O2 regulation of denitrification in Flexibacter canadensis

1994 ◽  
Vol 40 (11) ◽  
pp. 916-921 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Donald F. Niven
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Reductase Activity ◽  
Inhibitory Effect ◽  
Nitrate Transport ◽  
Intact Cells ◽  
Benzyl Viologen ◽  
Whole Cells ◽  
Nitrate And Nitrite

We studied the sensitivity to oxygen of the reductases involved in denitrification by whole cells and membrane fractions of Flexibacter canadensis. All of the nitrate reductase activity was found in the membrane fraction, suggesting that the nitrate reductase of F. canadensis is largely or entirely a membrane-bound enzyme. Methyl viologen and benzyl viologen were good electron donors to nitrate reductase in both whole cells and membrane fractions, whereas glucose and glycerol were effective in whole cells but, as expected, not in membrane fractions. Oxygen, generated by means of H2O2 plus catalase, inhibited the production of nitrite from nitrate by intact cells but not by membrane fractions, suggesting that O2 exerts its inhibitory effect at the level of nitrate transport rather than nitrate reduction. In intact cells, the rates of nitric oxide accumulation during reduction of nitrite in the presence of 20 μM carbonyl cyanide m-chlorophenylhydrazone, and consumption of nitric oxide and nitrous oxide, decreased as the concentration of H2O2 was increased. The concentrations of H2O2 giving 50% inhibition of reduction of nitrate and nitrite were 0.34 and 0.12 mM, respectively. In contrast, the rates of nitric oxide and nitrous oxide consumption were inhibited by only 36 and 32% at a concentration of H2O2 of 3.99 mM. These results indicate that the reduction of both nitric oxide and nitrous oxide is relatively tolerant to oxygen, and that nitrite reductase is much more sensitive to oxygen than the other reductases.Key words: nitrate reductase, nitrate transport, denitrification, O2 inhibition, Flexibacter canadensis.

Effect of ionophores on denitrification inFlexibacter canadensis

1995 ◽  
Vol 41 (3) ◽  
pp. 227-234 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Donald F. Niven
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Proton Motive Force ◽  
Nitrate Transport ◽  
Nitric Oxide Reductase ◽  
Anaerobic Conditions ◽  
Intact Cells ◽  
Nitrite Production ◽  
Carbonyl Cyanide ◽  
No Consumption

Denitrification by Flexibacter canadensis was investigated by measuring the production and (or) consumption of nitrite, nitric oxide (NO), and nitrous oxide (N2O) under anaerobic conditions. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2,4-dinitrophenol, and nigericin, but not valinomycin-K+inhibited the production of nitrite and N2O from nitrate by intact cells. However, CCCP, FCCP, 2,4-dinitrophenol, nigericin, and valinomycin-K+did not affect nitrite production from nitrate by cell-free extracts. These results suggest that nitrate transport was dependent on the transmembrane pH gradient but not on the membrane potential. CCCP, FCCP, and nigericin but not 2,4-dinitrophenol and valinomycin-K+caused NO accumulation during the reduction of nitrite, and also inhibited NO consumption and N2O production from nitrite by intact cells. These results preclude an explanation for NO accumulation based on the collapse of the proton motive force by ionophores, and imply that CCCP, FCCP, and nigericin perhaps dissociated a nitrite reductase–nitric oxide reductase complex, and (or) inhibited nitric oxide reductase specifically. 2,4-Dinitrophenol and CCCP did not inhibit the reduction of N2O to dinitrogen. Addition of ≤ 1.16 μM dissolved NO did not affect the production of nitrite from nitrate, or the disappearance of nitrite or N2O. The rate of NO consumption was linear with concentrations of dissolved NO up to 67 nM. Above 67 nM NO, NO consumption was inhibited, suggesting that NO is toxic to nitric oxide reductase.Key words: ionophores, denitrification, nitric oxide, Flexibacter canadensis.

scholarly journals Role of narK2X and narGHJI inHypoxic Upregulation of Nitrate Reduction byMycobacteriumtuberculosis

2003 ◽  
Vol 185 (24) ◽  
pp. 7247-7256 ◽  
Author(s):  
Charles D. Sohaskey ◽  
Lawrence G. Wayne
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Anaerobic Growth ◽  
Reporter System ◽  
Whole Cells ◽  
Hypoxic Conditions ◽  
Cell Extracts ◽  
Insertional Inactivation ◽  
Nitrate And Nitrite

ABSTRACT Mycobacterium tuberculosis is one of the strongest reducers of nitrate in the genus Mycobacterium. Under microaerobic conditions, whole cells exhibit upregulation of activity, producing approximately eightfold more nitrite than those of aerobic cultures of the same age. Assays of cell extracts from aerobic cultures and hypoxic cultures yielded comparable nitrate reductase activities. Mycobacterium bovis produced only low levels of nitrite, and this activity was not induced by hypoxia. M. tuberculosis has two sets of genes, narGHJI and narX of the narK2X operon, that exhibit some degree of homology to prokaryotic dissimilatory nitrate reductases. Each of these were knocked out by insertional inactivation. The narG mutant showed no nitrate reductase activity in whole culture or in cell-free assays, while the narX mutant showed wild-type levels in both assays. A knockout of the putative nitrite transporter narK2 gene produced a strain that had aerobic levels of nitrate reductase activity but failed to show hypoxic upregulation. Insertion of the M. tuberculosis narGHJI into a nitrate reductase Escherichia coli mutant allowed anaerobic growth in the presence of nitrate. Under aerobic and hypoxic conditions, transcription of narGHJI was constitutive, while the narK2X operon was induced under hypoxia, as measured with a lacZ reporter system and by quantitative real-time reverse PCR. This indicates that nitrate reductase activity in M. tuberculosis is due to the narGHJI locus with no detectable contribution from narX and that the hypoxic upregulation of activity is associated with the induction of the nitrate and nitrite transport gene narK2.

scholarly journals Effect of tungsten on nitrate and nitrite reductases in Azospirillum brasilense Sp7

1991 ◽  
Vol 37 (10) ◽  
pp. 744-750 ◽  
Author(s):  
Christian Chauret ◽  
Roger Knowles
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reductase ◽  
Azospirillum Brasilense ◽  
Nitrate Reductase Activity ◽  
Paracoccus Denitrificans ◽  
Reductase Activity ◽  
Whole Cells ◽  
Azospirillum Brasilense Sp7 ◽  
Nitrite Reductase Activity ◽  
Nitrate And Nitrite

Tungstate, at concentrations that completely suppressed nitrate reductase activity in Paracoccus denitrificans, caused only partial inhibition of nitrate reductase in Azospirillum brasilense Sp7. Nitrate reductase activity in cell-free extracts was much more sensitive than whole cells to tungstate, suggesting that there may be a barrier to its transport. Nitrite reductase activity was partially inhibited by tungstate in both whole cells and cell-free extracts. Azospirillum brasilense apparently scavenged enough contaminating molybdenum from molybdenum-limited medium to allow maximum nitrate reductase activity, which was not stimulated by added molybdate. Cells grown in molybdenum-depleted medium could not reduce nitrate. Nitrate concentrations less than 0.25 mM inhibited activity, but not synthesis, of nitrite reductase and caused significant accumulation of nitrite during reduction of nitrate. Key words: Azospirillum brasilense, Paracoccus denitrificans, nitrate reductase, nitrite reductase, tungsten, molybdenum, denitrification.

Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus: exploring the activity of a redox-balancing enzyme as a function of electrochemical potential

2007 ◽  
Vol 409 (1) ◽  
pp. 159-168 ◽  
Author(s):  
Andrew J. Gates ◽  
David J. Richardson ◽  
Julea N. Butt
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Reductase Activity ◽  
Electrochemical Potential ◽  
Intact Cells ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Voltammetric Studies ◽  
Paracoccus Pantotrophus

Paracoccus pantotrophus expresses two nitrate reductases associated with respiratory electron transport, termed NapABC and NarGHI. Both enzymes derive electrons from ubiquinol to reduce nitrate to nitrite. However, while NarGHI harnesses the energy of the quinol/nitrate couple to generate a transmembrane proton gradient, NapABC dissipates the energy associated with these reducing equivalents. In the present paper we explore the nitrate reductase activity of purified NapAB as a function of electrochemical potential, substrate concentration and pH using protein film voltammetry. Nitrate reduction by NapAB is shown to occur at potentials below approx. 0.1 V at pH 7. These are lower potentials than required for NarGH nitrate reduction. The potentials required for Nap nitrate reduction are also likely to require ubiquinol/ubiquinone ratios higher than are needed to activate the H+-pumping oxidases expressed during aerobic growth where Nap levels are maximal. Thus the operational potentials of P. pantotrophus NapAB are consistent with a productive role in redox balancing. A Michaelis constant (KM) of approx. 45 μM was determined for NapAB nitrate reduction at pH 7. This is in line with studies on intact cells where nitrate reduction by Nap was described by a Monod constant (KS) of less than 15 μM. The voltammetric studies also disclosed maximal NapAB activity in a narrow window of potential. This behaviour is resistant to change of pH, nitrate concentration and inhibitor concentration and its possible mechanistic origins are discussed.

scholarly journals The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans

1980 ◽  
Vol 192 (1) ◽  
pp. 231-240 ◽  
Author(s):  
P R Alefounder ◽  
S J Ferguson
Keyword(s):  
Plasma Membrane ◽  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Paracoccus Denitrificans ◽  
Osmotic Shock ◽  
Inhibitory Effect ◽  
Nitrate Respiration ◽  
Permeability Barrier ◽  
Intact Cells ◽  
Close Relationship

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.

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]

Effects of oxygen on each step of denitrification on Pseudomonas nautica

1989 ◽  
Vol 35 (11) ◽  
pp. 1061-1064 ◽  
Author(s):  
P. Bonin ◽  
M. Gilewicz ◽  
J. C. Bertrand
Keyword(s):  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Oxygen Concentration ◽  
Nitrite Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
A Value ◽  
Nitrite Reductase Activity ◽  
Effect Of Oxygen

Studies on the effect of oxygen on denitrification have shown that denitrification on Pseudomonas nautica 617 can take place in the presence of oxygen. The enzymes associated with denitrification are affected differently with respect to oxygen concentration. Nitrate reductase was less sensitive toward oxygen than nitrite and nitrous oxide reductases. Nitrate reductase activity was completely blocked at an oxygen concentration greater than 4.05 mg/L, compared with 2.15 and 0.25 mg/L for nitrite and nitrous oxide reductases, respectively. After an aerobic–anaerobic shift, nitrate reductase activity remained unchanged whereas the rate of nitrite reductase activity rose to a value only 20% that of the original rate.Key words: denitrification, oxygen, Pseudomonas.

scholarly journals Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1

2006 ◽  
Vol 189 (2) ◽  
pp. 656-662 ◽  
Author(s):  
Claribel Cruz-García ◽  
Alison E. Murray ◽  
Joel A. Klappenbach ◽  
Valley Stewart ◽  
James M. Tiedje
Keyword(s):  
Nitrous Oxide ◽  
Nitrate Reductase ◽  
Electron Acceptor ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Shewanella Oneidensis ◽  
Gene Encoding ◽  
Nitrate Ammonification ◽  
Sequential Reduction

ABSTRACT Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.

scholarly journals Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum)

2009 ◽  
Vol 104 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Chong Wei Jin ◽  
Shao Ting Du ◽  
Yong Song Zhang ◽  
Xian Yong Lin ◽  
Cai Xian Tang
Keyword(s):  
Nitric Oxide ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Regulatory Role

Regulation of nitrate reductase activity and its involvement in the production of nitric oxide in wheat leaves

2010 ◽  
Vol 435 (1) ◽  
pp. 457-460
Author(s):  
T. V. Trifonova ◽  
N. N. Maksyutova ◽  
L. V. Viktorova ◽  
E. I. Galeeva ◽  
G. G. Yafarova ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Wheat Leaves
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