scholarly journals Proton translocation and the respiratory nitrate reductase of Escherichia coli

1975 ◽  
Vol 152 (3) ◽  
pp. 547-559 ◽  
Author(s):  
P B Garland ◽  
J A Downie ◽  
B A Haddock
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Cytoplasmic Membrane ◽  
Proton Translocation ◽  
Hydrazoic Acid ◽  
Ph Gradients ◽  
Formate Oxidation ◽  
Outer Aspect ◽  
Inner Aspect

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.

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]

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Proton translocation and the respiratory nitrate reductase of Escherichia coli

1976 ◽  
Vol 154 (3) ◽  
pp. 561.b1-561.b1
Author(s):  
P. B. Garland ◽  
J. A. Downie ◽  
B. A. Haddock
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Proton Translocation ◽  
Respiratory Nitrate Reductase

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.

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.

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