scholarly journals Identification of periplasmic nitrate reductase Mo(V) EPR signals in intact cells of Paracoccus denitrificans

1995 ◽  
Vol 310 (1) ◽  
pp. 311-314 ◽  
Author(s):  
H J Sears ◽  
B Bennett ◽  
S Spiro ◽  
A J Thomson ◽  
D J Richardson
Keyword(s):  
Nitrate Reductase ◽  
Mutant Strain ◽  
Epr Spectroscopy ◽  
Ferric Iron ◽  
Paracoccus Denitrificans ◽  
Intact Cells ◽  
Periplasmic Nitrate Reductase ◽  
Haem Iron

EPR spectroscopy has been successfully used to detect signals due to molybdenum (V) and ferric iron in intact cells of aerobically grown Paracoccus denitrificans. The signals are ascribed to the catalytic molybdenum centre and to the haem iron of the periplasmic nitrate reductase. These signals are absent from a mutant strain deficient in this enzyme. The Mo(V) signal is due to the High-g Split species which has been well characterized in the purified enzyme. This confirms that the High-g Split is the physiologically relevant signal of a number observed in the previous work on the purified enzyme.

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 Characterization of the Reduction of Selenate and Tellurite by Nitrate Reductases

2001 ◽  
Vol 67 (11) ◽  
pp. 5122-5126 ◽  
Author(s):  
Monique Sabaty ◽  
Cécile Avazeri ◽  
David Pignol ◽  
André Vermeglio
Keyword(s):  
Nitrate Reductase ◽  
Ralstonia Eutropha ◽  
Paracoccus Denitrificans ◽  
Potassium Tellurite ◽  
Benzyl Viologen ◽  
Periplasmic Nitrate Reductase ◽  
Membrane Bound ◽  
Nitrate Reductases

ABSTRACT Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V max andKm were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min−1 · mg−1 and 0.12 mM were obtained for V max and Km , respectively, whereas the V max values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.

Molybdenum incorporation in denitrifying Azospirillum brasilense Sp7

1992 ◽  
Vol 38 (10) ◽  
pp. 1042-1047 ◽  
Author(s):  
Christian Chauret ◽  
Wilfredo L. Barraquio ◽  
Roger Knowles
Keyword(s):  
Nitrate Reductase ◽  
Key Words ◽  
Nitrate Reduction ◽  
Gel Electrophoresis ◽  
Azospirillum Brasilense ◽  
Paracoccus Denitrificans ◽  
Free Medium ◽  
Azospirillum Brasilense Sp7

Nondenaturating disc gel electrophoresis revealed that 99Mo was incorporated into the nitrate reductase of Azospirillum brasilense grown in the absence but not in the presence of tungstate. Under denitrifying conditions, A. brasilense grown in tungsten-free medium steadily accumulated 99Mo for 12 h. In contrast, Paracoccus denitrificans grown under the same conditions ceased uptake after 1 h. However, both bacteria were incapable of accumulating significant amounts of 99Mo in media containing 10 mM tungstate, even though nitrate was reduced by A. brasilense. Aerobically grown A. brasilense cells transported 99Mo more efficiently than anaerobically grown cells. Key words: Azospirillum brasilense, tungsten, molybdenum incorporation, nitrate reduction.

Non-haem iron centres of bacterioferritin by EPR spectroscopy

1991 ◽  
Vol 43 (2-3) ◽  
pp. 129
Author(s):  
M.R. Cheesman ◽  
F.H.A. Kadir ◽  
G.R. Moore ◽  
A.J. Thomson ◽  
C. Greenwood ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Haem Iron

The periplasmic nitrate reductase of

1993 ◽  
Vol 51 (1-2) ◽  
pp. 369
Author(s):  
B.C. Berks ◽  
D.J. Richardson ◽  
S. Marritt ◽  
A.J. Thomson ◽  
S.J. Ferguson
Keyword(s):  
Nitrate Reductase ◽  
Periplasmic Nitrate Reductase

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 Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222

2017 ◽  
Vol 365 (1) ◽  
Author(s):  
Alfonso Olaya-Abril ◽  
Víctor M Luque-Almagro ◽  
Isabel Manso ◽  
Andrew J Gates ◽  
Conrado Moreno-Vivián ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Paracoccus Denitrificans

Oxygen control of nitrogen oxide respiration, focusing on α-proteobacteria

2011 ◽  
Vol 39 (1) ◽  
pp. 179-183 ◽  
Author(s):  
James P. Shapleigh
Keyword(s):  
Nitrate Reductase ◽  
Nitrogen Oxide ◽  
Physiological Function ◽  
Physiological Role ◽  
Present Review ◽  
Oxygen Control ◽  
Anoxic Conditions ◽  
Periplasmic Nitrate Reductase

Denitrification is generally considered to occur under micro-oxic or anoxic conditions. With this in mind, the physiological function and regulation of several steps in the denitrification of model α-proteobacteria are compared in the present review. Expression of the periplasmic nitrate reductase is quite variable, with this enzyme being maximally expressed under oxic conditions in some bacteria, but under micro-oxic conditions in others. Expression of nitrite and NO reductases in most denitrifiers is more tightly controlled, with expression only occurring under micro-oxic conditions. A possible exception to this may be Roseobacter denitrificans, but the physiological role of these enzymes under oxic conditions is uncertain.

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