The Structure of the Dimeric State of IscU Harboring Two Adjacent [2Fe-2S] Clusters Provides Mechanistic Insights into Cluster Conversion to [4Fe-4S].

IscU serves as a scaffold for the de novo assembly of a [2Fe-2S] cluster prior to its delivery to recipient protein. It has also been proposed that on one dimer of bacterial IscU, two [2Fe-2S] clusters can be converted into a single [4Fe-4S] cluster. However, lack of structural information about the dimeric state of IscU has hindered our understanding of the underlying mechanisms. In this study, we determine the X-ray crystal structure of IscU from the thermophilic archaeon Methanothrix thermoacetophila and demonstrate a dimer structure of IscU in which two [2Fe-2S] clusters are facing each other in close proximity at the dimer interface. Our structure also reveals for the first time that Asp40 serves as a fourth ligand to the [2Fe-2S] cluster with three Cys ligands in each monomer, consistent with previous spectroscopic data. We confirm by EPR spectroscopic analysis that in solution two adjacent [2Fe-2S] clusters in the wild-type dimer are converted to a [4Fe-4S] cluster via reductive coupling. Furthermore, we find that the H106A substitution abolishes the reductive conversion to the [4Fe-4S] cluster without structural alteration, suggesting that His106 is functionally involved in this process. Overall, these findings provide a structural explanation for the assembly and conversion of Fe-S clusters on IscU and highlight a dynamic process that advances via association and dissociation of the IscU dimer.

Honoring Bacon Ke at 100: a legend among the many luminaries and a highly collaborative scientist in photosynthesis research.

Bacon Ke, who did pioneering research on the primary photochemistry of photosynthesis, was born in China on July 26, 1920, and currently, he is living in a senior home in San Francisco, California, and is a centenarian. To us, this is a very happy and unique occasion to honor him. After providing a brief account of his life, and a glimpse of his research in photosynthesis, we present here "messages" for Bacon Ke@ 100 from: Robert Alfano (USA), Charles Arntzen (USA), Sandor Demeter (Hungary), Richard A. Dilley (USA), John Golbeck (USA), Isamu Ikegami (Japan), Ting-Yun Kuang (China), Richard Malkin (USA), Hualing Mi (China), Teruo Ogawa (Japan), Yasusi Yamamoto (Japan), and Xin-Guang Zhu (China).

Making iron-sulfur cluster: structure, regulation and evolution of the bacterial ISC system.

Iron sulfur (Fe-S) clusters rank among the most ancient and conserved prosthetic groups. Fe-S clusters containing proteins are present in most, if not all, organisms. Fe-S clusters containing proteins are involved in a wide range of cellular processes, from gene regulation to central metabolism, via gene expression, RNA modification or bioenergetics. Fe-S clusters are built by biogenesis machineries conserved throughout both prokaryotes and eukaryotes. We focus mostly on bacterial ISC machinery, but not exclusively, as we refer to eukaryotic ISC system when it brings significant complementary information. Besides covering the structural and regulatory aspects of Fe-S biogenesis, this review aims to highlight Fe-S biogenesis facets remaining matters of discussion, such as the role of frataxin, or the link between fatty acid metabolism and Fe-S homeostasis. Last, we discuss recent advances on strategies used by different species to make and use Fe-S clusters in changing redox environmental conditions.

Magnetic Noise Enabled Biocompass.

The discovery of magnetic protein provides a new understanding of a biocompass at the molecular level. However, the mechanism by which magnetic protein enables a biocompass is still under debate, mainly because of the absence of permanent magnetism in the magnetic protein at room temperature. Here, based on a widely accepted radical pair model of a biocompass, we propose a microscopic mechanism that allows the biocompass to operate without a finite magnetization of the magnetic protein in a biological environment. With the structure of the magnetic protein, we show that the magnetic fluctuation, rather than the permanent magnetism, of the magnetic protein can enable geomagnetic field sensing. An analysis of the quantum dynamics of our microscopic model reveals the necessary conditions for optimal sensitivity. Our work clarifies the mechanism by which magnetic protein enables a biocompass.

Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.

Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.

Pseudomonas aeruginosa nfuA: Gene regulation and its physiological roles in sustaining growth under stress and anaerobic conditions and maintaining bacterial virulence.

The role of the nfuA gene encoding an iron-sulfur ([Fe-S]) cluster-delivery protein in the pathogenic bacterium Pseudomonas aeruginosa was investigated. The analysis of nfuA expression under various stress conditions showed that superoxide generators, a thiol-depleting agent and CuCl2 highly induced nfuA expression. The expression of nfuA was regulated by a global [2Fe-2S] cluster containing the transcription regulator IscR. Increased expression of nfuA in the ΔiscR mutant under uninduced conditions suggests that IscR acts as a transcriptional repressor. In vitro experiments revealed that IscR directly bound to a sequence homologous to the Escherichia coli Type-I IscR-binding motifs on a putative nfuA promoter that overlapped the -35 element. Binding of IscR prevented RNA polymerase from binding to the nfuA promoter, leading to repression of the nfuA transcription. Physiologically, deletion of nfuA reduced the bacterial ability to cope with oxidative stress, iron deprivation conditions and attenuated virulence in the Caenorhabditis elegans infection model. Site-directed mutagenesis analysis revealed that the conserved CXXC motif of the Nfu-type scaffold protein domain at the N-terminus was required for the NfuA functions in conferring the stress resistance phenotype. Furthermore, anaerobic growth of the ΔnfuA mutant in the presence of nitrate was drastically retarded. This phenotype was associated with a reduction in the [Fe-S] cluster containing nitrate reductase enzyme activity. However, NfuA was not required for the maturation of [Fe-S]-containing proteins such as aconitase, succinate dehydrogenase, SoxR and IscR. Taken together, our results indicate that NfuA functions in [Fe-S] cluster delivery to selected target proteins that link to many physiological processes such as anaerobic growth, bacterial virulence and stress responses in P. aeruginosa.

A novel complex neurological phenotype due to a homozygous mutation in FDX2.

Defects in iron-sulphur [Fe-S] cluster biogenesis are increasingly recognized as causing neurological disease. Mutations in a number of genes that encode proteins involved in mitochondrial [Fe-S] protein assembly lead to complex neurological phenotypes. One class of proteins essential in the early cluster assembly are ferredoxins. FDX2 is ubiquitously expressed and is essential in the de novo formation of [2Fe-2S] clusters in humans. We describe and genetically define a novel complex neurological syndrome identified in two Brazilian families, with a novel homozygous mutation in FDX2. Patients were clinically evaluated, underwent MRI, nerve conduction studies, EMG and muscle biopsy. To define the genetic aetiology, a combination of homozygosity mapping and whole exome sequencing was performed. We identified six patients from two apparently unrelated families with autosomal recessive inheritance of a complex neurological phenotype involving optic atrophy and nystagmus developing by age 3, followed by myopathy and recurrent episodes of cramps, myalgia and muscle weakness in the first or second decade of life. Sensory-motor axonal neuropathy led to progressive distal weakness. MRI disclosed a reversible or partially reversible leukoencephalopathy. Muscle biopsy demonstrated an unusual pattern of regional succinate dehydrogenase and cytochrome c oxidase deficiency with iron accumulation. The phenotype was mapped in both families to the same homozygous missense mutation in FDX2 (c.431C > T, p.P144L). The deleterious effect of the mutation was validated by real-time reverse transcription polymerase chain reaction and western blot analysis, which demonstrated normal expression of FDX2 mRNA but severely reduced expression of FDX2 protein in muscle tissue. This study describes a novel complex neurological phenotype with unusual MRI and muscle biopsy features, conclusively mapped to a mutation in FDX2, which encodes a ubiquitously expressed mitochondrial ferredoxin essential for early [Fe-S] cluster biogenesis.

Ultrafast Charge-Transfer Dynamics in the Iron-Sulfur Complex of Rhodobacter capsulatus Ferredoxin VI.

Iron-sulfur proteins play essential roles in various biological processes. Their electronic structure and vibrational dynamics are key to their rich chemistry but nontrivial to unravel. Here, the first ultrafast transient absorption and impulsive coherent vibrational spectroscopic (ICVS) studies on 2Fe-2S clusters in Rhodobacter capsulatus ferreodoxin VI are characterized. Photoexcitation initiated populations on multiple excited electronic states that evolve into each other in a long-lived charge-transfer state. This suggests a potential light-induced electron-transfer pathway as well as the possibility of using iron-sulfur proteins as photosensitizers for light-dependent enzymes. A tyrosine chain near the active site suggests potential hole-transfer pathways and affirms this electron-transfer pathway. The ICVS data revealed vibrational bands at 417 and 484 cm-1, with the latter attributed to an excited-state mode. The temperature dependence of the ICVS modes suggests that the temperature effect on protein structure or conformational heterogeneities needs to be considered during cryogenic temperature studies.

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme.

Protein folding and assembly into macromolecule complexes within the living cell are complex processes requiring intimate coordination. The biogenesis of complex iron sulfur molybdoenzymes (CISM) requires use of a system specific chaperone - a redox enzyme maturation protein (REMP) - to help mediate final folding and assembly. The CISM dimethyl sulfoxide (DMSO) reductase is a bacterial oxidoreductase that utilizes DMSO as a final electron acceptor for anaerobic respiration. The REMP DmsD strongly interacts with DMSO reductase to facilitate folding, cofactor-insertion, subunit assembly and targeting of the multi-subunit enzyme prior to membrane translocation and final assembly and maturation into a bioenergetic catalytic unit. In this article, we discuss the biogenesis of DMSO reductase as an example of the participant network for bacterial CISM maturation pathways.

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

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

Introduction to Metals in Biology 2017: Iron transport, storage, and the ramifications.

In this tenth Thematic Series in Metals in Biology, six Minireviews deal with aspects of iron metabolism. A number of important proteins control iron homeostasis, including hepcidin and ferroportin, in various cells. Other aspects of iron dealt with here include biogenesis of iron-sulfur proteins and chaperones that deliver iron cofactors in cells. Additionally, an iron-regulated metastasis suppressor interacts with the epidermal growth factor receptor and mediates its downstream signaling activity.

The iron-sulfur cluster sensor IscR is a negative regulator of Spi1 type III secretion system in Salmonella enterica.

Iron-sulfur (Fe-S)-containing proteins contribute to various biological processes, including redox reactions or regulation of gene expression. Living organisms have evolved by developing distinct biosynthetic pathways to assemble these clusters, including iron sulfur cluster (ISC) and sulfur mobilization (SUF). Salmonella enterica serovar Typhimurium is an intracellular pathogen responsible for a wide range of infections, from gastroenteritis to severe systemic diseases. Salmonella possesses all known prokaryotic systems to assemble Fe-S clusters, including ISC and SUF. Because iron starvation and oxidative stress are detrimental for Fe-S enzyme biogenesis and because such environments are often met by Salmonella during its intracellular life, we investigated the role of the ISC and SUF machineries during the course of the infection. The iscU mutant, which is predicted to have no ISC system functioning, was found to be defective for epithelial cell invasion and for mice infection, whereas the sufBC mutant, which is predicted to have no SUF system functioning, did not present any defect. Moreover, the iscU mutant was highly impaired in the expression of Salmonella pathogenicity island 1 (Spi1) type III secretion system that is essential for the first stage of Salmonella infection. The Fe-S cluster sensor IscR, a transcriptional regulator matured by the ISC machinery, was shown to bind the promoter of hilD, which encodes the master regulator of Spi1. IscR was also demonstrated to repress hilD and subsequently Spi1 gene expression, consistent with the observation that an IscR mutant is hyper-invasive in epithelial cells. Collectively, our findings indicate that the ISC machinery plays a central role in Salmonella virulence through the ability of IscR to down-regulate Spi1 gene expression. At a broader level, this model illustrates an adaptive mechanism used by bacterial pathogens to modulate their infectivity according to iron and oxygen availability.

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes.

Roles of the Nfu Fe-S targeting factors in the trypanosome mitochondrion.

Iron-sulphur clusters (ISCs) are protein co-factors essential for a wide range of cellular functions. The core iron-sulphur cluster assembly machinery resides in the mitochondrion, yet due to export of an essential precursor from the organelle, it is also needed for cytosolic and nuclear iron-sulphur cluster assembly. In mitochondria all [4Fe-4S] iron-sulphur clusters are synthesised and transferred to specific apoproteins by so-called iron-sulphur cluster targeting factors. One of these factors is the universally present mitochondrial Nfu1, which in humans is required for the proper assembly of a subset of mitochondrial [4Fe-4S] proteins. Although most eukaryotes harbour a single Nfu1, the genomes of Trypanosoma brucei and related flagellates encode three Nfu genes. All three Nfu proteins localise to the mitochondrion in the procyclic form of T. brucei, and TbNfu2 and TbNfu3 are both individually essential for growth in bloodstream and procyclic forms, suggesting highly specific functions for each of these proteins in the trypanosome cell. Moreover, these two proteins are functional in the iron-sulphur cluster assembly in a heterologous system and rescue the growth defect of a yeast deletion mutant.

Escherichia coli nfuA is essential for maintenance of Shiga toxin phage Min27 lysogeny under iron-depleted condition.

It has been earlier hypothesized that lysogenic infection with Stx-encoding phages influences protein expression in the bacterial host, and therefore, some differentially expressed proteins could affect survival characteristics and pathogenicity. We compared the protein expression profiles of the host MG1655 and lysogens by 2D electrophoresis. Four different genes identified were all related to Fe/S subunit production, namely, nfuA, fdoH, sdhB and ftnA. To explore the role of nfuA in the biology of Stx prophage lysogeny, gene knockout experiments and phage lysogenic conversion were performed. The inactivation of nfuA caused the prophage to enter its lytic life cycle, especially under an iron-depleted condition. A similar activity was also detected in the Escherichia coli O157:H7 strain from which the Stx phage Min 27 was originally isolated. NfuA might be the positive regulator of genes controlling lysogenic cycle such as cI, cII and cIII since their transcriptional level was significantly reduced in nfuA deletion mutant as shown by qRT-PCR. We conclude that NfuA is essential for maintenance of Stx phage lysogeny in host's genetic reservoir under iron-deficient condition.

Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+) Reductase Gene (fprB) Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response.

P. aeruginosa (PAO1) has two putative genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein's physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection.

Mammalian iron-sulphur proteins: novel insights into biogenesis and function.

Iron-sulphur (Fe-S) clusters are inorganic cofactors that are found in nearly all species and are composed of various combinations of iron and sulphur atoms. Fe-S clusters can accept or donate single electrons to carry out oxidation and reduction reactions and to facilitate electron transport. Many details of how these complex modular structures are assembled and ligated to cellular proteins in the mitochondrial, nuclear and cytosolic compartments of mammalian cells remain unclear. Recent evidence indicates that a Leu-Tyr-Arg (LYR) tripeptide motif found in some Fe-S recipient proteins may facilitate the direct and shielded transfer of Fe-S clusters from a scaffold to client proteins. Fe-S clusters are probably an unrecognized and elusive cofactor of many known proteins.

[Iron and sulfur in proteins. How does the cell build Fe-S clusters, cofactors essential for life?]. / Du fer et du soufre dans les protéines - Comment la cellule construit-elle les cofacteurs fer-soufre essentiels à son fonctionnement ?

Iron-sulfur clusters (Fe-S) are ubiquitous cofactors present in numerous proteins of most living organisms. By way of an example, the E. coli bacterium synthesizes more that 130 different types of Fe-S proteins. Fe-S proteins are involved in a great diversity of biological processes, ranging from respiration, photosynthesis, central metabolism, to genetic expression and genomic stability. Proteins can acquire spontaneously Fe-S clusters in vitro, but in vivo, dedicated molecular machineries are necessary. Dysfunction of these machineries alters cellular capacities leading to lethality in bacteria and severe pathologies in humans. In this review we will describe how cells make Fe-S clusters and deliver them to clients proteins. The importance of Fe-S clusters homeostasis will be illustrated by reporting a list of cellular dysfunctions associated with mutations altering either Fe-S proteins or Fe-S biogenesis machineries.

IscR regulation of capsular polysaccharide biosynthesis and iron-acquisition systems in Klebsiella pneumoniae CG43.

IscR, an Fe-S cluster-containing transcriptional factor, regulates genes involved in various cellular processes. In response to environmental stimuli such as oxidative stress and iron levels, IscR switches between its holo and apo forms to regulate various targets. IscR binding sequences are classified into two types: the type 1 IscR box that is specific for holo-IscR binding, and the type 2 IscR box that binds holo- and apo-IscR. Studying Klebsiella pneumoniae CG43S3, we have previously shown that iron availability regulates capsular polysaccharide (CPS) biosynthesis and iron-acquisition systems. The present study investigated whether IscR is involved in this regulation. Compared with that in CG43S3, the amount of CPS was decreased in AP001 (ΔiscR) or AP002 (iscR3CA), a CG43S3-derived strain expressing mutated IscR mimicked apo-IscR, suggesting that only holo-IscR activates CPS biosynthesis. Furthermore, a promoter-reporter assay verified that the transcription of cps genes was reduced in AP001 and AP002. Purified IscR::His6, but not IscR3CA::His6, was also found to bind the predicted type 1 IscR box specifically in the cps promoter. Furthermore, reduced siderophore production was observed in AP004 (Δfur-ΔiscR) but not in AP005 (Δfur-iscR3CA), implying that apo-IscR activates iron acquisition. Compared with those in AP004, mRNA levels of three putative iron acquisition systems (fhu, iuc, and sit) were increased in AP005, and both purified IscR::His6 and IscR3CA::His6 bound the predicted type 2 IscR box in the fhuA, iucA, and sitA promoters, whereas IscR3CA::His6 displayed a lower affinity. Finally, we analyzed the effect of external iron levels on iscR expression. The transcription of iscR was increased under iron-depleted conditions as well as in AP001 and AP002, suggesting an auto-repression exerted by apo-IscR. Our results show that in K. pneumoniae, IscR plays a dual role in the regulation of CPS biosynthesis and iron-acquisition systems in response to environmental iron availability.