FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells.

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global down-regulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53-deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids (PLs). Our results suggest that p53 status and PL homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

Benign prostatic hyperplasia nodules in patients treated with celecoxib and/or finasteride have reduced levels of NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, a mitochondrial protein essential for efficient function of the electron transport chain.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a condition generally associated with advanced age in men that can be accompanied by bothersome lower urinary tract symptoms (LUTS) including intermittency, weak stream, straining, urgency, frequency, and incomplete bladder voiding. Pharmacotherapies for LUTS/BPH include alpha-blockers, which relax prostatic and urethral smooth muscle and 5ɑ-reductase inhibitors such as finasteride, which can block conversion of testosterone to dihydrotestosterone thereby reducing prostate volume. Celecoxib is a cyclooxygenase-2 inhibitor that reduces inflammation and has shown some promise in reducing prostatic inflammation and alleviating LUTS for some men with histological BPH. However, finasteride and celecoxib can reduce mitochondrial function in some contexts, potentially impacting their efficacy for alleviating BPH-associated LUTS. METHODS: To determine the impact of these pharmacotherapies on mitochondrial function in prostate tissues, we performed immunostaining of mitochondrial Complex I (CI) protein NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3) and inflammatory cells on BPH specimens from patients naïve to treatment, or who were treated with celecoxib and/or finasteride for 28 days, as well as prostate tissues from male mice treated with celecoxib or vehicle control for 28 days. Quantification and statistical correlation analyses of immunostaining were performed. RESULTS: NDUFS3 immunostaining was decreased in BPH compared to normal adjacent prostate. Patients treated with celecoxib and/or finasteride had significantly decreased NDUFS3 in both BPH and normal tissues, and no change in inflammatory cell infiltration compared to untreated patients. Mice treated with celecoxib also displayed a significant decrease in NDUFS3 immunostaining and no change in inflammatory cell infiltration. CONCLUSIONS: These findings suggest that celecoxib and/or finasteride are associated with an overall decrease in NDUFS3 levels in prostate tissues but do not impact the presence of inflammatory cells, suggesting a decline in mitochondrial CI function in the absence of enhanced inflammation. Given that BPH has recently been associated with increased prostatic mitochondrial dysfunction, celecoxib and/or finasteride may exacerbate existing mitochondrial dysfunction in some BPH patients thereby potentially limiting their overall efficacy in providing metabolic stability and symptom relief.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol.

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis.

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays among the [FeFe]-hydrogenases; however, it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of enzymes. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII) exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric electron paramagnetic resonance, we resolved two electron paramagnetic resonance signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV versus normal hydrogen electrode, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Harnessing iron­sulfur enzymes for synthetic biology.

Reactions catalysed by iron-sulfur (Fe-S) enzymes appear in a variety of biosynthetic pathways that produce valuable natural products. Harnessing these biosynthetic pathways by expression in microbial cell factories grown on an industrial scale would yield enormous economic and environmental benefits. However, Fe-S enzymes often become bottlenecks that limits the productivity of engineered pathways. As a consequence, achieving the production metrics required for industrial application remains a distant goal for Fe-S enzyme-dependent pathways. Here, we identify and review three core challenges in harnessing Fe-S enzyme activity, which all stem from the properties of Fe-S clusters: 1) limited Fe-S cluster supply within the host cell, 2) Fe-S cluster instability, and 3) lack of specialized reducing cofactor proteins often required for Fe-S enzyme activity, such as enzyme-specific flavodoxins and ferredoxins. We highlight successful methods developed for a variety of Fe-S enzymes and electron carriers for overcoming these difficulties. We use heterologous nitrogenase expression as a grand case study demonstrating how each of these challenges can be addressed. We predict that recent breakthroughs in protein structure prediction and design will prove well-suited to addressing each of these challenges. A reliable toolkit for harnessing Fe-S enzymes in engineered metabolic pathways will accelerate the development of industry-ready Fe-S enzyme-dependent biosynthesis pathways.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Inhibition of CISD1 alleviates mitochondrial dysfunction and ferroptosis in mice with acute lung injury.

The NET family member, CDGSH iron-sulfur domain-containing protein 1 (CISD1), is located in theoutermembrane of mitochondria, where it regulates energy and iron metabolism. CISD1 has vital functions in certain human diseases; however, its function in acute lung injury (ALI) is unknown. ALI pathogenesis critically involves mitochondrial dysfunction and ferroptosis, which might be regulated by CISD1. Therefore, we investigated CISD1's function in mitochondrial dysfunction and ferroptosis regulation in lipopolysaccharide (LPS)-induced ALI. We found that CISD1 was upregulated in LPS-induced ALI,and silencing Cisd1 prevented cell apoptosis and increased cell viability. When CISD1was inhibited by mitoNEET ligand-1 (NL-1) there was a significant mitigation of pathological injury and lung edema, and reduced numbers of total cells, polymorphonuclear leukocytes, and a decreased protein content in the bronchoalveolar lavage fluid (BALF). Moreover, inhibition of CISD1 markedly decreased the interleukin (IL)6, IL-1ß, and tumor necrosis factor alpha (TNF-α) levels in the lungs and BALF of ALI-model mice. Silencing of Cisd1 prevented LPS-induced mitochondrial membrane potential depolarization, cellular ATP reduction, and reactive oxygen species (ROS) accumulation, suggesting mitochondrial protection. ALI activated ferroptosis, as evidenced by the increased lipid-ROS, intracellular Fe2+ level, reduced Gpx4 (glutathione peroxidase 4) expression, and the glutathione/glutathione disulfide ratio. Interestingly, inhibition of CISD1 reduced LPS-induced ferroptosis in vivo and in vitro. In conclusion, inhibition of CISD1 alleviated mitochondrial dysfunction and ferroptosis in LPS-induced ALI, identifying CISD1 as possible target for therapy of LPS-induced ALI.

Shedding light on the electron transfer chain of a moderately acidophilic iron oxidizer: characterization of recombinant HiPIP-41, CytC-18 and CytC-78 derived from Ferrovum sp. PN-J47-F6.

Efficient electron transfer from the donor to the acceptor couple presents a necessary requirement for acidophilic and neutrophilic iron oxidizers due to the low energy yield of aerobic ferrous iron oxidation. Involved periplasmic electron carriers are very diverse in these bacteria and show adaptations to the respective thermodynamic constraints such as a more positive redox potential reported for extreme acidophilic Acidithiobacillus spp. Respiratory chain candidates of moderately acidophilic members of the genus Ferrovum share similarities with both their neutrophilic iron oxidizing relatives and the more distantly related Acidithiobacillus spp. We examined our previous omics-based conclusions on the potential electron transfer chain in Ferrovum spp. by characterizing the three redox protein candidates CytC-18, CytC-78 and HiPIP-41 of strain PN-J47-F6 which were produced as recombinant proteins in Eschericha coli. UV/Vis-based redox assays suggested that HiPIP-41 has a very positive redox potential while redox potentials of CytC-18 and CytC-78 are more negative than their counterparts in Acidithiobacillus spp. Far Western dot blotting demonstrated interactions between all three recombinant redox proteins while redox assays showed the electron transfer from HiPIP-41 to either of the cytochromes. Altogether, CytC-18, CytC-78 and HiPIP-41 indeed represent very likely candidates of the electron transfer in Ferrovum sp. PN-J4-F6.

The cytochrome b6f complex: plastoquinol oxidation and regulation of electron transport in chloroplasts.

In oxygenic photosynthetic systems, the cytochrome b6f (Cytb6f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb6f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH2). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH2 from PSII to the Cytb6f does not limit the intersystem electron transport. The overall rate of PQH2 turnover is determined mainly by the first step of the bifurcated oxidation of PQH2 at the catalytic site Qo, i.e., the reaction of electron transfer from PQH2 to the Fe2S2 cluster of the high-potential Rieske iron-sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH2 oxidation within the framework of a model system including the Fe2S2 cluster of the ISP and surrounding amino acids, the low-potential heme b6L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH2). Other structure-function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb6f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

NCOA4 requires a [3Fe-4S] to sense and maintain the iron homeostasis.

NCOA4 is a selective cargo receptor for ferritinophagy, the autophagic turnover of ferritin (FTH), a process critical for regulating intracellular iron bioavailability. However, how ferritinophagy flux is controlled through NCOA4 in iron-dependent processes needs to be better understood. Here, we show that the C-terminal FTH-binding domain of NCOA4 harbors a [3Fe-4S]-binding site with a stoichiometry of approximately one labile [3Fe-4S] cluster per NCOA4 monomer. By analyzing the interaction between NCOA4 and HERC2 ubiquitin ligase or NCOA4 and FTH, we demonstrate that NCOA4 regulates ferritinophagy by sensing the intracellular iron-sulfur cluster levels. Under iron-repletion conditions, HERC2 recognizes and recruits holo-NCOA4 as a substrate for polyubiquitination and degradation, favoring ferritin iron storage. Under iron-depletion conditions, NCOA4 exists in the form of apo-protein and binds ferritin to promote the occurrence of ferritinophagy and release iron. Thus, we identify an iron-sulfur cluster [3Fe-4S] as a critical cofactor in determining the fate of NCOA4 in favoring iron storage in ferritin or iron release via ferritinophagy and provide a dual mechanism for selective interaction between HERC2 and [3Fe-4S]-NCOA4 for proteasomal degradation or between ferritin and apo-NCOA4 for ferritinophagy in the control of iron homeostasis.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide is present at the C-terminus of more than a quarter of clients or their adaptors. When present, this targeting complex recognition (TCR) motif is necessary and sufficient for binding to the CTC in vitro and for directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR signal enables engineering of cluster maturation on a nonnative protein via recruitment of the CIA machinery. Our study advances our understanding of Fe-S protein maturation and paves the way for bioengineering novel pathways containing Fe-S enzymes.

Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the ß-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator.

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of dinitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at noncryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the fumarate and nitrate reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR-binding sites. By coupling size-exclusion chromatography-anSAXS with full-spectrum UV-Vis analysis, we show that the [4Fe-4S] cluster-containing dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

Exploring substrate interaction in respiratory alternative complex III from Rhodothermus marinus.

Rhodothermus marinus is a thermohalophilic organism that has optimized its microaerobic metabolism at 65 °C. We have been exploring its respiratory chain and observed the existence of a quinone:cytochrome c oxidoreductase complex, named Alternative Complex III, structurally different from the bc1 complex. In the present work, we took profit from nanodiscs and liposomes technology to investigate ACIII activity in membrane-mimicking systems. In addition, we studied the interaction of ACIII with menaquinone, its potential electron acceptors (HiPIP and cytochrome c) and the caa3 oxygen reductase.

Research progress on the biosynthesis and delivery of iron-sulfur clusters in the plastid.

Iron-sulfur (Fe-S) clusters are ancient protein cofactors ubiquitously exist in organisms. They are involved in many important life processes. Plastids are semi-autonomous organelles with a double membrane and it is believed to originate from a cyanobacterial endosymbiont. By learning form the research in cyanobacteria, a Fe-S cluster biosynthesis and delivery pathway has been proposed and partly demonstrated in plastids, including iron uptake, sulfur mobilization, Fe-S cluster assembly and delivery. Fe-S clusters are essential for the downstream Fe-S proteins to perform their normal biological functions. Because of the importance of Fe-S proteins in plastid, researchers have made a lot of research progress on this pathway in recent years. This review summarizes the detail research progress made in recent years. In addition, the scientific problems remained in this pathway are also discussed.

Structural Plasticity of NFU1 Upon Interaction with Binding Partners: Insights into the Mitochondrial [4Fe-4S] Cluster Pathway.

In humans, the biosynthesis and trafficking of mitochondrial [4Fe-4S]2+ clusters is a highly coordinated process that requires a complex protein machinery. In a mitochondrial pathway among various proposed to biosynthesize nascent [4Fe-4S]2+ clusters, two [2Fe-2S]2+ clusters are converted into a [4Fe-4S]2+ cluster on a ISCA1-ISCA2 complex. Along this pathway, this cluster is then mobilized from this complex to mitochondrial apo recipient proteins with the assistance of accessory proteins. NFU1 is the accessory protein that first receives the [4Fe-4S]2+ cluster from ISCA1-ISCA2 complex. A structural view of the protein-protein recognition events occurring along the [4Fe-4S]2+ cluster trafficking as well as how the globular N-terminal and C-terminal domains of NFU1 act in such process is, however, still elusive. Here, we applied small-angle X-ray scattering coupled with on-line size-exclusion chromatography and paramagnetic NMR to disclose structural snapshots of ISCA1-, ISCA2- and NFU1-containing apo complexes as well as the coordination of [4Fe-4S]2+ cluster bound to the ISCA1-NFU1 complex, which is the terminal stable species of the [4Fe-4S]2+ cluster transfer pathway involving ISCA1-, ISCA2- and NFU1 proteins. The structural modelling of ISCA1-ISCA2, ISCA1-ISCA2-NFU1 and ISCA1-NFU1 apo complexes, here reported, reveals that the structural plasticity of NFU1 domains is crucial to drive protein partner recognition and modulate [4Fe-4S]2+ cluster transfer from the cluster-assembly site in the ISCA1-ISCA2 complex to a cluster-binding site in the ISCA1-NFU1 complex. These structures allowed us to provide a first rational for the molecular function of the N-domain of NFU1, which can act as a modulator in the [4Fe-4S]2+ cluster transfer.

Antibiotic resistance in Neisseria gonorrhoeae: broad-spectrum drug target identification using subtractive genomics.

Neisseria gonorrhoeae is a Gram-negative aerobic diplococcus bacterium that primarily causes sexually transmitted infections through direct human sexual contact. It is a major public health threat due to its impact on reproductive health, the widespread presence of antimicrobial resistance, and the lack of a vaccine. In this study, we used a bioinformatics approach and performed subtractive genomic methods to identify potential drug targets against the core proteome of N. gonorrhoeae (12 strains). In total, 12,300 protein sequences were retrieved, and paralogous proteins were removed using CD-HIT. The remaining sequences were analyzed for non-homology against the human proteome and gut microbiota, and screened for broad-spectrum analysis, druggability, and anti-target analysis. The proteins were also characterized for unique interactions between the host and pathogen through metabolic pathway analysis. Based on the subtractive genomic approach and subcellular localization, we identified one cytoplasmic protein, 2Fe-2S iron-sulfur cluster binding domain-containing protein (NGFG RS03485), as a potential drug target. This protein could be further exploited for drug development to create new medications and therapeutic agents for the treatment of N. gonorrhoeae infections.