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Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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spectral deconvolution of redox species in the crotonyl coa dependent nadh ferredoxin oxidoreductase from megasphaera elsdenii a flavin dependent bifurcating enzyme
Archives of Biochemistry and Biophysics, 2021Co-Authors: Wayne Vigil, Nilanjan Pal Chowdhury, Wolfgang Buckel, Dimitri Niks, Sophie Franzbadur, Russ HilleAbstract:We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.
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rapid kinetics reveal surprising flavin chemistry in the bifurcating electron transfer flavoprotein from acidaminococcus fermentans
Journal of Biological Chemistry, 2021Co-Authors: Jeerus Sucharitakul, Wolfgang Buckel, Pimchai ChaiyenAbstract:Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. β-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD•−). The resultant α-FAD hydroquinone (α-FADH−) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce Crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD•− to monitor formation of α-FAD•− or α-FADH−, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to β-FAD at a rate of 920 s−1, yielding the charge–transfer complex NAD+:β-FADH− with an absorbance maximum at 650 nm. β-FADH− bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD•−. With α-FAD•−, the reduction of β-FAD with NADH was 1500 times slower. Reduction of β-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD•−. These data indicate that a nearby radical (14 A apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf–Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.
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modulations of the reduction potentials of flavin based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow
FEBS Journal, 2020Co-Authors: Jeerus Sucharitakul, Supacha Buttranon, Thanyaporn Wongnate, Methinee Prongjit, Nilanjan Pal Chowdhury, Wolfgang Buckel, Pimchai ChaiyenAbstract:: The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, β-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces β-FAD to β-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces Crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of β-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from β-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.
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Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review
Frontiers Media S.A., 2018Co-Authors: Wolfgang Buckel, Rudolf K ThauerAbstract:Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of Crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
Nilanjan Pal Chowdhury - One of the best experts on this subject based on the ideXlab platform.
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spectral deconvolution of redox species in the crotonyl coa dependent nadh ferredoxin oxidoreductase from megasphaera elsdenii a flavin dependent bifurcating enzyme
Archives of Biochemistry and Biophysics, 2021Co-Authors: Wayne Vigil, Nilanjan Pal Chowdhury, Wolfgang Buckel, Dimitri Niks, Sophie Franzbadur, Russ HilleAbstract:We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.
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modulations of the reduction potentials of flavin based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow
FEBS Journal, 2020Co-Authors: Jeerus Sucharitakul, Supacha Buttranon, Thanyaporn Wongnate, Methinee Prongjit, Nilanjan Pal Chowdhury, Wolfgang Buckel, Pimchai ChaiyenAbstract:: The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, β-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces β-FAD to β-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces Crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of β-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from β-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.
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the semiquinone swing in the bifurcating electron transferring flavoprotein butyryl coa dehydrogenase complex from clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Publishing Group, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron-transferring flavoprotein / butyryl-CoA dehydrogenase (EtfAB/Bcd) complex catalyzes the reduction of Crotonyl-CoA and ferredoxins by NADH in anaerobic microbes. Here, the authors present the crystal structure of Clostridium difficile EtfAB/Bcd and discuss the bifurcation mechanism for electron flow
Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.
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Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review
Frontiers Media S.A., 2018Co-Authors: Wolfgang Buckel, Rudolf K ThauerAbstract:Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0′ = −420 mV) with NADH (−320 mV) to the exergonic reduction of Crotonyl-CoA to butyryl-CoA (−10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms—in which they have been found—are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered
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energy conservation via electron bifurcating ferredoxin reduction and proton na translocating ferredoxin oxidation
Biochimica et Biophysica Acta, 2013Co-Authors: Wolfgang Buckel, Rudolf K ThauerAbstract:Abstract The review describes four flavin-containing cytoplasmatic multienzyme complexes from anaerobic bacteria and archaea that catalyze the reduction of the low potential ferredoxin by electron donors with higher potentials, such as NAD(P)H or H 2 at ≤ 100 kPa. These endergonic reactions are driven by concomitant oxidation of the same donor with higher potential acceptors such as Crotonyl-CoA, NAD + or heterodisulfide (CoM-S-S-CoB). The process called flavin-based electron bifurcation (FBEB) can be regarded as a third mode of energy conservation in addition to substrate level phosphorylation (SLP) and electron transport phosphorylation (ETP). FBEB has been detected in the clostridial butyryl-CoA dehydrogenase/electron transferring flavoprotein complex (BcdA-EtfBC), the multisubunit [FeFe]hydrogenase from Thermotoga maritima (HydABC) and from acetogenic bacteria, the [NiFe]hydrogenase/heterodisulfide reductase (MvhADG–HdrABC) from methanogenic archaea, and the transhydrogenase (NfnAB) from many Gram positive and Gram negative bacteria and from anaerobic archaea. The Bcd/EtfBC complex that catalyzes electron bifurcation from NADH to the low potential ferredoxin and to the high potential Crotonyl-CoA has already been studied in some detail. The bifurcating protein most likely is EtfBC, which in each subunit (βγ) contains one FAD. In analogy to the bifurcating complex III of the mitochondrial respiratory chain and with the help of the structure of the human ETF, we propose a conformational change by which γ-FADH − in EtfBC approaches β-FAD to enable the bifurcating one-electron transfer. The ferredoxin reduced in one of the four electron bifurcating reactions can regenerate H 2 or NADPH, reduce CO 2 in acetogenic bacteria and methanogenic archaea, or is converted to ΔμH + /Na + by the membrane-associated enzyme complexes Rnf and Ech, whereby NADH and H 2 are recycled, respectively. The mainly bacterial Rnf complexes couple ferredoxin oxidation by NAD + with proton/sodium ion translocation and the more diverse e nergy c onverting [NiFe] h ydrogenases (Ech) do the same, whereby NAD + is replaced by H + . Many organisms also use Rnf and Ech in the reverse direction to reduce ferredoxin driven by ΔμH + /Na + . Finally examples are shown, in which the four bifurcating multienzyme complexes alone or together with Rnf and Ech are integrated into energy metabolisms of nine anaerobes. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.
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NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri
Journal of Bacteriology, 2010Co-Authors: Shuning Wang, Johanna Moll, Haiyan Huang, Rudolf K ThauerAbstract:It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of Crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP+ with reduced ferredoxin (Fdred) and the endergonic reduction of NADP+ with NADH in a reversible reaction: Fdred2− + NADH + 2 NADP+ + H+ = Fdox + NAD+ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.
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coupled ferredoxin and crotonyl coenzyme a coa reduction with nadh catalyzed by the butyryl coa dehydrogenase etf complex from clostridium kluyveri
Journal of Bacteriology, 2008Co-Authors: Fuli Li, Julia Hinderberger, Henning Seedorf, Wolfgang Buckel, Jin Zhang, Rudolf K ThauerAbstract:Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via Crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of Crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 Crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper.
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Coupled ferredoxin- and Crotonyl-CoA reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri
2008Co-Authors: H. Hinderberger, Henning Seedorf, Wolfgang Buckel, Jin Zhang, Rudolf K ThauerAbstract:Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via Crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0' = -410 mV) with NADH (E0' = -320 mV) to the exergonic reduction of Crotonyl-CoA to butyryl-CoA (E0' = -10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 Crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper
Ulrich Ermler - One of the best experts on this subject based on the ideXlab platform.
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the semiquinone swing in the bifurcating electron transferring flavoprotein butyryl coa dehydrogenase complex from clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Publishing Group, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron-transferring flavoprotein / butyryl-CoA dehydrogenase (EtfAB/Bcd) complex catalyzes the reduction of Crotonyl-CoA and ferredoxins by NADH in anaerobic microbes. Here, the authors present the crystal structure of Clostridium difficile EtfAB/Bcd and discuss the bifurcation mechanism for electron flow
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studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein etf and butyryl coa dehydrogenase bcd of acidaminococcus fermentans
Journal of Biological Chemistry, 2014Co-Authors: Nilanjan Pal Chowdhury, Jörg Kahnt, Julius K Demmer, Amr M Mowafy, Vikrant Upadhyay, Sebastian Koelzer, Ulrike Demmer, Elamparithi Jayamani, Marco Hornung, Ulrich ErmlerAbstract:Abstract Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the two [4Fe-4S] cluster-containing ferredoxin, an energy rich compound that is used to conduct difficult reductions as well as to increase the efficiency of substrate level and electron transport phosphorylations (SLP and ETP). Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) from Acidaminococcus fermentans which couple the exergonic reduction of Crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Angstrom. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH− is considered as the bifurcating electron donor. Due to a conformational change, α-FAD is able to approach β-FADH− by ca. 5 Angstrom and take up one electron yielding a stable anionic semiquinone, α-FAD●−, which due to a second conformational change donates this electron further to FAD of BcdAf. The remaining non-stabilized neutral semiquinone, β-FADH●, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and FADH− of BcdAf that converts Crotonyl-CoA to butyryl-CoA.
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the semiquinone swing in the bifurcating electron transferring flavoprotein butyryl coa dehydrogenase complex from clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Communications, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one Crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 A apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 A apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces Crotonyl-CoA.
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The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile
Nature Publishing Group, 2017Co-Authors: Julius K Demmer, Ulrich Ermler, Thorsten Selmer, Nilanjan Pal Chowdhury, Wolfgang BuckelAbstract:The electron-transferring flavoprotein / butyryl-CoA dehydrogenase (EtfAB/Bcd) complex catalyzes the reduction of Crotonyl-CoA and ferredoxins by NADH in anaerobic microbes. Here, the authors present the crystal structure of Clostridium difficile EtfAB/Bcd and discuss the bifurcation mechanism for electron flow
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studies on the mechanism of electron bifurcation catalyzed by electron transferring flavoprotein etf and butyryl coa dehydrogenase bcd of acidaminococcus fermentans
Journal of Biological Chemistry, 2014Co-Authors: Nilanjan Pal Chowdhury, Jörg Kahnt, Julius K Demmer, Amr M Mowafy, Vikrant Upadhyay, Sebastian Koelzer, Ulrike Demmer, Elamparithi Jayamani, Marco Hornung, Ulrich ErmlerAbstract:Abstract Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the two [4Fe-4S] cluster-containing ferredoxin, an energy rich compound that is used to conduct difficult reductions as well as to increase the efficiency of substrate level and electron transport phosphorylations (SLP and ETP). Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) from Acidaminococcus fermentans which couple the exergonic reduction of Crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Angstrom. The EtfAf-NAD+ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH− is considered as the bifurcating electron donor. Due to a conformational change, α-FAD is able to approach β-FADH− by ca. 5 Angstrom and take up one electron yielding a stable anionic semiquinone, α-FAD●−, which due to a second conformational change donates this electron further to FAD of BcdAf. The remaining non-stabilized neutral semiquinone, β-FADH●, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and FADH− of BcdAf that converts Crotonyl-CoA to butyryl-CoA.