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

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.

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

Effects of Vanadate on the Molybdoproteins Xanthine Oxidase and Nitrate Reductase: Kinetic Evidence for Multiple Site Interaction

1980 ◽  
Vol 35 (9-10) ◽  
pp. 702-707 ◽  
Author(s):  
Candadai S. Ramadoss
Keyword(s):  
Catalytic Activity ◽  
Nitrate Reductase ◽  
Xanthine Oxidase ◽  
Oxidase Activity ◽  
Metal Ion ◽  
Nadh Oxidase ◽  
Multiple Site ◽  
Vanadate Inhibition ◽  
Nadh Oxidase Activity

Abstract The inhibition of the activity of xanthine oxidase by vanadate was strikingly similar to vanadate inhibition of another molybdoprotein nitrate reductase. Although the main catalytic activity of both enzymes was inhibited, the partial NADH oxidase activity associated with these enzymes was stimulated several fold. It appears that the metal ion binds at multiple site in both enzymes. In the absence of any enzymes a combination of vanadium (V) and molybdenum (V) in air was found to oxide NADH rapidly.

Very Rapid, Cooperative Two-Electron/Two-Proton Redox Reactions of [3Fe−4S] Clusters:  Detection and Analysis by Protein-Film Voltammetry

1998 ◽  
Vol 120 (46) ◽  
pp. 11994-11999 ◽  
Author(s):  
Judy Hirst ◽  
Guy N. L. Jameson ◽  
James W. A. Allen ◽  
Fraser A. Armstrong
Keyword(s):  
Redox Reactions ◽  
Protein Film ◽  
Protein Film Voltammetry

NITRATE REDUCTASE MUTANTS IN EUDORINA ELEGANS (CHLOROPHYCEAE)1

1977 ◽  
Vol 13 (4) ◽  
pp. 368-372 ◽  
Author(s):  
Anne L. Toby ◽  
C. Lindley Kemp
Keyword(s):  
Nitrate Reductase ◽  
Nitrate Reductase Mutants

scholarly journals Redox (In)activations of Metalloenzymes: A Protein Film Voltammetry Approach

2019 ◽  
Vol 6 (19) ◽  
pp. 4949-4962 ◽  
Author(s):  
Melisa Barrio ◽  
Vincent Fourmond
Keyword(s):  
Protein Film ◽  
Protein Film Voltammetry

scholarly journals Opportunities for mesoporous nanocrystalline SnO2 electrodes in kinetic and catalytic analyses of redox proteins

2009 ◽  
Vol 37 (2) ◽  
pp. 368-372 ◽  
Author(s):  
Gemma L. Kemp ◽  
Sophie J. Marritt ◽  
Li Xiaoe ◽  
James R. Durrant ◽  
Myles R. Cheesman ◽  
...  
Keyword(s):  
Magnetic Circular Dichroism ◽  
High Surface ◽  
High Surface Area ◽  
Electrical Current ◽  
Protein Films ◽  
Protein Film ◽  
Protein Film Voltammetry ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Visible Wavelengths ◽  
Chemical Events

PFV (protein film voltammetry) allows kinetic analysis of redox and coupled-chemical events. However, the voltammograms report on the electron transfer through a flow of electrical current such that simultaneous spectroscopy is required for chemical insights into the species involved. Mesoporous nanocrystalline SnO2 electrodes provide opportunities for such ‘spectroelectrochemical’ analyses through their high surface area and optical transparency at visible wavelengths. Here, we illustrate kinetic and mechanistic insights that may be afforded by working with such electrodes through studies of Escherichia coli NrfA, a pentahaem cytochrome with nitrite and nitric oxide reductase activities. In addition, we demonstrate that the ability to characterize electrocatalytically active protein films by MCD (magnetic circular dichroism) spectroscopy is an advance that should ultimately assist our efforts to resolve catalytic intermediates in many redox enzymes.

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