RESUMEN
Ironsulfur (FeS) clusters are inorganic protein cofactors that perform essential functions in many physiological processes. Spectroscopic techniques have historically been used to elucidate details of FeS cluster type, their assembly and transfer, and changes in redox and ligand binding properties. Structural probes of protein topology, complex formation, and conformational dynamics are also necessary to fully understand these FeS protein systems. Recent developments in mass spectrometry (MS) instrumentation and methods provide new tools to investigate FeS cluster and structural properties. With the unique advantage of sampling all species in a mixture, MS-based methods can be utilized as a powerful complementary approach to probe native dynamic heterogeneity, interrogate protein folding and unfolding equilibria, and provide extensive insight into protein binding partners within an entire proteome. Here, we highlight key advances in FeS protein studies made possible by MS methodology and contribute an outlook for its role in the field.
RESUMEN
Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.
Asunto(s)
Cobre , Complejo IV de Transporte de Electrones , Hidrazinas , Ionóforos , Ferredoxinas/metabolismoRESUMEN
Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Proteínas Hierro-Azufre/química , Temperatura , Espectrometría de Masa por Ionización de Electrospray , Liasas de Carbono-Azufre/metabolismo , Azufre/metabolismo , Hierro/química , Proteínas de Escherichia coli/químicaRESUMEN
Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated. Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from â¼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold- and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.
Asunto(s)
Proteínas , Espectrometría de Masa por Ionización de Electrospray , Ligandos , Transición de Fase , TemperaturaRESUMEN
Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.
Asunto(s)
Liasas de Carbono-Azufre/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Hierro-Azufre/química , Liasas de Carbono-Azufre/metabolismo , Dominio Catalítico , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Hierro/química , Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Espectrometría de Masas , Oxidación-Reducción , Multimerización de Proteína , Zinc/químicaRESUMEN
The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to the incurable neurodegenerative disease Friedreich's ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondrial function. The structural and functional core of the Fe-S cluster assembly complex is a low-activity pyridoxal 5'-phosphate (PLP)-dependent cysteine desulfurase enzyme that consists of catalytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) subunits. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. Here, we develop a radiolabeling assay and use stopped-flow kinetics to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. Our results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. Our resulting architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.
Asunto(s)
Liasas de Carbono-Azufre/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Azufre/metabolismo , Regulación Alostérica , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/genética , Dominio Catalítico , Cisteína/metabolismo , Humanos , Cinética , Complejos Multiproteicos/metabolismo , Mutación , Unión Proteica , Fosfato de Piridoxal/metabolismo , Sulfuros/metabolismo , FrataxinaRESUMEN
LigU from Novosphingobium sp. strain KA1 catalyzes the isomerization of (4E)-oxalomesaconate (OMA) to (3Z)-2-keto-4-carboxy-3-hexenedioate (KCH) as part of the protocatechuate (PCA) 4,5-cleavage pathway during the degradation of lignin. The three-dimensional structure of the apo form of the wild-type enzyme was determined by X-ray crystallography, and the structure of the K66M mutant enzyme was determined in the presence of the substrate OMA. LigU is a homodimer requiring no cofactors or metal ions with a diaminopimelate epimerase structural fold, consisting of two domains with similar topologies. Each domain has a central α-helix surrounded by a ß-barrel composed of antiparallel ß-strands. The active site is at the cleft of the two domains. 1H nuclear magnetic resonance spectroscopy demonstrated that the enzyme catalyzes the exchange of the pro-S hydrogen at C5 of KCH with D2O during the isomerization reaction. Solvent-deuterium exchange experiments demonstrated that mutation of Lys-66 eliminated the isotope exchange at C5 and that mutation of C100 abolished exchange at C3. The positioning of these two residues in the active site of LigU is consistent with a reaction mechanism that is initiated by the abstraction of the pro-S hydrogen at C3 of OMA by the thiolate anion of Cys-100 and the donation of a proton at C5 of the proposed enolate anion intermediate by the side chain of Lys-66 to form the product KCH. The 1,3-proton transfer is suprafacial.
Asunto(s)
Proteínas Bacterianas/metabolismo , Isomerasas/metabolismo , Lignina/metabolismo , Modelos Moleculares , Sphingomonadaceae/enzimología , Carbohidrato Epimerasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hidroxibenzoatos/metabolismo , Cinética , Conformación ProteicaRESUMEN
In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia. Recently, a mutation that suppressed the loss of the FXN homolog in Saccharomyces cerevisiae was identified that encodes an amino acid substitution equivalent to the human variant ISCU2 M140I. Here, we developed iron-sulfur cluster synthesis and transfer functional assays and determined that the human ISCU2 M140I variant can substitute for FXN in accelerating the rate of iron-sulfur cluster formation on the monothiol glutaredoxin (GRX5) acceptor protein. Incorporation of both FXN and the M140I substitution had an additive effect, suggesting an acceleration of distinct steps in iron-sulfur cluster biogenesis. In contrast to the canonical role of FXN in stimulating the formation of [2Fe-2S]-ISCU2 intermediates, we found here that the M140I substitution in ISCU2 promotes the transfer of iron-sulfur clusters to GRX5. Together, these results reveal an unexpected mechanism that replaces FXN-based stimulation of the iron-sulfur cluster biosynthetic pathway and suggest new strategies to overcome the loss of cellular FXN that may be relevant to the development of therapeutics for Friedreich's ataxia.
Asunto(s)
Ataxia de Friedreich/patología , Proteínas de Unión a Hierro/metabolismo , Regulación Alostérica , Liasas de Carbono-Azufre/metabolismo , Ataxia de Friedreich/metabolismo , Glutarredoxinas/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Unión Proteica , FrataxinaRESUMEN
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.
Asunto(s)
Hipoxia/metabolismo , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Femenino , Ataxia de Friedreich/metabolismo , Células HEK293 , Humanos , Hipoxia/fisiopatología , Hierro/metabolismo , Proteína 2 Reguladora de Hierro/metabolismo , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/fisiología , Células K562 , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Saccharomyces cerevisiae/metabolismo , Azufre/metabolismo , FrataxinaRESUMEN
In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.
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Proteína Transportadora de Acilo/metabolismo , Liasas de Carbono-Azufre/metabolismo , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Sitios de Unión , Liasas de Carbono-Azufre/química , Dominio Catalítico , Cromatografía de Gases y Espectrometría de Masas , Humanos , Proteínas de Unión a Hierro/química , Proteínas Reguladoras del Hierro/química , Cinética , Lípidos/química , Mitocondrias/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , FrataxinaRESUMEN
Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.
Asunto(s)
Glutarredoxinas/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Sondas Moleculares , Compuestos de Sulfhidrilo/metabolismo , Proteínas Hierro-Azufre/metabolismo , CinéticaRESUMEN
Iron-sulfur (Fe-S) clusters are essential protein cofactors for most life forms. In human mitochondria, the core Fe-S biosynthetic enzymatic complex (called SDUF) consists of NFS1, ISD11, ISCU2, and frataxin (FXN) protein components. Few mechanistic details about how this complex synthesizes Fe-S clusters and how these clusters are delivered to targets are known. Here circular dichroism and Mössbauer spectroscopies were used to reveal details of the Fe-S cluster assembly reaction on the SDUF complex. SDUF reactions generated [2Fe-2S] cluster intermediates that readily converted to stable [2Fe-2S] clusters bound to uncomplexed ISCU2. Similar reactions that included the apo Fe-S acceptor protein human ferredoxin (FDX1) resulted in formation of [2Fe-2S]-ISCU2 rather than [2Fe-2S]-FDX1. Subsequent addition of dithiothreitol (DTT) induced transfer of the cluster from ISCU2 to FDX1, suggesting that [2Fe-2S]-ISCU2 is an intermediate. Reactions that initially included DTT rapidly generated [2Fe-2S]-FDX1 and bypassed formation of [2Fe-2S]-ISCU2. In the absence of apo-FDX1, incubation of [2Fe-2S]-ISCU2 with DTT generated [4Fe-4S]-ISCU2 species. Together, these results conflict with a recent report of stable [4Fe-4S] cluster formation on the SDUF complex. Rather, they support a model in which SDUF builds transient [2Fe-2S] cluster intermediates that generate clusters on sulfur-containing molecules, including uncomplexed ISCU2. Additional small molecule or protein factors are required for the transfer of these clusters to Fe-S acceptor proteins or the synthesis of [4Fe-4S] clusters.
Asunto(s)
Proteínas Hierro-Azufre/metabolismo , Biocatálisis , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Dicroismo Circular , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/genética , Proteínas de Unión a Hierro/metabolismo , Proteínas Reguladoras del Hierro/química , Proteínas Reguladoras del Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Estructura Molecular , Azufre/metabolismo , FrataxinaRESUMEN
Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.
Asunto(s)
Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Dicroismo Circular , Humanos , Hierro/metabolismo , Proteínas de Unión a Hierro/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Cinética , Azufre/metabolismo , FrataxinaRESUMEN
Iron-sulfur (Fe-S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe-S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe-S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe-2S] and [4Fe-4S] clusters), ligand environments ([2Fe-2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe-S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe-S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe-2S]-DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe-S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe-S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. We anticipate that this cluster detection methodology will transform the study of Fe-S cluster pathways and potentially other metal cofactor biosynthetic pathways.
Asunto(s)
Vías Biosintéticas , Colorantes Fluorescentes/análisis , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Catálisis , Ditiotreitol/química , Ditiotreitol/metabolismo , Fluorescencia , Colorantes Fluorescentes/química , CinéticaRESUMEN
Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.
Asunto(s)
Proteínas de Unión a Hierro/química , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Azufre/química , Cisteína/química , Humanos , Proteínas de Unión a Hierro/fisiología , Proteínas Hierro-Azufre/biosíntesis , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/fisiología , Unión Proteica/fisiología , Especificidad por Sustrato/fisiología , Azufre/metabolismo , FrataxinaRESUMEN
Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily "young" enzyme, and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate-degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans ), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to 5 orders of magnitude, achieving absolute values of catalytic efficiencies up to 10(6) M(-1) s(-1). Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.
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Organofosfatos/metabolismo , Hidrolasas de Triéster Fosfórico/genética , Hidrolasas de Triéster Fosfórico/metabolismo , Pseudomonas/enzimología , Pseudomonas/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Cristalografía por Rayos X , Hidrólisis , Modelos Moleculares , Mutagénesis , Hidrolasas de Triéster Fosfórico/química , Conformación Proteica , Pseudomonas/química , Pseudomonas/genética , Estereoisomerismo , Especificidad por SustratoRESUMEN
Phosphotriesterase (PTE) from soil bacteria is known for its ability to catalyze the detoxification of organophosphate pesticides and chemical warfare agents. Most of the organophosphate chemical warfare agents are a mixture of two stereoisomers at the phosphorus center, and the S(P)-enantiomers are significantly more toxic than the R(P)-enantiomers. In previous investigations, PTE variants were created through the manipulation of the substrate binding pockets and these mutants were shown to have greater catalytic activities for the detoxification of the more toxic S(P)-enantiomers of nerve agent analogues for GB, GD, GF, VX, and VR than the less toxic R(P)-enantiomers. In this investigation, alternate strategies were employed to discover additional PTE variants with significant improvements in catalytic activities relative to that of the wild-type enzyme. Screening and selection techniques were utilized to isolate PTE variants from randomized libraries and site specific modifications. The catalytic activities of these newly identified PTE variants toward the S(P)-enantiomers of chromophoric analogues of GB, GD, GF, VX, and VR have been improved up to 15000-fold relative to that of the wild-type enzyme. The X-ray crystal structures of the best PTE variants were determined. Characterization of these mutants with the authentic G-type nerve agents has confirmed the expected improvements in catalytic activity against the most toxic enantiomers of GB, GD, and GF. The values of k(cat)/K(m) for the H257Y/L303T (YT) mutant for the hydrolysis of GB, GD, and GF were determined to be 2 × 10(6), 5 × 10(5), and 8 × 10(5) M(-1) s(-1), respectively. The YT mutant is the most proficient enzyme reported thus far for the detoxification of G-type nerve agents. These results support a combinatorial strategy of rational design and directed evolution as a powerful tool for the discovery of more efficient enzymes for the detoxification of organophosphate nerve agents.
Asunto(s)
Proteínas Bacterianas/química , Sustancias para la Guerra Química/química , Compuestos Organofosforados/química , Hidrolasas de Triéster Fosfórico/química , Proteínas Bacterianas/genética , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Enterobacter aerogenes/enzimología , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Hidrólisis , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas de Triéster Fosfórico/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sarín/química , Soman/química , EstereoisomerismoRESUMEN
Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.
Asunto(s)
Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Azufre/metabolismo , Liasas de Carbono-Azufre/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de Unión a Hierro/química , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Homología de Secuencia de Aminoácido , FrataxinaRESUMEN
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.
Asunto(s)
Ataxia de Friedreich/metabolismo , Proteínas de Unión a Hierro/química , Proteínas de Unión a Hierro/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Mutantes/metabolismo , Mutación Missense/genética , Sulfuros/metabolismo , Regulación Alostérica , Liasas de Carbono-Azufre/genética , Liasas de Carbono-Azufre/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Ataxia de Friedreich/genética , Humanos , Proteínas de Unión a Hierro/genética , Proteínas Reguladoras del Hierro/metabolismo , Proteínas Hierro-Azufre/genética , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , FrataxinaRESUMEN
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the â¼15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn â¼ I154F > W155F > W155A â¼ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.