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![NO is rapidly consumed by the IlvD [4Fe-4S] cluster under anaerobic conditions](profile/Guoqiang-Tan/publication/23409225/figure/fig1/AS:302046705143812@1449024954293/NO-is-rapidly-consumed-by-the-IlvD-4Fe-4S-cluster-under-anaerobic-conditions.png)
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![Figure 1. NO is rapidly consumed by the IlvD [4Fe-4S] cluster under...](profile/Guoqiang-Tan/publication/23409225/figure/fig1/AS:302046705143812@1449024954293/NO-is-rapidly-consumed-by-the-IlvD-4Fe-4S-cluster-under-anaerobic-conditions_Q320.jpg)
![Figure 2. Sensitivity of the IlvD [4Fe-4S] cluster to NO](profile/Guoqiang-Tan/publication/23409225/figure/fig2/AS:302046705143816@1449024954530/Sensitivity-of-the-IlvD-4Fe-4S-cluster-to-NO_Q320.jpg)
![Figure 3. Relative reactivity of NO with the IlvD [4Fe-4S] cluster and O 2](profile/Guoqiang-Tan/publication/23409225/figure/fig3/AS:302046705143818@1449024954588/Relative-reactivity-of-NO-with-the-IlvD-4Fe-4S-cluster-and-O-2_Q320.jpg)
![Figure 4. GSH fails to protect the IlvD [4Fe-4S] cluster from being...](profile/Guoqiang-Tan/publication/23409225/figure/fig4/AS:302046705143820@1449024954677/GSH-fails-to-protect-the-IlvD-4Fe-4S-cluster-from-being-modified-by-NO-with-or-without_Q320.jpg)
Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the...
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... was chosen because its [4Fe-4S] cluster is relatively stable under aerobic conditions [35]. Figure 1A shows the UV-visible absorption spectra of apo-IlvD and IlvD with the [4Fe-4S] cluster. The absorption peaks at 320 nm and 411 nm represent the charge transfer bands of the [4Fe-4S] cluster in the protein [35]. ...
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... the NO concentration reached a plateau in the sealed vial, a pre-degassed solution containing the IlvD [4Fe-4S] cluster was immediately injected using a gas-tight Hamilton syringe. As shown in Figure 1B, NO was rapidly consumed by the IlvD [4Fe-4S] cluster with a half-life time of less than 30 seconds. In contrast, injection of a pre-degassed solution containing apo-IlvD had no effect on the NO concentration in the solution. ...
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... reaction between NO and the IlvD [4Fe-4S] cluster was also traced by EPR spectroscopy and an enzyme activity assay. Figure 1C shows that the EPR signal at g=2.04 representing the protein-bound DNIC [22][23][24][25][26][27][28] quickly appeared (within 15 s) after injection of the IlvD [4Fe-4S] cluster into the NO solution anaerobically. Using the glutathione-bound DNIC as a standard [30], we estimated that the maximum amount of 1.8±0.5 molecules of DNIC per each IlvD [4Fe-4S] cluster were formed by NO under anaerobic conditions. ...
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... the glutathione-bound DNIC as a standard [30], we estimated that the maximum amount of 1.8±0.5 molecules of DNIC per each IlvD [4Fe-4S] cluster were formed by NO under anaerobic conditions. The parallel enzyme activity measurements revealed that IlvD was also rapidly inactivated upon the injection to the NO solution anaerobically ( Figure 1D). ...
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... observed rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster ( Figure 1B) Figure 3A shows that the NO consumption was evidently faster by the IlvD [4Fe-4S] cluster (10 μM) than by O 2 (10 μM). When equal amounts of the IlvD [4Fe-4S] cluster and O 2 were injected together into the NO solution, the NO consumption became even faster ( Figure 3A), suggesting that both the IlvD [4Fe-4S] cluster and O 2 may simultaneously consume NO. ...
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... EPR measurements of purified IlvD samples demonstrated that the IlvD [4Fe-4S] cluster was efficiently converted to the IlvD-bound DNIC by NO under both anaerobic ( Figure 6C) and aerobic conditions ( Figure 6D). Similar results were obtained when the E. coli cells containing the recombinant aconitase B [4Fe-4S] cluster were exposed to NO under aerobic and anaerobic conditions (see Supplementary Figure 1). However, the same NO exposure of the E. coli cells containing the recombinant endonuclease III produced only a small amount of the endonuclease III-bound DNIC (results not shown), consistent with its slow reaction kinetics with NO ( Figure 5A). ...
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... we were unable to simulate the observed kinetics of the NO consumption using simple algorithms, as the redox reactions underlying the NO-mediated modifications of iron- sulfur clusters are still not fully understood. In addition, the iron and sulfide released from the disrupted iron-sulfur clusters could potentially react with NO, thus further complicating the NO consumption kinetics in the reaction ( Figure 1B). Although our results clearly suggest that iron-sulfur clusters are the primary targets of the NO cytotoxicity, it should be pointed out that not all iron-sulfur proteins have the same reactivity with NO. ...
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... is considered an effective anti-bacteria and anti-tumor cytotoxic agent [7][8][9]. Unlike reversible binding of NO to hemes in soluble guanylate cyclase [11] or the binuclear heme a 3 /Cu B center in cytochrome c oxidase [12][13][14], the reaction of NO with iron-sulfur clusters is irreversible with the concomitant formation of the protein-bound DNIC [22][23][24][25][26][27][28]38] (Figure 1). Since iron-sulfur proteins are broadly distributed [16,38], modification of iron-sulfur clusters by NO is expected to inactivate multiple physiological processes. ...
Citations
... Although RNS inhibit amino acid biosynthetic mechanisms in bacteria, this has never been identified in fungi until now. Incubating E. coli with NO inactivates the iron-sulfur enzyme dihydroxy acid dehydratase, which is involved in the synthesis of branched-chain amino acids and leads to their depletion [30,31]. Nitric oxide inactivates the iron-sulfur enzyme aconitase and other TCA cycle enzymes that synthesize the precursors of methionine and lysine in Salmonella typhimurium, and results in auxotrophy [32]. ...
Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.
... Nitric oxide (NO) forms complexes with iron atoms. The nitrosylation of [4Fe-4S] clusters has been detected in studies of dihydroxyacid dehydratase [57], aconitase [58], and endonuclease III [59]. To our knowledge NO reactivity with RSEs has not been tested. ...
Radical S-adenosylmethionine enzymes (RSEs) drive diverse biological processes by catalyzing chemically difficult reactions. Each of these enzymes uses a solvent-exposed [4Fe–4S] cluster to coordinate and cleave its SAM co-reactant. This cluster is destroyed during oxic handling, forcing investigators to work with these enzymes under anoxic conditions. Analogous substrate-binding [4Fe–4S] clusters in dehydratases are similarly sensitive to oxygen in vitro; they are also extremely vulnerable to reactive oxygen species (ROS) in vitro and in vivo. These observations suggested that ROS might similarly poison RSEs. This conjecture received apparent support by the observation that when E. coli experiences hydrogen peroxide stress, it induces a cluster-free isozyme of the RSE HemN. In the present study, surprisingly, the purified RSEs viperin and HemN proved quite resistant to peroxide and superoxide in vitro. Furthermore, pathways that require RSEs remained active inside E. coli cells that were acutely stressed by hydrogen peroxide and superoxide. Viperin, but not HemN, was gradually poisoned by molecular oxygen in vitro, forming an apparent [3Fe–4S]⁺ form that was readily reactivated. The modest rate of damage, and the known ability of cells to repair [3Fe–4S]⁺ clusters, suggest why these RSEs remain functional inside fully aerated organisms. In contrast, copper(I) damaged HemN and viperin in vitro as readily as it did fumarase, a known target of copper toxicity inside E. coli. Excess intracellular copper also impaired RSE-dependent biosynthetic processes. These data indicate that RSEs may be targets of copper stress but not of reactive oxygen species.
... The NO concentration in the prepared NO solution was measured using a nitric oxide electrode (World Precision Instrument. co) (Duan et al., 2009). Aliquot of the prepared NO solution was added to the pre-degassed protein samples in a sealed vial using a gas-tight Hamilton syringe, followed by the UV-Vis absorption measurements. ...
MitoNEET is a mitochondrial outer membrane protein that regulates energy metabolism, iron homeostasis, and production of reactive oxygen species in cells. Aberrant expression of mitoNEET in tissues has been linked to type II diabetes, neurodegenerative diseases, and several types of cancer. Structurally, the N-terminal domain of mitoNEET has a single transmembrane alpha helix that anchors the protein to mitochondrial outer membrane. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine and one histidine residues. Here we report that the reduced [2Fe-2S] cluster in the C-terminal cytosolic domain of mitoNEET (mitoNEET 45-108 ) is able to bind nitric oxide (NO) without disruption of the cluster. Importantly, binding of NO at the reduced [2Fe-2S] cluster effectively inhibits the redox transition of the cluster in mitoNEET 45-108 . While the NO-bound [2Fe-2S] cluster in mitoNEET 45-108 is stable, light excitation releases NO from the NO-bound [2Fe-2S] cluster and restores the redox transition activity of the cluster in mitoNEET 45-108 . The results suggest that NO may regulate the electron transfer activity of mitoNEET in mitochondrial outer membrane via reversible binding to its reduced [2Fe-2S] cluster.
... While NO is a general bacterial inhibitor, it does so by inactivating specific prosthetic groups in specific proteins. Solvent-exposed iron-sulfur clusters are particularly sensitive to NO, such as the [4Fe-4S] cluster in dihydroxyacid dehydratase, an essential enzyme in the biosynthesis of branched-chain amino acids (34,(43)(44)(45)(46). Thiols are also key targets of NO, particularly when they coordinate metal cofactors, such as zinc in the cases of DksA, a transcriptional regulator, and the glycolytic enzyme fructose bisphosphate aldolase (36,(47)(48)(49). ...
Rickettsia rickettsii , the causative agent of Rocky Mountain spotted fever, is an enzootic, obligate intracellular bacterial pathogen. Nitric oxide (NO) synthesized by the inducible nitric oxide synthase (iNOS) is a potent antimicrobial component of innate immunity and has been implicated in the control of virulent Rickettsia spp. in diverse cell types. In this study, we examined the antibacterial role of NO on R. rickettsii . Our results indicate that NO challenge dramatically reduces R. rickettsii adhesion through the disruption of bacterial energetics. Additionally, NO-treated R. rickettsii were unable to synthesize protein or replicate in permissive cells. Activated, NO-producing macrophages restricted R. rickettsii infections, but inhibition of iNOS ablated the inhibition of bacterial growth. These data indicate that NO is a potent anti-rickettsial effector of innate immunity that targets energy generation in these pathogenic bacteria to prevent growth and subversion of infected host cells.
... These genes have been studied in metabolic processes or tumor development. ISCU is an important scaffold protein and can assemble iron and sulfur into iron-sulfur clusters These clusters participate in the maturation of [2Fe-2S] and [4Fe-4S] proteins in the mitochondria and cytoplasm and the regulation of iron metabolism (Duan et al., 2009). As an important redox center, iron-sulfur clusters participated in many physiological functions such especially in metabolism (Xu and Møller, 2011). ...
Background: Every year, nearly 170,000 people die from bladder cancer worldwide. A major problem after transurethral resection of bladder tumor is that 40–80% of the tumors recur. Ferroptosis is a type of regulatory necrosis mediated by iron-catalyzed, excessive oxidation of polyunsaturated fatty acids. Increasing the sensitivity of tumor cells to ferroptosis is a potential treatment option for cancer. Establishing a diagnostic and prognostic model based on ferroptosis-related genes may provide guidance for the precise treatment of bladder cancer.
Methods: We downloaded mRNA data in Bladder Cancer from The Cancer Genome Atlas and analyzed differentially expressed genes based on and extract ferroptosis-related genes. We identified relevant pathways and annotate the functions of ferroptosis-related DEGs using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and Gene Ontology functions. On the website of Search Tool for Retrieving Interacting Genes database (STRING), we downloaded the protein-protein interactions of DEGs, which were drawn by the Cytoscape software. Then the Cox regression analysis were performed so that the prognostic value of ferroptosis-related genes and survival time are combined to identify survival- and ferroptosis-related genes and establish a prognostic formula. Survival analysis and receiver operating characteristic curvevalidation were then performed. Risk curves and nomograms were generated for both groups to predict survival. Finally, RT-qPCR was applied to analyze gene expression.
Results: Eight ferroptosis-related genes with prognostic value (ISCU, NFE2L2, MAFG, ZEB1, VDAC2, TXNIP, SCD, and JDP2) were identified. With clinical data, we established a prognostic model to provide promising diagnostic and prognostic information of bladder cancer based on the eight ferroptosis-related genes. RT-qPCR revealed the genes that were differentially expressed between normal and cancer tissues.
Conclusion: This study found that the ferroptosis-related genes is associated with bladder cancer, which may serve as new target for the treatment of bladder cancer.
... This activity occurs rapidly in the enzyme active site, thereby changing its conformation [100]. Nitric oxide alone rapidly reacts with cluster-forming dinitrosyl iron complexes (DNICs) and even more complicated structures [101] or is responsible for thiol nitrosylation [102,103]. The radicals NO and • OH can bind to Fe atoms of the Fe-S clusters [104]. ...
... N-acetyl-L-cysteine and GSH were able to repair clusters to a much lower degree [125]. However, GSH failed to protect the E. coli [4Fe-4S] cluster of dihydroxyacid dehydratase from NO-mediated transformation [102]. ...
Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity.
... Genes regulated by the σ 70 -dependent NsrR provide a first line of defense against the very low concentrations of NO that occur in the bacterial cytoplasm ( Figure 1, range 1). The housekeeping function of NsrR-regulated gene products is to protect vulnerable cytoplasmic proteins such as the dehydratase family against the very low concentrations of NO encountered during normal growth (Duan et al., 2009;Hyduke et al., 2007;Ren et al., 2008;Varghese et al., 2003). When the intracellular [NO] increases toward the concentration that would overwhelm NsrR-regulated defenses, NorR is activated and the NorVW | 35 ...
... Dinitrosylation of some iron-sulfur proteins, for example, the dehydratase family, results in chemical damage and the slow release of iron atoms (D'Autreaux et al., 2002;Duan et al., 2009;Hyduke et al., 2007). ...
How anaerobic bacteria protect themselves against nitric oxide‐induced stress is controversial, not least because far higher levels of stress were used in the experiments on which most of the literature is based than bacteria experience in their natural environments. This results in chemical damage to enzymes that inactivates their physiological function. Bacteria are exposed to nitric oxide from many sources, especially in anaerobic environments including the human body. How it is detoxified anaerobically is a major source of disagreement, largely because the literature is based upon experiments that fail to reflect conditions encountered by bacteria in their natural environments. This review shows how chemical damage inactivates proteins, preventing their physiological roles from being revealed
... [2][3][4][5] NO also interacts with iron to form dinitrosyl-iron complexes (DNICs), which can impair the activity of a variety of regulatory proteins and enzymes. [6][7][8][9][10][11] A microarray to identify NO -responsive genes in Salmonella enterica serovar Typhimurium (S. Typhimurium) identified manganese transporters mntH and sitABCD among the most highly up-regulated genes, suggesting that manganese may play a role in cellular resistance to nitrosative stress. ...
Nitric oxide (NO˙) is a radical molecule produced by mammalian phagocytic cells as part of the innate immune response to bacterial pathogens. It exerts its antimicrobial activity in part by impairing the function of metalloproteins, particularly those containing iron and zinc cofactors. The pathogenic Gram-negative bacterium Salmonella enterica serovar typhimurium undergoes dynamic changes in its cellular content of the four most common metal cofactors following exposure to NO˙ stress. Zinc, iron and magnesium all decrease in response to NO˙ while cellular manganese increases significantly. Manganese acquisition is driven primarily by increased expression of the mntH and sitABCD transporters following derepression of MntR and Fur. ZupT also contributes to manganese acquisition in response to nitrosative stress. S. Typhimurium mutants lacking manganese importers are more sensitive to NO˙, indicating that manganese is important for resistance to nitrosative stress.
... Unlike aconitase, E. coli DHAD (Ec-DHAD) is not reactivated by addition of Fe 2+ and thiols under reducing conditions, implying that the cluster is degraded completely. The cluster also reacts rapidly with nitric oxide (NO) to generate a nitrosyl form of the cluster that is irreversibly inactivated (10). ...
Iron-sulfur (Fe-S) clusters are protein cofactors with an ancient evolutionary origin. These clusters are best known for their roles in redox proteins such as ferredoxins, but some Fe-S clusters have non-redox roles in the active sites of enzymes. Such clusters are often prone to oxidative degradation, making the enzymes difficult to characterize. Here we report a structural and functional characterization of dihydroxyacid dehydratase (DHAD) from Mycobacterium tuberculosis (Mtb), an essential enzyme in the biosynthesis of branched-chain amino acids. Conducting this analysis under fully anaerobic conditions, we solved the DHAD crystal structure, at 1.88 Å resolution, revealing a 2Fe-2S cluster in which one Fe ligand is a potentially-exchangeable water molecule or hydroxide. UV and EPR spectroscopy both suggested that the substrate binds directly to the cluster, or very close to it. Kinetic analysis implicated two ionizable groups in the catalytic mechanism, which we postulate to be Ser491 and the iron-bound water/hydroxide. Site-directed mutagenesis showed that Ser491 is essential for activity, and substrate docking indicated that this residue is perfectly placed for proton abstraction. We found that a bound Mg2+ ion 6.5 Å from the 2Fe-2S cluster plays a key role in substrate binding. We also identified a putative entry channel that enables access to the cluster, and show that Mtb-DHAD is inhibited by a recently-discovered herbicide, aspterric acid, that, given the essentiality of DHAD for Mtb survival, is a potential lead compound for the design of novel anti-TB drugs.
... DHAD activity loss resulting from O 2 -induced [Fe 4 S 4 ] cluster degradation has been studied both in vivo and in vitro (4,10). Moreover, the NO-induced cluster degradation of bacterial DHADs has been implicated in the mammalian immune response against pathogens (11)(12)(13). In contrast, DHAD as-isolated from spinach has been shown to accommodate a [Fe 2 S 2 ] 2ϩ cluster, which is rare among the large family of Fe-S cluster-containing (de)hydratases (2). ...
Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe4S4] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe2S2] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism, resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from Arabidopsis thaliana (At). Our results indicated that AtDHAD can accommodate [Fe2S2] and [Fe4S4] clusters. However, only the [Fe2S2] cluster-bound form is catalytically active. We found that the [Fe2S2] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. In vitro cluster transfer and reconstitution reactions revealed that [Fe2S2] cluster-containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of AtDHAD. In summary, AtDHAD binds either one [Fe4S4] or one [Fe2S2] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe2S2] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant AtDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.
