A Study of the Permeability of the Cytoplasmic Membrane of Escherichia coli to Reduced and Oxidized Benzyl Viologen and Methyl Viologen Cations: Complications in the Use of Viologens as Redox Mediators for Membrane-Bound Enzymes

1976 ◽  
Vol 4 (4) ◽  
pp. 671-673 ◽  
Author(s):  
ROBERT W. JONES ◽  
TREVOR A. GRAY ◽  
PETER B. GARLAND
Keyword(s):  
Escherichia Coli ◽  
Methyl Viologen ◽  
Cytoplasmic Membrane ◽  
Redox Mediators ◽  
Benzyl Viologen ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

scholarly journals The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli

1980 ◽  
Vol 188 (2) ◽  
pp. 345-350 ◽  
Author(s):  
R W Jones
Keyword(s):  
Escherichia Coli ◽  
Electron Acceptor ◽  
Cytoplasmic Membrane ◽  
Proton Translocation ◽  
Terminal Electron Acceptor ◽  
Benzyl Viologen ◽  
Carbonyl Cyanide ◽  
Energy Conserving ◽  
Membrane Bound ◽  
Hydroxy Quinoline

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.

scholarly journals Purification of the membrane-bound hydrogenase of Escherichia coli

1979 ◽  
Vol 183 (1) ◽  
pp. 11-22 ◽  
Author(s):  
M W W Adams ◽  
D O Hall
Keyword(s):  
Escherichia Coli ◽  
Methyl Viologen ◽  
Hydrophobic Interaction Chromatography ◽  
H2 Evolution ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Electron Carrier ◽  
Membrane Bound ◽  
High Concentrations ◽  
Electron Carriers

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.

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.

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]

Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes

1975 ◽  
Vol 148 (2) ◽  
pp. 329-333 ◽  
Author(s):  
M B Kemp ◽  
B A Haddock ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytochrome B ◽  
Cytoplasmic Membrane ◽  
Coli Strain ◽  
Respiratory Pathway ◽  
Aminolaevulinic Acid ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

The synthesis of nitrate reductase and its incorporation into the cytoplasmic membrane of Escherichia coli strain A1004a (5-aminolaevulinic acid auxotroph) does not require synthesis of cytochrome b. The synthesis of the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to nitrate appears to be inhibited by the absence of haem. No member of the respiratory pathway from NADH to oxygen is capable of reducing nitrate reductase directly. The site on nitrate reductase that oxidizes FMNH2 is located on the cytoplasmic aspect of the cytoplasmic membrane.

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).

scholarly journals Localization of hydrogenase in Thiocapsa roseopersicina photosynthetic membrane

1982 ◽  
Vol 202 (1) ◽  
pp. 255-258 ◽  
Author(s):  
Csaba Bagyinka ◽  
Kornel L. Kovács ◽  
Erika Rak
Keyword(s):  
Cell Membrane ◽  
Methyl Viologen ◽  
Photosynthetic Membrane ◽  
Benzyl Viologen ◽  
Thiocapsa Roseopersicina ◽  
Membrane Bound ◽  
Reduced Forms

The photosynthetic cell membrane is impermeable to the oxidized redox dyes Methyl Viologen and Benzyl Viologen, whereas the reduced forms easily penetrate into the cells. By exploiting this permeability difference, the orientation of the membrane-bound hydrogenase has been determined.

scholarly journals The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 197 (2) ◽  
pp. 283-291 ◽  
Author(s):  
A Graham
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Labelling Pattern ◽  
F1 Atpase ◽  
Crossed Immunoelectrophoresis ◽  
Cytoplasmic Surface ◽  
Membrane Bound ◽  
Inside Out

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.

Binding of mutagenic pyrolyzates to fractions of intestinal bacterial cells

1992 ◽  
Vol 38 (7) ◽  
pp. 614-617 ◽  
Author(s):  
Xue Bin Zhang ◽  
Yoshiyuki Ohta
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Outer Membrane ◽  
Bacterial Strain ◽  
Cytoplasmic Membrane ◽  
Intestinal Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Membrane Bound ◽  
Cell Fractions

The binding of mutagenic pyrolyzates to cell fractions from some gram-negative intestinal bacteria and to thermally treated bacterial cells was investigated. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) were effectively bound by several of the bacterial cells. The cell wall skeletons of all bacteria effectively bound Trp-P-1 and Trp-P-2. Their cytoplasmic fractions retained Trp-P-1 and Trp-P-2, but to a lesser extent than the cell wall skeletons. 2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) was not found in their cytoplasmic fractions. These cell wall skeletons also bound 2-amino-6-methyldipyrido[1,2-a:3′2′-d] imidazole (Glu-P-1), 2-amino-5-phenylpyridine (Phe-P-1), IQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQX). The amount of each mutagen bound differed with the type of mutagen and the bacterial strain used. The outer membrane of Escherichia coli IFO 14249 showed binding of about 123.7 μg/mg of Trp-P-2, and its cytoplasmic membrane bound 57.14 μg/mg. Trp-P-2 bound to the bacterial cells was extracted with ammonia (5%), methanol, and ethanol but not with water. Key words: cell wall skeletons, outer membrane, cytoplasmic membrane, binding of mutagenic pyrolyzates.

scholarly journals Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

1977 ◽  
Vol 164 (1) ◽  
pp. 199-211 ◽  
Author(s):  
Robert W. Jones ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Respiratory Enzymes ◽  
Radical Species ◽  
Benzyl Viologen ◽  
E Coli ◽  
Nitrite Reductase Activity

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.

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