The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
Nigel S Scrutton - One of the best experts on this subject based on the ideXlab platform.
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probing the dynamic interface between trimethylamine dehydrogenase tmadh and Electron Transferring Flavoprotein etf in the tmadh 2etf complex role of the arg alpha237 etf and tyr 442 tmadh residue pair
Biochemistry, 2008Co-Authors: Selena G Burgess, David Leys, Hanan L Messiha, Gergely Katona, Stephen E J Rigby, Nigel S ScruttonAbstract:We have used multiple solution state techniques and crystallographic analysis to investigate the importance of a putative transient interaction formed between Arg-a237 in Electron Transferring Flavoprotein (ETF) and Tyr-442 in trimethylamine dehydrogenase (TMADH) in complex assembly, Electron transfer, and structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF altered in the identity of the residue at position 237 (αR237A, αR237K, αR237C, and αR237E) and with each form studied Electron transfer from TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor, and analyzed complex formation. We show that mutation of Arg-a237 substantially destabilizes the semiquinone couple of the bound FAD and impedes Electron transfer from TMADH to ETF. Crystallographic structures of the mutant ETF proteins indicate that mutation does not perturb the overall structure of ETF, but leads to disruption of an electrostatic network at an ETF domain boundary that likely affects the dynamic properties of ETF in the crystal and in solution. We show that Arg-a237 is required for TMADH to structurally imprint the as-purified semiquinone form of wild-type ETF and that the ability of TMADH to facilitate this structural reorganization is lost following (i) redox cycling of ETF, or simple conversion to the oxidized form, and (ii) mutagenesis of Arg-a237. We discuss this result in light of recent apparent conflict in the literature relating to the structural imprinting of wild-type ETF. Our studies support a mechanism of Electron transfer by conformational sampling as advanced from our previous analysis of the crystal structure of the TMADH-2ETF complex [Leys, D., Basran, J., Sutcliffe, M. J., and Scrutton, N. S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the Tyr-442 (TMADH) and Arg-a237 (ETF) residue pair in transiently stabilizing productive Electron transfer configurations. Our work also points to the importance of Arg-a237 in controlling the thermodynamics of Electron transfer, the dynamics of ETF, and the protection of reducing equivalents following disassembly of the TMADH-2ETF complex.
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stabilization of non productive conformations underpins rapid Electron transfer to Electron Transferring Flavoprotein
Journal of Biological Chemistry, 2005Co-Authors: Helen S Toogood, Nigel S Scrutton, Adam Van Thiel, David LeysAbstract:Abstract Crystal structures of protein complexes with Electron-Transferring Flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165β in human ETF leads to drastically modulated rates of interprotein Electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165βA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165βA ETF·medium chain acyl-CoA dehydrogenase complex reveals clear Electron density for the FAD domain in a position optimal for fast interprotein Electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF· medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165β plays a key role in stabilizing positions incompatible with fast interprotein Electron transfer, thus ensuring high rates of complex dissociation.
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extensive domain motion and Electron transfer in the human Electron Transferring Flavoprotein medium chain acyl coa dehydrogenase complex
Journal of Biological Chemistry, 2004Co-Authors: Helen S Toogood, Jaswir Basran, Michael J Sutcliffe, Nigel S Scrutton, Adam Van Thiel, David LeysAbstract:Abstract The crystal structure of the human Electron Transferring Flavoprotein (ETF)·medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and Electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF·MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein Electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD·ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu212 and ETF Argα249 are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced Electron transfer rates between both partners.
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flavin radicals conformational sampling and robust design principles in interprotein Electron transfer the trimethylamine dehydrogenase Electron Transferring Flavoprotein complex
Biochemical Society Symposia, 2004Co-Authors: David Leys, Jaswir Basran, Kamaldeep K Chohan, Michael J Sutcliffe, Francois Talfournier, Andrew W Munro, Nigel S ScruttonAbstract:TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur Flavoprotein that forms a soluble Electron-transfer complex with ETF (Electron-Transferring Flavoprotein). The mechanism of Electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 9conformational sampling9 in the Electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced Electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of Electron transfer provides for a more robust and accessible design principle for interprotein Electron transfer compared with simpler models that invoke the collision of redox partners followed by Electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in Electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.
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Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and Electron Transferring Flavoprotein
Journal of Biological Chemistry, 2002Co-Authors: Matthew Jones, Michael J Sutcliffe, Francois Talfournier, Anton Bobrov, Gunter J Grossmann, Nikolai Vekshin, Nigel S ScruttonAbstract:Abstract The trimethylamine dehydrogenase-Electron Transferring Flavoprotein (TMADH·ETF) Electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH·ETF Electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (αR237A) complexes are much slower than are the corresponding rates of Electron transfer in these complexes. This suggests that Electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in Transferring Electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH·ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of Electron transfer to the artificial Electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH·ETF Electron transfer complex, and that the properties of Electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological Electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitrointerprotein Electron transfer reactions to those occurring in vivo.
Wolfgang Buckel - 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, Nilanjan Pal Chowdhury, Thorsten Selmer, Ulrich Ermler, 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, Nilanjan Pal Chowdhury, Thorsten Selmer, Ulrich Ermler, 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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effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase Electron Transferring Flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
Journal of Bacteriology, 2013Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two Electron-Transferring Flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables Electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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energy conservation via Electron Transferring Flavoprotein in anaerobic bacteria
Journal of Bacteriology, 2008Co-Authors: Gloria Herrmann, Elamparithi Jayamani, Wolfgang BuckelAbstract:Energy conservation in chemotrophic organisms is generally coupled to redox reactions in catabolic pathways. In the oxidative part or branch, “energy-rich” compounds are formed, from which ATP is generated via substrate-level phosphorylation (SLP). In the reductive branch the Electron carriers
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acryloyl coa reductase from clostridium propionicum an enzyme complex of propionyl coa dehydrogenase and Electron Transferring Flavoprotein
FEBS Journal, 2003Co-Authors: Marc Hetzel, Thorsten Selmer, Matthias Brock, Antonio J Pierik, Bernard T Golding, Wolfgang BuckelAbstract:Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow–greenish enzyme complex [(α2βγ)4; molecular mass 600 ± 50 kDa] composed of a propionyl-CoA dehydrogenase (α2, 2 × 40 kDa) and an Electron-Transferring Flavoprotein (ETF; β, 38 kDa; γ, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per α2βγ subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 ± 1 µm; kcat = 4.5 s−1 at 100 µm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 µm; kcat = 29 s−1 at 300 µm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 µm; kcat = 2.0 s−1) or butyryl-CoA (Km = 100 µm; kcat = 3.5 s−1) as Electron donor and 200 µm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the β and γ subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as Electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
Kiyoshi Shiga - One of the best experts on this subject based on the ideXlab platform.
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decomposition of the fluorescence spectra of two fad molecules in Electron Transferring Flavoprotein from megasphaera elsdenii
Journal of Biochemistry, 2013Co-Authors: Kyosuke Sato, Yasuzo Nishina, Kiyoshi ShigaAbstract:Electron-Transferring Flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.
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Interaction between NADH and Electron-Transferring Flavoprotein from Megasphaera elsdenii
Journal of Biochemistry, 2013Co-Authors: Kyosuke Sato, Yasuzo Nishina, Kiyoshi ShigaAbstract:Electron-Transferring Flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-Electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives Electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-Electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives Electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.
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isomers in the excited state of Electron Transferring Flavoprotein from megasphaera elsdenii spectral resolution from the time resolved fluorescence spectra
Journal of Photochemistry and Photobiology B-biology, 2008Co-Authors: Kyosuke Sato, Yasuzo Nishina, Kiyoshi Shiga, Fumio TanakaAbstract:Abstract Electron-Transferring Flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD’s, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 °C and 25 °C. Wavelength-dependent fluorescence decays of the both ETF at 5 °C and 25 °C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 °C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 °C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 °C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 °C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.
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purification of Electron Transferring Flavoprotein from megasphaera elsdenii and binding of additional fad with an unusual absorption spectrum
Journal of Biochemistry, 2003Co-Authors: Kyosuke Sato, Yasuzo Nishina, Kiyoshi ShigaAbstract:Electron-Transferring Flavoprotein (ETF), its redox partner Flavoproteins, i.e., D-lactate dehydrogenase and butyryl-CoA dehydrogenase, and another well-known Flavoprotein, flavodoxin, were purified from the same startingcell paste of an anaerobic bacterium, Megasphaera elsdenii. The purified ETF contained one mol FAD/mol ETF as the sole non-protein component and bound almost one mol of additional FAD. This preparation is a better subject for investigations of M. elsdenii ETF than the previously isolated ETF, which contains varying amounts of FAD and varying percentages of modified flavins such as 6-OH-FAD and 8-OH-FAD. The additionally bound FAD shows an anomalous absorption spectrum with strong absorption around 400 nm. This spectral change is not due to a chemical modification of the flavin ring because the flavin released by KBr or guanidine hydrochloride is normal FAD. It is also not due to unknown small molecules because the same spectrum appears when ETF is reconstituted from its guanidine-denatured subunits and FAD. A similar anomalous spectrum was observed for AMP-free pig ETF under acidic conditions, suggesting a common flavin environment between pig and M. elsdenii ETFs.
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hydrogen bonding dynamics of free flavins in benzene and fad in Electron Transferring Flavoprotein upon excitation
Journal of Photochemistry and Photobiology B-biology, 2003Co-Authors: Kyousuke Sato, Kiyoshi Shiga, Yasuzou Nishina, Fumio TanakaAbstract:Abstract The dynamic natures of two hydrogen-bonding model systems, riboflavin tetrabutylate (RFTB)–trichloroacetic acid (TCA) and RFTB–phenol in benzene, and of Electron-Transferring Flavoprotein (ETF) from pig kidney upon excitation of flavins was investigated by means of steady state and time-resolved fluorescence spectroscopy. In both model systems fluorescence intensities of RFTB decreased as TCA or phenol was added. The spectral characteristics of ETF under steady state excitation were quite similar to those of the RFTB–TCA system, but not to those of the RFTB–phenol system. The observed fluorescence decay curves of ETF fit well with the calculated decay curves with two lifetime components, as in the model systems. Averaged lifetime was 0.9 ns. The time-resolved fluorescence spectrum of ETF shifted toward longer wavelength with time after pulsed excitation, which was also observed in the RFTB–TCA system. In the RFTB–phenol system the emission spectrum did not shift at all with time. These results reveal that the dynamic nature of ETF can be ascribed to aliphatic hydrogen-bonding(s) of the isoalloxazine ring with surrounding amino acid(s). From the fluorescence characteristics of ETF in comparison with the model systems, human ETF and other Flavoproteins, it was suggested that ETF from pig kidney does not contain Tyr-16 in the β subunit, unlike human ETF.
Jerry Vockley - One of the best experts on this subject based on the ideXlab platform.
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mammalian Electron Transferring Flavoprotein Flavoprotein dehydrogenase complexes observed by microelectrospray ionization mass spectrometry and surface plasmon resonance
Journal of Biological Chemistry, 2004Co-Authors: Heidi M Hoardfruchey, Eric S Goetzman, Linda Benson, Stephen Naylor, Jerry VockleyAbstract:Abstract Microelectrospray ionization-mass spectrometry was used to directly observe Electron Transferring Flavoprotein·Flavoprotein dehydrogenase interactions. When Electron Transferring Flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the Flavoprotein·Electron Transferring Flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and Electron Transferring Flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase·Electron Transferring Flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to Electron Transferring Flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 μm for the dimethylglycine dehydrogenase-Electron Transferring Flavoprotein and short chain acyl-CoA dehydrogenase-Electron Transferring Flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on Electron Transferring Flavoprotein for dehydrogenase interaction.
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microelectrospray ionization analysis of noncovalent interactions within the Electron Transferring Flavoprotein
Biochemical and Biophysical Research Communications, 2001Co-Authors: Heidi M Hoard, Jerry Vockley, Linda M Benson, Stephen NaylorAbstract:Abstract Cofactor associations within the Electron Transferring Flavoprotein (ETF) were studied in real time using microelectrospray ionization-mass spectrometry (μESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) revealed only the holoprotein. When μESI-MS source energies were increased, both pETF and hETF readily lost AMP. Analysis of hETF and pETF in methanol revealed intact α- and β-subunits, and β-subunit with AMP. The pETF also contained β-subunit with FAD and β-subunit with both cofactors. In contrast to crystal structure predictions, AMP dissociates more readily than FAD, and the pETF β-subunit has an intimate association with FAD. This work demonstrates the complementarity of μESI-MS with NMR X-ray and optical spectroscopy in the analysis of noncovalent complexes.
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Identification of Caenorhabditis elegans isovaleryl-CoA dehydrogenase and structural comparison with other Acyl-CoA dehydrogenases
Molecular Genetics and Metabolism, 2001Co-Authors: Al Walid A Mohsen, Bryan Navarette, Jerry VockleyAbstract:Isovaleryl-CoA dehydrogenase (IVD) is a flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway and transfers Electrons to the Electron-Transferring Flavoprotein (ETF). IVDs from human and rat have been identified and characterized previously. In this study, the gene coding for Caenorhabditis elegans IVD has been identified from a published cDNA sequence and molecular modeling has been performed using the human IVD atomic coordinates. The coding sequence for the mature form of the enzyme was expressed in Escherichia coli, and the recombinant nematode IVD enzyme was purified to essential homogeneity. Its spectrum is typical of recombinant FAD-containing acyl-CoA dehydrogenases and shows a minor broad absorption band at 650-700 nm characteristic of an IVD:CoA persulfide charge-transfer complex. Following treatment of the enzyme with sodium dithionite to remove the bound CoA persulfide, the Km values for isovaleryl-, butyryl-, valeryl-, and hexanoyl-CoA were estimated to be 2.5, 36.2, 10.5, and 33.8 μM, respectively, using the ETF fluorescence reduction assay. The catalytic efficiency (kcat/Km) for these substrates was 56.9, 1.3, 13.7, and 3.2 μM-1 min-1 per mole of FAD, respectively. The apparent binding constant (KD app) of the recombinant IVD determined spectrally for isovaleryl-CoA was 0.34 μM. These kinetic parameters confirm that isovaleryl-CoA is the preferred substrate for the purified enzyme. The variability in the protein structure among known and putative IVDs from various species is discussed in the context of possible mechanisms for modulating enzyme activity. © 2001 Academic Press.
Thorsten Selmer - 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, Nilanjan Pal Chowdhury, Thorsten Selmer, Ulrich Ermler, 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, Nilanjan Pal Chowdhury, Thorsten Selmer, Ulrich Ermler, 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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effect of an oxygen tolerant bifurcating butyryl coenzyme a dehydrogenase Electron Transferring Flavoprotein complex from clostridium difficile on butyrate production in escherichia coli
Journal of Bacteriology, 2013Co-Authors: Wolfgang Buckel, E A Aboulnaga, Olaf Pinkenburg, Johannes Schiffels, Ahmed Elrefai, Thorsten SelmerAbstract:ABSTRACT The butyrogenic genes from Clostridium difficile DSM 1296 T have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two Electron-Transferring Flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro . While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD + -oxidoreductase enables Electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases ( R hodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.
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acryloyl coa reductase from clostridium propionicum an enzyme complex of propionyl coa dehydrogenase and Electron Transferring Flavoprotein
FEBS Journal, 2003Co-Authors: Marc Hetzel, Thorsten Selmer, Matthias Brock, Antonio J Pierik, Bernard T Golding, Wolfgang BuckelAbstract:Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow–greenish enzyme complex [(α2βγ)4; molecular mass 600 ± 50 kDa] composed of a propionyl-CoA dehydrogenase (α2, 2 × 40 kDa) and an Electron-Transferring Flavoprotein (ETF; β, 38 kDa; γ, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per α2βγ subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 ± 1 µm; kcat = 4.5 s−1 at 100 µm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 µm; kcat = 29 s−1 at 300 µm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 µm; kcat = 2.0 s−1) or butyryl-CoA (Km = 100 µm; kcat = 3.5 s−1) as Electron donor and 200 µm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the β and γ subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as Electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
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an enzyme complex of propionyl coa dehydrogenase and Electron Transferring Flavoprotein
2003Co-Authors: Marc Hetzel, Thorsten Selmer, Matthias Brock, Antonio J Pierik, Bernard T Golding, Wolfgang BuckelAbstract:dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the b and c subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as Electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloylCoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.