids (canker) and birds of prey (frounce) that predate on them, and has recently emerged as an important cause of passerine disease. An archived panel of DNA from North American (USA) birds used initially to establish the ITS ribotypes was reanalysed using Iron hydrogenase (FeHyd) gene sequences to provide an alphanumeric subtyping scheme with improved resolution for strain discrimination. Thirteen novel subtypes of T. gallinae using FeHyd gene as the subtyping locus are described. Although the phylogenetic topologies derived from each single marker are complementary, they are not entirely congruent. This may reflect the complex genetic histories of the isolates analysed which appear to contain two major lineages and several that are hybrid. This new analysis consolidates much of the phylogenetic signal generated from the ITS ribotype and provides additional resolution for discrimination of T. gallinae strains. The single copy FeHyd gene provides higher resolution genotyping than ITS ribotype alone. It should be used where possible as an additional, single-marker subtyping tool for cultured isolates.

Rhodiola crenulata attenuates apoptosis and mitochondrial energy metabolism disorder in rats with hypobaric hypoxia-induced brain injury by regulating the HIF-1α/microRNA 210/ISCU1/2(COX10) signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata, a traditional Tibetan medicine, has shown promise in the treatment of hypobaric hypoxia (HH)-induced brain injury. However, the underlying mechanisms remain unclear. This study investigated the protective effects of R. crenulata aqueous extract (RCAE) on HH-induced brain injury in rats. MATERIALS AND METHODS: An animal model of high-altitude hypoxic brain injury was established in SD rats using an animal decompression chamber for 24 h. Serum and hippocampus levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and lactate dehydrogenase (LDH) were then determined using commercial biochemical kits. Neuron morphology and vitality were also evaluated using H&E and Nissl staining, and TUNEL staining was used to examine apoptosis. Gene and protein expression of HIF-1α, microRNA 210, ISCU1/2, COX10, Apaf-1, cleaved Caspase-3, Caspase-3, Bax, Bcl-2, and Cyto-c were determined by western blot, immunohistochemical and qRT-PCR analysis. RESULTS: RCAE administration attenuated HH-induced brain injury as evidenced by decreased levels of MDA, LDH, and GSSG, increased GSH and SOD, improvements in hippocampus histopathological changes, increased cell vitality and ATP level, and reduced apoptotic cell numbers. RCAE treatment also enhanced HIF-1α, ISCU1/2, COX10, and Bcl-2 protein expression, while dramatically inhibiting expression of Apaf-1, Bax, Cyto-c, and cleaved Caspase-3. Treatment also increased gene levels of HIF-1α, microRNA 210, ISCU1/2, and COX10, and decreased Caspase-3 gene production. CONCLUSIONS: RCAE attenuated HH-induced brain injury by regulating apoptosis and mitochondrial energy metabolism via the HIF-1α/microRNA 210/ISCU1/2 (COX10) signaling pathway.

BOLA (BolA Family Member 3) Deficiency Controls Endothelial Metabolism and Glycine Homeostasis in Pulmonary Hypertension.

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

Characterization of two ETFDH mutations in a novel case of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

BACKGROUND: Deficiency of electron transfer flavoprotein dehydrogenase (ETFDH) is associated with multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder is an autosomal recessive lipid storage myopathy (LSM) that exhibits a wide range of clinical features, including myopathy, weakness and multisystem dysfunctions. Many patients with late onset of MADD improve when treated with riboflavin and are also referred to as RR-MADD (riboflavin-responsive multiple Acyl-CoA dehydrogenase disorder). METHODS: In this study, we report the clinical and genetic characterization of a novel RR-MADD patient. Biochemical data were obtained from analysis of muscle and plasma samples. DNA and RNA were extracted from peripheral blood, and sequence analysis and expression study of ETFDH gene were performed. Finally, the impact of mutations on ETFDH folding was evaluated using bioinformatic tools. RESULTS: Patient initially presented with vomiting, muscle weakness, and acidosis. Muscle biopsy revealed typical myopathological patterns of lipid storage myopathy and blood acylcarnitine profiles showed a combined elevation of long and medium chain acylcarnitines, supporting the diagnosis of RR-MADD. Molecular analysis of ETFDH gene revealed two heterozygous mutations, a novel splice variation in intron 10, c.1285 + 1G > A, and the previously reported c.560C > T missense mutation. RT-PCR analysis showed an alteration of ETFDH RNA splicing which in turn should lead to the production of a truncated protein. The in silico prediction analysis of ETFDH tridimensional structure demonstrated that the missense mutation resulted in instability and loss of protein activation, while the splice site variation induced a dramatic conformational change of the truncated protein. After MCT diet supplemented with carnitine and riboflavin, the patient showed significant biochemical and clinical improvement, in spite of severe molecular defect. CONCLUSION: This case report extends the spectrum of ETFDH mutations in MADD, providing further evidence that patients presenting at least one missense mutation in the FAD-binding domain may respond to either carnitine or riboflavin treatment, due to the recovery of some enzymatic activity.

Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site.

Mobilization of iron from bacterioferritin (BfrB) requires specific interactions with a [2Fe-2S] ferredoxin (Bfd). Blocking the BfrB:Bfd interaction results in irreversible iron accumulation in BfrB and iron deficiency in the cytosol [Eshelman, K., et al. (2017) Metallomics 9, 646-659]. The only known Bfd structure, which was obtained in complex with BfrB (Protein Data Bank entry 4E6K ), indicated a new fold and suggested that the stability of Bfd is aided by an anion binding site consisting of R26, R29, and K46. We investigated the Bfd fold using site-directed mutagenesis, X-ray crystallography, and biochemistry in solution. The X-ray structure, which is nearly identical to that of Bfd in the BfrB:Bfd complex, shows that the [2Fe-2S] cluster preorganizes residues at the BfrB:Bfd interface into a structure complementary to the Bfd binding site on BfrB. Studies in solution showed rapid loss of the [2Fe-2S] cluster at a low ionic strength but higher stability with an increasing ionic strength, thus supporting a structural anion binding site. Structures of the R26E and R26E/K46Y mutants are nearly identical to that of Bfd, except for a new network of hydrogen bonds stabilizing the region encompassing the former anion binding site. The stability of the R26E and R26E/K46Y mutants, which is weakly and completely independent of solution ionic strength, respectively, corroborates that Bfd requires an anion binding site. The mutations, which caused only small changes to the strength of the BfrB:Bfd interaction and mobilization of iron from BfrB, indicate that the anion binding site in Bfd serves primarily a structural role.

The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O2 and dimer stability. Around physiological cytosolic pH, the ability of mNT to transfer its cluster is tightly regulated by the pH. Finally, mNT is extremely resistant to H2O2 compared to ISCU and SufB, two other Fe-S cluster transfer proteins, which is consistent with its involvement in a repair pathway of stress-damaged Fe-S proteins. Taken together, our results suggest that the ability of mNT to transfer its cluster to recipient proteins is not only controlled by the redox state of its cluster but also tightly modulated by the pH of the cytosol. We propose that when pathophysiological conditions such as cancer and neurodegenerative diseases dysregulate cellular pH homeostasis, this pH-dependent regulation of mNT is lost, as is the regulation of cellular pathways under the control of mNT.

Coenzyme Q10 serves to couple mitochondrial oxidative phosphorylation and fatty acid ß-oxidation, and attenuates NLRP3 inflammasome activation.

Multiple acyl-CoA dehydrogenase deficiency (MADD), an autosomal recessive metabolic disorder of fatty acid metabolism, is mostly caused by mutations in the ETFA, ETFB or ETFDH genes that result in dysfunctions in electron transfer flavoprotein (ETF) or electron transfer flavoprotein-ubiquinone dehydrogenase (ETFDH). In ß-oxidation, fatty acids are processed to generate acyl-CoA, which is oxidised by flavin adenine dinucleotide and transfers an electron to ETF and, through ETFDH, to mitochondrial respiratory complex III to trigger ATP synthesis. Coenzyme Q10 (CoQ10) is believed to be a potential treatment that produces symptom relief in some MADD patients. CoQ10 acts as a key regulator linking ETFDH and mitochondrial respiratory complex III. Our aim is to investigate the effectiveness of CoQ10 in serving in the ETF/ETFDH system to improve mitochondrial function and to reduce lipotoxicity. In this study, we used lymphoblastoid cells with an ETFDH mutation from MADD patients. ETFDH dysfunction caused insufficient ß-oxidation, leading to increasing lipid droplet and lipid peroxide accumulation. In contrast, supplementation with CoQ10 significantly recovered mitochondrial function and concurrently decreased the generation of reactive oxygen species and lipid peroxides, inhibited the accumulation of lipid droplets and the formation of the NOD-like receptor family pyrin domain-containing three (NLRP3) inflammasome, and reduced interleukin-1ß release and cell death. These results clarify the causal role of CoQ10 in coupling the electron transport chain with ß-oxidation, which may promote the development of CoQ10-directed therapies for MADD patients.

Involvement of Arabidopsis glutaredoxin S14 in the maintenance of chlorophyll content.

Plant class-II glutaredoxins (GRXs) are oxidoreductases carrying a CGFS active site signature and are able to bind iron-sulfur clusters in vitro. In order to explore the physiological functions of the 2 plastidial class-II isoforms, GRXS14 and GRXS16, we generated knockdown and overexpression Arabidopsis thaliana lines and characterized their phenotypes using physiological and biochemical approaches. Plants deficient in one GRX did not display any growth defect, whereas the growth of plants lacking both was slowed. Plants overexpressing GRXS14 exhibited reduced chlorophyll content in control, high-light, and high-salt conditions. However, when exposed to prolonged darkness, plants lacking GRXS14 showed accelerated chlorophyll loss compared to wild-type and overexpression lines. We observed that the GRXS14 abundance and the proportion of reduced form were modified in wild type upon darkness and high salt. The dark treatment also resulted in decreased abundance of proteins involved in the maturation of iron-sulfur proteins. We propose that the phenotype of GRXS14-modified lines results from its participation in the control of chlorophyll content in relation with light and osmotic conditions, possibly through a dual action in regulating the redox status of biosynthetic enzymes and contributing to the biogenesis of iron-sulfur clusters, which are essential cofactors in chlorophyll metabolism.