Molecular properties of fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli

1982 ◽  
Vol 60 (8) ◽  
pp. 811-816 ◽  
Author(s):  
John J. Robinson ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Rhodospirillum Rubrum ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Sulfhydryl Group ◽  
Fumarate Reductase ◽  
Molar Ratio ◽  
Heart Mitochondrion ◽  
Visible Absorption ◽  
Nitrobenzoic Acid

Fumarate reductase, purified from the cytoplasmic membrane of Escherichia coli, has been cross-linked with the bifunctional reagent dimethylsuberimidate and shown to exist as an αβ dimer of polypeptides of molecular weights 69 000 and 25 000 in a 1:1 molar ratio. The protein has an s20,W of 7.67S and a D20,W of 6.5 × 10−7 cm2/s. The purified enzyme contained 4–5 mol of nonheme iron and 4–5 mol of acid labile sulfur while the visible absorption spectrum showed a broad peak between 400 and 470 nm owing to the presence of an Fe–S centre and 8α[N-3]histidyl FAD. Fumarate reductase activity was readily inhibited by the sulfhydryl reagents 5,5′-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoate, and iodoacetamide. Using 5,5′-dithiobis-(2-nitrobenzoic acid) sulfhydryl group modification was followed as a function of enzyme activity. A single cysteine residue was shown to be required for activity and this essential sulfhydryl group was located in the 69 000 dalton subunit. The amino acid composition of E. coli fumarate reductase was similar to the succinate dehydrogenases from beef heart mitochondrion and Rhodospirillum rubrum.

scholarly journals The effects of anions on fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 473-477 ◽  
Author(s):  
J J Robinson ◽  
J H Weiner
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Chemical Properties ◽  
Fumarate Reductase ◽  
Maximal Velocity ◽  
Nitrobenzoic Acid ◽  
Thiol Reagent ◽  
Membrane Bound ◽  
Physical And Chemical ◽  
And Chemical Properties

A broad range of anions was shown to stimulate the maximal velocity of purified fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli, while leaving the Km for fumarate unaffected. Reducing agents potentiate the effects of anions on the activity, but have no effect by themselves. Thermal stability, conformation as monitored by circular dichroism and susceptibility to the thiol reagent 5,5′-dithiobis-(2-nitrobenzoic acid) are also altered by anions. The apparent Km for succinate in the reverse reaction (succinate dehydrogenase activity) varies as a function of anion concentration, but the maximal velocity is not affected. The membrane-bound activity is not stimulated by anions and its properties closely resemble those of the purified enzyme in the presence of anions. Thus it appears that anions alter the physical and chemical properties of fumarate reductase, so that it more closely resembles the membrane-bound form.

A model for the structure of fumarate reductase in the cytoplasmic membrane of escherichia coli

1984 ◽  
Vol 24 (3) ◽  
pp. 207-216 ◽  
Author(s):  
Joel H. Weiner ◽  
Bernard D. Lemire ◽  
Robert W. Jones ◽  
Wayne F. Anderson ◽  
Douglas G. Scraba
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Fumarate Reductase

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

1979 ◽  
Vol 57 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sodium Cholate ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Fumarate Reductase ◽  
Molar Ratios ◽  
Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

scholarly journals The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli

1980 ◽  
Vol 190 (1) ◽  
pp. 79-94 ◽  
Author(s):  
Robert W. Jones ◽  
Alan Lamont ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Proton Translocation ◽  
Deficient Mutant ◽  
Benzyl Viologen ◽  
Low Concentrations ◽  
Nitrite Reductase Activity

Low concentrations (1–50μm) of ubiquinol1 were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol1 drove an outward translocation of protons with a corrected →H+/2e− stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309–323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol1 was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA−menA−), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol1. Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ+) to the oxidized species (DQ++) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV+) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV++. In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol1 are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate→H+/2e− stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol1 (QH2), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed→H+/2e− stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]

Cloning and expression of the fumarate reductase gene of Escherichia coli

1981 ◽  
Vol 59 (3) ◽  
pp. 158-164 ◽  
Author(s):  
Elke Lohmeier ◽  
D. Scott Hagen ◽  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Reductase Activity ◽  
Fumarate Reductase ◽  
Cloning And Expression ◽  
Kinetic Properties ◽  
Structural Genes ◽  
E Coli ◽  
Reductase Gene ◽  
Mutant Host

Mutants of Escherichia coli deficient in fumarate reductase activity and therefore unable to grow anaerobically with fumarate as an electron acceptor have been isolated. By F+-mediated conjugation and complementation with the mutant host, two E. coli: Col E1 recombinant DNA plasmids have been identified from the Clarke and Carbon Colony Bank which carry the structural genes for fumarate reductase. Bacteria harboring either of these plasmids express about ten times the normal level of fumarate reductase. Enzyme purified from the two sources, plasmid-carrying and plasmidless E. coli, have identical physical and kinetic properties indicating that both the 69 000 and 25 000 dalton polypeptides are amplified. Regulation of plasmid-encoded enzyme, like the chromosomally encoded enzyme, is dependent upon the presence of fumarate and anaerobiosis.

scholarly journals Effects of dicyclohexylcarbodi-imide on proton translocation coupled to fumarate reduction in anaerobically grown cells of Escherichia coli K-12

1976 ◽  
Vol 160 (3) ◽  
pp. 813-816 ◽  
Author(s):  
S J Gutowski ◽  
H Rosenberg
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Proton Translocation ◽  
Fumarate Reductase ◽  
Escherichia Coli K12 ◽  
Fumarate Reduction ◽  
Cell Extracts ◽  
Endogenous Substrates ◽  
K 12

The addition of dicyclohexylcarbodi-imide to anaerobic cells of Escherichia coli K12 decreases both the observed extent of proton translocation coupled to fumarate reduction by endogenous substrates and the t1/2 of proton re-entry after such translocation, but does not affect fumarate uptake. Dicyclohexylcarbodi-imide also inhibits fumarate reductase activity in cell extracts.

scholarly journals Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase

1979 ◽  
Vol 184 (1) ◽  
pp. 45-50 ◽  
Author(s):  
E Cadenas ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Selection Method ◽  
Separation Procedure ◽  
Physical Separation ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93–101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).

The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5′-dithiobis-(2-nitrobenzoic acid) and 4,4′-dithiodipyridine

1979 ◽  
Vol 57 (6) ◽  
pp. 822-833 ◽  
Author(s):  
Michael M. Talgoy ◽  
Alexander W. Bell ◽  
Harry W. Duckworth
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Nitrobenzoic Acid ◽  
Nadh Fluorescence ◽  
Close Relationship ◽  
Polypeptide Chains ◽  
Pt Modification

Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4′-dithiodipyridine (4,4′-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4′-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4′-PDS, subsequent reactivity toward the other reagent is greatly decreased, (iv) Following modification, the DTNB and 4,4′-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4′-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4′-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4′-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4′-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4′-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.

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