Catalytic Protein Film Voltammetry from a Respiratory Nitrate Reductase Provides Evidence for Complex Electrochemical Modulation of Enzyme Activity†

Biochemistry ◽  
2001 ◽  
Vol 40 (38) ◽  
pp. 11294-11307 ◽  
Author(s):  
Lee J. Anderson ◽  
David J. Richardson ◽  
Julea N. Butt
Keyword(s):  
Enzyme Activity ◽  
Nitrate Reductase ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Respiratory 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.

Dependence of Catalytic Activity on Driving Force in Solution Assays and Protein Film Voltammetry: Insights from the Comparison of Nitrate Reductase Mutants

Biochemistry ◽  
2010 ◽  
Vol 49 (11) ◽  
pp. 2424-2432 ◽  
Author(s):  
Vincent Fourmond ◽  
Bénédicte Burlat ◽  
Sébastien Dementin ◽  
Monique Sabaty ◽  
Pascal Arnoux ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Nitrate Reductase ◽  
Driving Force ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Nitrate Reductase Mutants

Respiratory nitrate and nitrite pathway in the denitrifier haloarchaeon Haloferax mediterranei

2006 ◽  
Vol 34 (1) ◽  
pp. 115-117 ◽  
Author(s):  
R.M. Martínez-Espinosa ◽  
D.J. Richardson ◽  
J.N. Butt ◽  
M.J. Bonete
Keyword(s):  
Electron Acceptors ◽  
Open Reading Frames ◽  
Transcriptional Level ◽  
Haloferax Mediterranei ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Nitrate And Nitrite ◽  
Reading Frames ◽  
Respiratory Nitrate Reductase

Haloferax mediterranei cells are able to use high nitrate or nitrite concentrations as electron acceptors under anoxic conditions. The nar operon, which has eight open reading frames, has been sequenced and its regulation has been characterized at the transcriptional level. The narG and narH genes encode the Nar (respiratory nitrate reductase) catalytic subunit (NarG) and the electron transfer Nar subunit (NarH) respectively. Nar has been purified and characterized in vitro. This characterization has included protein-film voltammetry and preliminary EPR studies.

Protein Film Voltammetry ofRhodobacterCapsulatusXanthine Dehydrogenase

2003 ◽  
Vol 125 (50) ◽  
pp. 15352-15358 ◽  
Author(s):  
Kondo François Aguey-Zinsou ◽  
Paul V. Bernhardt ◽  
Silke Leimkühler
Keyword(s):  
Protein Film ◽  
Protein Film Voltammetry

Acid phosphatase, alkaline phosphatase, and nitrate reductase activity of selected ectomycorrhizal fungi

1989 ◽  
Vol 67 (3) ◽  
pp. 750-753 ◽  
Author(s):  
Iwan Ho
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Nitrate Reductase ◽  
Phosphatase Activity ◽  
Ectomycorrhizal Fungi ◽  
Acid Phosphatase Activity ◽  
Nitrate Reductase Activity ◽  
Reductase Activity ◽  
Phosphatase Alkaline

Seventeen isolates, encompassing five genera and eight species of ectomycorrhizal fungi, were compared for acid phosphatase, alkaline phosphatase, and nitrate reductase activity. Isolates within species differed in enzyme activity and isozyme patterns by host specificity and site (as exemplified by the genus Suillus). Host and site may have affected phosphatase enzyme activity. Generally, the Douglas-fir associates, which dominate in mesic sites, have higher acid phosphatase activity than pine associates, which mostly occupy xeric sites; however, pine associates from mesic sites also have higher acid phosphatase activity (e.g., S. tomentosus). In four isolates of Amanita muscaria, the effect of site was also apparent. Two of them, which have significantly higher acid phosphatase activity than the others, were isolated from mesic sites. The isozyme pattern of the genus Suillus appeared to be separated by host groups. Other isolates with only one species also differed more or less by host groups. They shared at least one band within host groups, except for the two isolates of Paxillus involutus from different hosts. The P. involutus S-403 isolated from an orchard showed much higher nitrate reductase activity than all other isolates. No apparent differences in nitrate reductase activity were found between the other isolates.

scholarly journals Activity of Spore-Specific Respiratory Nitrate Reductase 1 ofStreptomyces coelicolorA3(2) Requires a Functional Cytochromebcc-aa3Oxidase Supercomplex

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Dörte Falke ◽  
Bianca Biefel ◽  
Alexander Haase ◽  
Stefan Franke ◽  
Marco Fischer ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Nitrate Reductase ◽  
Nitrate Reduction ◽  
Streptomyces Coelicolor ◽  
Nitrate Respiration ◽  
Nitrate Transport ◽  
Developmental Cycle ◽  
Content Type ◽  
Resting Spores ◽  
Respiratory Nitrate Reductase

ABSTRACTSpores have strongly reduced metabolic activity and are produced during the complex developmental cycle of the actinobacteriumStreptomyces coelicolor. Resting spores can remain viable for decades, yet little is known about how they conserve energy. It is known, however, that they can reduce either oxygen or nitrate using endogenous electron sources.S. coelicoloruses either a cytochromebdoxidase or a cytochromebcc-aa3oxidase supercomplex to reduce oxygen, while nitrate is reduced by Nar-type nitrate reductases, which typically oxidize quinol directly. Here, we show that in resting spores the Nar1 nitrate reductase requires a functionalbcc-aa3supercomplex to reduce nitrate. Mutants lacking the completeqcr-ctagenetic locus encoding thebcc-aa3supercomplex showed no Nar1-dependent nitrate reduction. Recovery of Nar1 activity was achieved by genetic complementation but only when the completeqcr-ctalocus was reintroduced to the mutant strain. We could exclude that the dependence on the supercomplex for nitrate reduction was via regulation of nitrate transport. Moreover, the catalytic subunit, NarG1, of Nar1 was synthesized in theqcr-ctamutant, ruling out transcriptional control. Constitutive synthesis of Nar1 in mycelium revealed that the enzyme was poorly active in this compartment, suggesting that the Nar1 enzyme cannot act as a typical quinol oxidase. Notably, nitrate reduction by the Nar2 enzyme, which is active in growing mycelium, was not wholly dependent on thebcc-aa3supercomplex for activity. Together, our data suggest that Nar1 functions together with the proton-translocatingbcc-aa3supercomplex to increase the efficiency of energy conservation in resting spores.IMPORTANCEStreptomyces coelicolorforms spores that respire with either oxygen or nitrate, using only endogenous electron donors. This helps maintain a membrane potential and, thus, viability. Respiratory nitrate reductase (Nar) usually receives electrons directly from reduced quinone species; however, we show that nitrate respiration in spores requires a respiratory supercomplex comprising cytochromebccoxidoreductase andaa3oxidase. Our findings suggest that the Nar1 enzyme in theS. coelicolorspore functions together with the proton-translocatingbcc-aa3supercomplex to help maintain the membrane potential more efficiently. Dissecting the mechanisms underlying this survival strategy is important for our general understanding of bacterial persistence during infection processes and of how bacteria might deal with nutrient limitation in the natural environment.

scholarly journals The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

2011 ◽  
Vol 13 (17) ◽  
pp. 7720 ◽  
Author(s):  
Andrew J. Gates ◽  
Gemma L. Kemp ◽  
Chun Yip To ◽  
James Mann ◽  
Sophie J. Marritt ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Electrochemical Potential ◽  
Redox Enzyme ◽  
Protein Film ◽  
The Relationship
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