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.

Electrocatalytic reduction of nitrate and selenate by NapAB

2011 ◽  
Vol 39 (1) ◽  
pp. 236-242 ◽  
Author(s):  
Andrew J. Gates ◽  
Clive S. Butler ◽  
David J. Richardson ◽  
Julea N. Butt
Keyword(s):  
Cytoplasmic Membrane ◽  
Current Knowledge ◽  
Reductase Activity ◽  
Protonmotive Force ◽  
Metabolic Diversity ◽  
Protein Film ◽  
Carbon Substrates ◽  
Redox Poise ◽  
Paracoccus Pantotrophus ◽  
High Level

Bacterial cellular metabolism is renowned for its metabolic diversity and adaptability. However, certain environments present particular challenges. Aerobic metabolism of highly reduced carbon substrates by soil bacteria such as Paracoccus pantotrophus presents one such challenge since it may result in excessive electron delivery to the respiratory redox chain when compared with the availability of terminal oxidant, O2. The level of a periplasmic ubiquinol-dependent nitrate reductase, NAP, is up-regulated in the presence of highly reduced carbon substrates. NAP oxidizes ubiquinol at the periplasmic face of the cytoplasmic membrane and reduces nitrate in the periplasm. Thus its activity counteracts the accumulation of excess reducing equivalents in ubiquinol, thereby maintaining the redox poise of the ubiquinone/ubiquinol pool without contributing to the protonmotive force across the cytoplasmic membrane. Although P. pantotrophus NapAB shows a high level of substrate specificity towards nitrate, the enzyme has also been reported to reduce selenate in spectrophotometric solution assays. This transaction draws on our current knowledge concerning the bacterial respiratory nitrate reductases and extends the application of PFE (protein film electrochemistry) to resolve and quantify the selenate reductase activity of NapAB.

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.

Enzyme-catalysed nitrate reduction—themes and variations as revealed by protein film voltammetry

2002 ◽  
Vol 56 (1-2) ◽  
pp. 17-18 ◽  
Author(s):  
Julea N. Butt ◽  
Lee J. Anderson ◽  
Luis M. Rubio ◽  
David J. Richardson ◽  
Enrique Flores ◽  
...  
Keyword(s):  
Nitrate Reduction ◽  
Protein Film ◽  
Protein Film Voltammetry

Differential effects of ozone on in vivo nitrate reduction in soybean cultivars. I. Response to exogenous sugars

1978 ◽  
Vol 56 (13) ◽  
pp. 1540-1544 ◽  
Author(s):  
Albert C. Purvis
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Foliar Injury ◽  
Glycolytic Intermediates ◽  
Glycine Max L ◽  
Growth Chambers ◽  
Visible Foliar Injury

Two cultivars of soybeans (Glycine max (L.) Merr.) differing widely in their resistance to ozone were exposed to 0.5 μl/ℓ ozone for 2 h in growth chambers. In vivo nitrate reduction was depressed by more than 50% in the primary leaves of Dare, the ozone-sensitive cultivar, but was not significantly altered in Hood, the ozone-resistant cultivar. Sucrose, up to 1.5% (w/v), added to excised seedlings of the Dare cultivar during exposure to ozone eliminated the ozone depression of in vivo nitrate reductase activity and also reduced foliar injury. Addition of two glycolytic intermediates, glyceraldehyde-3-phosphate and fructose-1,6-diphosphate, to the infiltration medium recovered some in vivo nitrate reduction in treated Dare leaves. The levels of extractable nitrate reductase and glyceraldehyde-3-phosphate dehydrogenase in the primary leaves of both cultivars were unaltered by ozone fumigations. These observations led to the conclusion that ozone depression of in vivo nitrate reduction is not due to ozone inactivation of nitrate reductase or of the enzymes coupling nitrate reduction to glycolysis, but may be caused by an inadequate supply of photosynthetic sugars. It was also noted that ozone depression of in vivo nitrate reduction only occurred with treatments which subsequently caused the development of visible foliar injury.

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.

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