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Jeffrey A Gralnick - One of the best experts on this subject based on the ideXlab platform.
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Flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of Flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that Flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that Flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a Flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of Flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
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Flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of Flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that Flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that Flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a Flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of Flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboFlavin and Flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that Flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled Flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in Flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
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the mtr respiratory pathway is essential for reducing Flavins and electrodes in shewanella oneidensis
Journal of Bacteriology, 2010Co-Authors: Dan Coursolle, Daniel B Baron, Daniel R Bond, Jeffrey A GralnickAbstract:The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboFlavin and Flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated Flavins in supernatants to an extent similar to that of MR-1, suggesting that Flavin secretion is a general trait of the species. External Flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external Flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of Flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external Flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled Flavin concentrations. While mutants lacking MtrC were able to reduce Flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in Flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.
M. Fontecave - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of NAD(P)H:Flavin oxidoreductase from Escherichia coli.
Biochemistry, 1999Co-Authors: Margareta Ingelman, M. Fontecave, V Nivière, S. Ramaswamy, Hans EklundAbstract:Flavin reductases use Flavins as substrates and are distinct from flavoenzymes which have tightly bound Flavins. The reduced Flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced Flavins produced by Flavin reductase. The crystal structure of E. coli Flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between Flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the Flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for Flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboFlavin in a substrate complex.
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The NAD(P)H:Flavin oxidoreductase from Escherichia coli as a source of superoxide radicals.
Journal of Biological Chemistry, 1994Co-Authors: P Gaudu, V Nivière, D Touati, M. FontecaveAbstract:The NAD(P)H:Flavin oxidoreductase (encoded by the fre gene) of Escherichia coli is a soluble enzyme which, under aerobic conditions and together with NAD(P)H and Flavins, generates superoxide radicals selectively. This was demonstrated from spin trapping experiments and from the ability of the Flavin reductase to achieve a superoxide dismutase (SOD)-sensitive reduction of cytochrome c. The participation of the Flavin reductase to O2-. generation in E. coli cells has been studied. Superoxide production in dialyzed cytosolic fraction of SOD-deficient E. coli was stimulated by the addition of Flavins. There was no stimulation in soluble extracts of Flavin reductase-deficient strains. Moreover, using fusions of sodA promoter to lacZ, we showed that sodA transcription was diminished in Flavin reductase-deficient E. coli and that the induction of MnSOD by Flavin reductase was SoxRS-independent. These results suggest that the Flavin reductase might: (i) in vivo, be an important cytosolic site of O2-. generation; (ii) in vitro, serve as a simple, efficient, and selective O2-. generator.
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Ferric Reductases in Escherichia coli: The Contribution of the Hemoglobin-like Protein
Biochemical and biophysical research communications, 1994Co-Authors: Michel Eschenbrenner, Jacques Covès, M. FontecaveAbstract:Abstract The haemoglobin-like protein (HMP) of E. coli previously isolated as a dihydropteridine reductase was shown to be also a ferric citrate reductase. We demonstrate that, in fact, HMP is a Flavin reductase and that its ferric reductase activity is a result of its ability to reduce free Flavins. However, when compared to the two main ferric/Flavin reductases of E. coli, i.e.,the NAD(P)H : Flavin oxidoreductase and the sulfite reductase, one can conclude that the contribution of HMP to iron reduction is negligible.
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Ferric reductases or Flavin reductases?
Biometals, 1994Co-Authors: M. Fontecave, Jacques Covès, Jean-louis PierreAbstract:Assimilation of iron by microorganisms requires the presence of ferric reductases which participate in the mobilization of iron from ferrisiderophores. The common structural and catalytic properties of these enzymes are described and shown to be identical to those of Flavin reductases. This strongly suggests that, in general, the reduction of iron depends on reduced Flavins provided by Flavin reductases.
Annefrances Miller - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic evidence for direct Flavin Flavin contact in a bifurcating electron transfer flavoprotein
Journal of Biological Chemistry, 2020Co-Authors: Diessel H Duan, Nishya Mohamedraseek, Annefrances MillerAbstract:A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both Flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both Flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-Flavin and Bf-Flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of Flavin dimers. Although such a CT complex has been demonstrated for free Flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct Flavin-Flavin contact.
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distinct properties underlie Flavin based electron bifurcation in a novel electron transfer flavoprotein fixab from rhodopseudomonas palustris
Journal of Biological Chemistry, 2018Co-Authors: Diessel H Duan, Carolyn E Lubner, Monika Tokminalukaszewska, George H Gauss, Brian Bothner, Paul W King, John W Peters, Annefrances MillerAbstract:A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin Flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different Flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual Flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° Flavin displays sequential one-electron (1-e-) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° Flavin displayed a single two-electron (2-e-) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two Flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two Flavins.
Nicholas J Kotloski - One of the best experts on this subject based on the ideXlab platform.
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Flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of Flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that Flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that Flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a Flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of Flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
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Flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis
Mbio, 2013Co-Authors: Nicholas J Kotloski, Jeffrey A GralnickAbstract:ABSTRACT Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of Flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete Flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe ( b acterial f lavin adenine dinucleotide [FAD] e xporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular Flavins, while overexpression of bfe increased the concentration of extracellular Flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous Flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δ bfe mutant. With the same concentration of supplemented Flavins, the two strains produced similar amounts of current. However, when exogenous Flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that Flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that Flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a Flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of Flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete Flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.
Dennis J Stuehr - One of the best experts on this subject based on the ideXlab platform.
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characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast pichia pastoris calmodulin response is complete within the reductase domain itself
Journal of Biological Chemistry, 1996Co-Authors: Ratan Gachhui, Anthony Presta, Dennis F Bentley, Husam M Abusoud, Ryan Mcarthur, Gary W Brudvig, Dipak K Ghosh, Dennis J StuehrAbstract:Abstract Rat neuronal NO synthase (nNOS) is comprised of a Flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its Flavins, triggers electron transfer from Flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2′,5′-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable Flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the Flavin semiquinone re-formed after addition of 1-electron equivalent and the Flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent Flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and Flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.