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Achim Kroger - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Δψ and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Δψ generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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Fumarate respiration of wolinella succinogenes enzymology energetics and coupling mechanism
Biochimica et Biophysica Acta, 2002Co-Authors: Achim Kroger, Jörg Simon, Roland Gross, Simone Biel, Gottfried Unden, Roy C D LancasterAbstract:Wolinella succinogenes performs oxidative phosphorylation with Fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('Fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of Fumarate Reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by Fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of Fumarate Reductase suggests an electrogenic mechanism for the latter process.
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structure of Fumarate Reductase from wolinella succinogenes at 2 2 a resolution
Nature, 1999Co-Authors: C. R. D. Lancaster, Manfred Auer, Achim Kroger, Hartmut MichelAbstract:Fumarate Reductase couples the reduction of Fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalysed by the related complex II of the respiratory chain (succinate dehydrogenase). Here we describe the crystal structure at 2.2 A resolution of the three protein subunits containing Fumarate Reductase from the anaerobic bacterium Wolinella succinogenes. Subunit A contains the site of Fumarate reduction and a covalently bound flavin adenine dinucleotide prosthetic group. Subunit B contains three iron–sulphur centres. The menaquinol-oxidizing subunit C consists of five membrane-spanning, primarily helical segments and binds two haem b molecules. On the basis of the structure, we propose a pathway of electron transfer from the dihaem cytochrome b to the site of Fumarate reduction and a mechanism of Fumarate reduction. The relative orientations of the soluble and membrane-embedded subunits of succinate:quinone oxidoReductases appear to be unique.
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a periplasmic flavoprotein in wolinella succinogenes that resembles the Fumarate Reductase of shewanella putrefaciens
Archives of Microbiology, 1998Co-Authors: Jörg Simon, Oliver Klimmek, Roland Gross, Michael Ringel, Achim KrogerAbstract:During growth with Fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic Fumarate Reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze Fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of Fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens Fumarate Reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens Fumarate Reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with Fumarate and formate or sulfide, suggesting that the deleted proteins were not required for Fumarate respiration with either electron donor.
Jörg Simon - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Δψ and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Δψ generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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Fumarate respiration of wolinella succinogenes enzymology energetics and coupling mechanism
Biochimica et Biophysica Acta, 2002Co-Authors: Achim Kroger, Jörg Simon, Roland Gross, Simone Biel, Gottfried Unden, Roy C D LancasterAbstract:Wolinella succinogenes performs oxidative phosphorylation with Fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('Fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of Fumarate Reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by Fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of Fumarate Reductase suggests an electrogenic mechanism for the latter process.
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a third crystal form of wolinella succinogenes quinol Fumarate Reductase reveals domain closure at the site of Fumarate reduction
FEBS Journal, 2001Co-Authors: Roy C D Lancaster, Roland Gros, Jörg SimonAbstract:Quinol:Fumarate Reductase (QFR) is a membrane protein complex that couples the reduction of Fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of Fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kroger, A., Auer, M., & Michel, H. (1999) Nature402, 377–385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14° relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble Fumarate Reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for Fumarate reduction in this superfamily of enzymes.
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a third crystal form of wolinella succinogenes quinol Fumarate Reductase reveals domain closure at the site of Fumarate reduction
FEBS Journal, 2001Co-Authors: Roy C D Lancaster, Roland Gros, Jörg SimonAbstract:Quinol:Fumarate Reductase (QFR) is a membrane protein complex that couples the reduction of Fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of Fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kroger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble Fumarate Reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for Fumarate reduction in this superfamily of enzymes.
Roy C D Lancaster - One of the best experts on this subject based on the ideXlab platform.
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design synthesis and biological testing of novel naphthoquinones as substrate based inhibitors of the quinol Fumarate Reductase from wolinella succinogenes
Journal of Medicinal Chemistry, 2013Co-Authors: Hamid R Nasiri, Roy C D Lancaster, Gregor M Madej, Robin Panisch, Jan W. Bats, Michael Lafontaine, Harald SchwalbeAbstract:Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/Fumarate Reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure–activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b–k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a–c) and 2-methoxy-1,4-naphthoquinone (5a–b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in lo...
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crystallization of wolinella succinogenes quinol Fumarate Reductase
Membrane Protein Purification and Crystallization (Second Edition)#R##N#A Practical Guide, 2003Co-Authors: Roy C D LancasterAbstract:Quinol:Fumarate Reductases (QFR) and succinate:quinone Reductases (SQR) catalyze the reduction of Fumarate to succinate with concomitant oxidation of hydroquinone (quinol) to quinone, as well as the reverse reaction. SQR (respiratory complex II) is involved in aerobic metabolism as part of the citric acid cycle and the aerobic respiratory chain. QFR is involved in anaerobic respiration with Fumarate as the terminal electron acceptor. QFR and SQR complexes are collectively referred to as succinate:quinone oxidoReductases and are predicted to share similar structures. The complexes consist of two hydrophilic and one or two hydrophobic membrane-integrated subunits. QFR of Wolinella succinogenes and SQR of Bacillus subtilis contain only one hydrophobic subunit (C) with two haem b groups. In contrast, SQR and QFR of Escherichia coli contain two hydrophobic subunits (C and D) that bind either one (SQR) or no haem b group (QFR). There are two principle types of membrane protein crystals. Type I crystals can be thought of as ordered stacks of two-dimensional crystals formed in the planes of the membrane. Type II crystals contain detergents bound in a micellar manner. They are held together via polar interactions between the polar surfaces of the membrane protein.
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Fumarate respiration of wolinella succinogenes enzymology energetics and coupling mechanism
Biochimica et Biophysica Acta, 2002Co-Authors: Achim Kroger, Jörg Simon, Roland Gross, Simone Biel, Gottfried Unden, Roy C D LancasterAbstract:Wolinella succinogenes performs oxidative phosphorylation with Fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('Fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of Fumarate Reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by Fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of Fumarate Reductase suggests an electrogenic mechanism for the latter process.
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a third crystal form of wolinella succinogenes quinol Fumarate Reductase reveals domain closure at the site of Fumarate reduction
FEBS Journal, 2001Co-Authors: Roy C D Lancaster, Roland Gros, Jörg SimonAbstract:Quinol:Fumarate Reductase (QFR) is a membrane protein complex that couples the reduction of Fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of Fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kroger, A., Auer, M., & Michel, H. (1999) Nature402, 377–385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14° relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble Fumarate Reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for Fumarate reduction in this superfamily of enzymes.
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a third crystal form of wolinella succinogenes quinol Fumarate Reductase reveals domain closure at the site of Fumarate reduction
FEBS Journal, 2001Co-Authors: Roy C D Lancaster, Roland Gros, Jörg SimonAbstract:Quinol:Fumarate Reductase (QFR) is a membrane protein complex that couples the reduction of Fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of Fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kroger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble Fumarate Reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for Fumarate reduction in this superfamily of enzymes.
James A. Imlay - One of the best experts on this subject based on the ideXlab platform.
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The Fumarate Reductase of Bacteroides thetaiotaomicron, unlike That of Escherichia coli, Is Configured so that It Does Not Generate Reactive Oxygen Species
Mbio, 2017Co-Authors: James A. ImlayAbstract:: The impact of oxidative stress upon organismal fitness is most apparent in the phenomenon of obligate anaerobiosis. The root cause may be multifaceted, but the intracellular generation of reactive oxygen species (ROS) likely plays a key role. ROS are formed when redox enzymes accidentally transfer electrons to oxygen rather than to their physiological substrates. In this study, we confirm that the predominant intestinal anaerobe Bacteroides thetaiotaomicron generates intracellular ROS at a very high rate when it is aerated. Fumarate Reductase (Frd) is a prominent enzyme in the anaerobic metabolism of many bacteria, including B. thetaiotaomicron, and prior studies of Escherichia coli Frd showed that the enzyme is unusually prone to ROS generation. Surprisingly, in this study biochemical analysis demonstrated that the B. thetaiotaomicron Frd does not react with oxygen at all: neither superoxide nor hydrogen peroxide is formed. Subunit-swapping experiments indicated that this difference does not derive from the flavoprotein subunit at which ROS normally arise. Experiments with the related enzyme succinate dehydrogenase discouraged the hypothesis that heme moieties are responsible. Thus, resistance to oxidation may reflect a shift of electron density away from the flavin moiety toward the iron-sulfur clusters. This study shows that the autoxidizability of a redox enzyme can be suppressed by subtle modifications that do not compromise its physiological function. One implication is that selective pressures might enhance the oxygen tolerance of an organism by manipulating the electronic properties of its redox enzymes so they do not generate ROS. IMPORTANCE: Whether in sediments or pathogenic biofilms, the structures of microbial communities are configured around the sensitivities of their members to oxygen. Oxygen triggers the intracellular formation of reactive oxygen species (ROS), and the sensitivity of a microbe to oxygen likely depends upon the rates at which ROS are formed inside it. This study supports that idea, as an obligate anaerobe was confirmed to generate ROS very rapidly upon aeration. However, the suspected source of the ROS was disproven, as the Fumarate Reductase of the anaerobe did not display the high oxidation rate of its E. coli homologue. Evidently, adjustments in its electronic structure can suppress the tendency of an enzyme to generate ROS. Importantly, this outcome suggests that evolutionary pressure may succeed in modifying redox enzymes and thereby diminishing the stress that an organism experiences in oxic environments. The actual source of ROS in the anaerobe remains to be discovered.
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The Fumarate Reductase of Bacteroides thetaiotaomicron, unlike That of Escherichia coli, Is Configured so that It Does Not Generate Reactive Oxygen Species
American Society for Microbiology, 2017Co-Authors: James A. Imlay, Matthew R. ChapmanAbstract:The impact of oxidative stress upon organismal fitness is most apparent in the phenomenon of obligate anaerobiosis. The root cause may be multifaceted, but the intracellular generation of reactive oxygen species (ROS) likely plays a key role. ROS are formed when redox enzymes accidentally transfer electrons to oxygen rather than to their physiological substrates. In this study, we confirm that the predominant intestinal anaerobe Bacteroides thetaiotaomicron generates intracellular ROS at a very high rate when it is aerated. Fumarate Reductase (Frd) is a prominent enzyme in the anaerobic metabolism of many bacteria, including B. thetaiotaomicron, and prior studies of Escherichia coli Frd showed that the enzyme is unusually prone to ROS generation. Surprisingly, in this study biochemical analysis demonstrated that the B. thetaiotaomicron Frd does not react with oxygen at all: neither superoxide nor hydrogen peroxide is formed. Subunit-swapping experiments indicated that this difference does not derive from the flavoprotein subunit at which ROS normally arise. Experiments with the related enzyme succinate dehydrogenase discouraged the hypothesis that heme moieties are responsible. Thus, resistance to oxidation may reflect a shift of electron density away from the flavin moiety toward the iron-sulfur clusters. This study shows that the autoxidizability of a redox enzyme can be suppressed by subtle modifications that do not compromise its physiological function. One implication is that selective pressures might enhance the oxygen tolerance of an organism by manipulating the electronic properties of its redox enzymes so they do not generate ROS
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mechanism of superoxide and hydrogen peroxide formation by Fumarate Reductase succinate dehydrogenase and aspartate oxidase
Journal of Biological Chemistry, 2002Co-Authors: Kevin R Messner, James A. ImlayAbstract:Abstract Oxidative stress is created in aerobic organisms when molecular oxygen chemically oxidizes redox enzymes, forming superoxide (O ) and hydrogen peroxide (H2O2). Prior work identified several flavoenzymes from Escherichia coli that tend to autoxidize. Of these, Fumarate Reductase (Frd) is notable both for its high turnover number and for its production of substantial O in addition to H2O2. We have sought to identify characteristics of Frd that predispose it to this behavior. The ability of excess succinate to block autoxidation and the inhibitory effect of lowering the flavin potential indicate that all detectable autoxidation occurs from its FAD site, rather than from iron-sulfur clusters or bound quinones. The flavin adenine dinucleotide (FAD) moiety of Frd is unusually solvent-exposed, as evidenced by its ability to bind sulfite, and this may make it more likely to react adventitiously with O2. The autoxidizing species is apparently fully reduced flavin rather than flavosemiquinone, since treatments that more fully reduce the enzyme do not slow its turnover number. They do, however, switch the major product from O to H2O2. A similar effect is achieved by lowering the potential of the proximal [2Fe-2S] cluster. These data suggest that Frd releases O into bulk solution if this cluster is available to sequester the semiquinone electron; otherwise, that electron is rapidly transferred to the nascent superoxide, and H2O2 is the product that leaves the active site. This model is supported by the behavior of “aspartate oxidase” (aspartate:Fumarate oxidoReductase), an Frd homologue that lacks Fe-S clusters. Its dihydroflavin also reacts avidly with oxygen, and H2O2 is the predominant product. In contrast, succinate dehydrogenase, with high potential clusters, generates O exclusively. The identities of enzyme autoxidation products are significant because O and H2O2 damage cells in different ways.
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a metabolic enzyme that rapidly produces superoxide Fumarate Reductase of escherichia coli
Journal of Biological Chemistry, 1995Co-Authors: James A. ImlayAbstract:Abstract Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of Fumarate Reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of Fumarate Reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant Fumarate Reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that Fumarate Reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of Fumarate Reductase (1600 min), additional steric or electronic factors must accelerate its autoxidation.
Roland Gross - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Δψ = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Δψ and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Δψ. Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Δψ of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Δψ generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Δψ generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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reconstitution of coupled Fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and Fumarate Reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed Fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by Fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by Fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without Fumarate Reductase. Proteoliposomes containing menaquinone and Fumarate Reductase with or without hydrogenase catalyzed Fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with Fumarate Reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of Fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled Fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by Fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
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Fumarate respiration of wolinella succinogenes enzymology energetics and coupling mechanism
Biochimica et Biophysica Acta, 2002Co-Authors: Achim Kroger, Jörg Simon, Roland Gross, Simone Biel, Gottfried Unden, Roy C D LancasterAbstract:Wolinella succinogenes performs oxidative phosphorylation with Fumarate instead of O2 as terminal electron acceptor and H2 or formate as electron donors. Fumarate reduction by these donors ('Fumarate respiration') is catalyzed by an electron transport chain in the bacterial membrane, and is coupled to the generation of an electrochemical proton potential (Deltap) across the bacterial membrane. The experimental evidence concerning the electron transport and its coupling to Deltap generation is reviewed in this article. The electron transport chain consists of Fumarate Reductase, menaquinone (MK) and either hydrogenase or formate dehydrogenase. Measurements indicate that the Deltap is generated exclusively by MK reduction with H2 or formate; MKH2 oxidation by Fumarate appears to be an electroneutral process. However, evidence derived from the crystal structure of Fumarate Reductase suggests an electrogenic mechanism for the latter process.
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a periplasmic flavoprotein in wolinella succinogenes that resembles the Fumarate Reductase of shewanella putrefaciens
Archives of Microbiology, 1998Co-Authors: Jörg Simon, Oliver Klimmek, Roland Gross, Michael Ringel, Achim KrogerAbstract:During growth with Fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic Fumarate Reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze Fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of Fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens Fumarate Reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens Fumarate Reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with Fumarate and formate or sulfide, suggesting that the deleted proteins were not required for Fumarate respiration with either electron donor.
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a periplasmic flavoprotein in wolinella succinogenes that resembles the Fumarate Reductase of shewanella putrefaciens
Archives of Microbiology, 1998Co-Authors: Jörg Simon, Oliver Klimmek, Roland Gross, Michael Ringel, Achim KrogerAbstract:During growth with Fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic Fumarate Reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze Fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of Fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens Fumarate Reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens Fumarate Reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The DeltafccABC mutant of W. succinogenes grew with Fumarate and formate or sulfide, suggesting that the deleted proteins were not required for Fumarate respiration with either electron donor.
