Mechanism of [4Fe-4S](Cys)4 Cluster Nitrosylation Is Conserved among NO-responsive Regulators

Crack, Jason C.; Stapleton, Melanie R.; Green, Jeffrey; Thomson, Andrew J.; Brun, Nick E. Le

doi:10.1074/jbc.m112.439901

Cited by 67 publications

(121 citation statements)

References 46 publications

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“…The UV-visible spectrum with absorption maxima at 360 and 405 nm indicates the presence of 4Fe-4S cluster in the enzyme and 78 % reconstitution efficiency using an Ɛ value of 16,000 M -1 cm -1 at 410 nm (30). The addition of sodium dithionite lowered the absorption intensity at 405 nm indicating a reduction of the cluster (Fig 3b) (31)(32)(33)(34) (21) and Trypanosoma cruzi (22) while for those from bacteria and archaea, the values are in the micromolar range. The catalytic efficiency (k cat /K m ) of PfFH∆40 is similar to L. major FH but 10-100 fold lower than that reported for other class I FHs ( Table 2).…”

Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 95%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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Section: Pffh Complements Fumarase Deficiency In E Coli-mentioning

confidence: 95%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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“…2). [95][96][97] Reaction with NO is extremely rapid, multiphasic and results in the formation of a proteinbound nitrosylated ironsulfur cluster that resembles a pair of Roussin's red esters. 97 Reaction with NO inhibits FNR DNA-binding activity in vitro and FNR-dependent transcription in vivo.…”

Section: Fnrmentioning

confidence: 99%

“…[95][96][97] Reaction with NO is extremely rapid, multiphasic and results in the formation of a proteinbound nitrosylated ironsulfur cluster that resembles a pair of Roussin's red esters. 97 Reaction with NO inhibits FNR DNA-binding activity in vitro and FNR-dependent transcription in vivo. 95,97 Thus, in addition to its well-established role as an O 2 -responsive regulator of anaerobic functions, the inactivation of FNR by NO was suggested to be a final safeguard against NO toxicity by switching off transcription of genes involved in nitrate and nitrite respiration, thereby minimizing endogenous NO production when the dedicated NO-responsive regulators and detoxification systems are overwhelmed.…”

Section: Fnrmentioning

confidence: 99%

Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

2014

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Molecular oxygen (O 2 ) and nitric oxide (NO) are diatomic gases that play major roles in infection. The host innate immune system generates reactive oxygen species and NO as bacteriocidal agents and both require O 2 for their production. Furthermore, the ability to adapt to changes in O 2 availability is crucial for many bacterial pathogens, as many niches within a host are hypoxic. Pathogenic bacteria have evolved transcriptional regulatory systems that perceive these gases and respond by reprogramming gene expression. Direct sensors possess iron-containing co-factors (iron-sulfur clusters, mononuclear iron, heme) or reactive cysteine thiols that react with O 2 and/or NO. Indirect sensors perceive the physiological effects of O 2 starvation. Thus, O 2 and NO act as environmental cues that trigger the coordinated expression of virulence genes and metabolic adaptations necessary for survival within a host. Here, the mechanisms of signal perception by key O 2 -and NO-responsive bacterial transcription factors and the effects on virulence gene expression are reviewed, followed by consideration of these aspects of gene regulation in two major pathogens, Staphylococcus aureus and Mycobacterium tuberculosis.

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“…Considering that anamorsin traffics between the cytoplasm and the nucleus [45] and is involved in intracellular antiapoptotic signaling pathways [46], the [2Fe-2S] cluster bound to the M2 motif might play a regulatory role in these processes. Indeed, besides the electron transfer function, Fe/S clusters are known to function as signaling and regulating agents because of their tunable sensitivity to various oxidants or reductants [47].…”

Section: Reduced Fl Constructmentioning

confidence: 99%

Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

et al. 2013

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The eukaryotic anamorsin protein family, which has recently been proposed to be part of an electron transfer chain functioning in the early steps of cytosolic iron-sulfur (Fe/S) protein biogenesis, is characterized by a largely unstructured domain (CIAPIN1) containing two conserved cysteine-rich motifs (CX 8 CX 2 CXC and CX 2 CX 7 CX 2 C) whose Fe/S binding properties and electronic structures are not well defined. Here, we found that (1) each motif in human anamorsin is able to bind independently a [2Fe-2S] cluster through its four cysteine residues, the binding of one cluster mutually excluding the binding of the second, (2) the reduced [2Fe-2S] ? clusters exhibit a unique electronic structure with considerable anisotropy in their coordination environment, different from that observed in reduced, plant-type and vertebratetype [2Fe-2S] ferredoxin centers, (3) the reduced cluster bound to the CX 2 CX 7 CX 2 C motif reveals an unprecedented valence localization-to-delocalization transition as a function of temperature, and (4) only the [2Fe-2S] cluster bound to the CX 8 CX 2 CXC motif is involved in the electron transfer with its physiological protein partner Ndor1. The unique electronic properties of both [2Fe-2S] centers can be interpreted by considering that both cysteine-rich motifs are located in a highly unstructured and flexible protein region, whose local conformational heterogeneity can induce anisotropy in metal coordination. This study contributes to the understanding of the functional role of the CIAPIN1 domain in the anamorsin family, suggesting that only the [2Fe-2S] cluster bound to the CX 8 CX 2 CXC motif is indispensable in the electron transfer chain assembling cytosolic Fe/S proteins.

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Mechanism of [4Fe-4S](Cys)4 Cluster Nitrosylation Is Conserved among NO-responsive Regulators

Cited by 67 publications

References 46 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Transcriptional regulation of bacterial virulence gene expression by molecular oxygen and nitric oxide

Human anamorsin binds [2Fe–2S] clusters with unique electronic properties

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