Dependence of Catalytic Activity on Driving Force in Solution Assays and Protein Film Voltammetry: Insights from the Comparison of Nitrate Reductase Mutants
Abstract
Rhodobacter sphaeroides periplasmic nitrate reductase (Rs NapAB) is one of the enzymes whose assays give odd results: in spectrophotometric assays with methyl viologen as the electron donor, the activity increases as the reaction progresses, whereas the driving force provided by the soluble redox partner decreases; in protein film voltammetry (PFV), whereby the enzyme directly exchanges electrons with an electrode, the activity of NapAB decreases at large overpotential, whereas a monotonic increase is expected [Elliott, S. J., et al. (2002) Biochim. Biophys. Acta 1555, 54-59]. The relations between these phenomena and the catalytic mechanism are still debated. By studying NapAB mutants, we found that the peculiar dependences of electrochemical and solution activities on driving force are greatly affected by substituting certain amino acids that are located in the vicinity of the active site (M153, Q384, R392); this led us to establish and discuss the relation between the experimental parameters of the electrochemical and spectrophotometric assays: we show that the rate of reduction of the enzyme (which depends on the electrode potential or on the concentration of reduced MV) modulates the activity of the enzyme, but the "solution potential" does not. Our results also support the view that the complex profiles of activity versus potential are fingerprints of the active site chemistry, rather than direct consequences of changes in the redox states of relays that are remote from the active site.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
PFV experiments on Nap and Nar enzymes reveal different kinetic parameters.
“PFV experiments on Nap and Nar enzymes reveal different kinetic parameters.217–220”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [220]
In R. sphaeroides NapAB, interfacial electron transfer is much faster than catalytic turnover.
“Furthermore, the same group reported the interfacial electron transfer is much faster than the catalytic turnover.205,220”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [220]
Cites
EasySpin, a comprehensive software package for spectral simulation and analysis in EPR
Stoll, S., & Schweiger, A. (2006) · Journal of Magnetic Resonance · doi:10.1016/j.jmr.2005.08.013 · Cited by 6,557 · see citing works
Brock Biology of Microorganisms
Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (1996) · Cited by 5,759 · see citing works
Electron Paramagnetic Resonance of d Transition Metal Compounds
(1992) · Studies in inorganic chemistry · doi:10.1016/c2009-0-09084-9 · Cited by 869 · see citing works
Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies
Léger, C., & Bertrand, P. (2008) · Chemical Reviews · doi:10.1021/cr0680742 · Cited by 781 · see citing works
Self-citationMolybdenum and tungsten in biology
Hille, R. (2002) · Trends in Biochemical Sciences · doi:10.1016/s0968-0004(02)02107-2 · Cited by 430 · see citing works
Léger, C., Elliott, S. J., Hoke, K. R., Jeuken, L. J. C., Jones, A. K., & Armstrong, F. A. (2003) · Biochemistry · doi:10.1021/bi034789c · Cited by 289 · see citing works
Self-citationPershad, H. R., Duff, J. L. C., Heering, H. A., Duin, E. C., Albracht, S. P. J., & Armstrong, F. A. (1999) · Biochemistry · doi:10.1021/bi990108v · Cited by 247 · see citing works
Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase
Liebgott, P.-P., Leroux, F., Burlat, B., Dementin, S., Baffert, C., Lautier, T., Fourmond, V., Ceccaldi, P., Cavazza, C., Meynial‐Salles, I., et al. (2009) · Nature Chemical Biology · doi:10.1038/nchembio.276 · Cited by 216 · see citing works
Self-citationCharacterization of the Reduction of Selenate and Tellurite by Nitrate Reductases
Sabaty, M., Avazéri, C., Pignol, D., & Verméglio, A. (2001) · Applied and Environmental Microbiology · doi:10.1128/aem.67.11.5122-5126.2001 · Cited by 210 · see citing works
Self-citationDiode-like behaviour of a mitochondrial electron-transport enzyme
Sucheta, A., Ackrell, B. A. C., Cochran, B. C., & Armstrong, F. A. (1992) · Nature · doi:10.1038/356361a0 · Cited by 183 · see citing works
Léger, C., Dementin, S., Bertrand, P., Rousset, M., & Guigliarelli, B. (2004) · Journal of the American Chemical Society · doi:10.1021/ja046548d · Cited by 166 · see citing works
Self-citationStructural and redox plasticity in the heterodimeric periplasmic nitrate reductase
Arnoux, P., Sabaty, M., Alric, J., Frangioni, B., Guigliarelli, B., Adriano, J.-M., & Pignol, D. (2003) · Nature Structural & Molecular Biology · doi:10.1038/nsb994 · Cited by 149 · see citing works
Self-citationMo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases
Moura, J. J. G., Brondino, C. D., Trinc�o, J., & Rom�o, M. J. (2004) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-004-0573-9 · Cited by 141 · see citing works
Enzyme Electrokinetics: Hydrogen Evolution and Oxidation byAllochromatium vinosum[NiFe]-Hydrogenase
Léger, C., Jones, A. K., Roseboom, W., Albracht, S. P. J., & Armstrong, F. A. (2002) · Biochemistry · doi:10.1021/bi026586e · Cited by 127 · see citing works
Self-citationRoldán, M. D., Sears, H. J., Cheesman, M. R., Ferguson, S. J., THOMSON, A. J., Berks, B. C., & Richardson, D. J. (1998) · Journal of Biological Chemistry · doi:10.1074/jbc.273.44.28785 · Cited by 124 · see citing works
Dementin, S., Belle, V., Bertrand, P., Guigliarelli, B., Adryanczyk‐Perrier, G., Lacey, A. L. D., Fernández, V. M., Rousset, M., & Léger, C. (2006) · Journal of the American Chemical Society · doi:10.1021/ja060233b · Cited by 116 · see citing works
Self-citationSOAS: A free program to analyze electrochemical data and other one-dimensional signals
Fourmond, V., Hoke, K. R., Heering, H. A., Baffert, C., Leroux, F., Bertrand, P., & Léger, C. (2009) · Bioelectrochemistry · doi:10.1016/j.bioelechem.2009.02.010 · Cited by 115 · see citing works
Self-citationHirst, J., Sucheta, A., Ackrell, B. A. C., & Armstrong, F. A. (1996) · Journal of the American Chemical Society · doi:10.1021/ja9534361 · Cited by 114 · see citing works
Hydrogen‐Activating Enzymes: Activity Does Not Correlate with Oxygen Sensitivity
Baffert, C., Demuez, M., Cournac, L., Burlat, B., Guigliarelli, B., Bertrand, P., Girbal, L., & Léger, C. (2008) · Angewandte Chemie International Edition · doi:10.1002/anie.200704313 · Cited by 98 · see citing works
Self-citationAngove, H. C., Cole, J. A., Richardson, D. J., & Butt, J. N. (2002) · Journal of Biological Chemistry · doi:10.1074/jbc.m200495200 · Cited by 91 · see citing works
Elliott, S. J., Hoke, K. R., Heffron, K., Palak, M., Rothery, R. A., Weiner, J. H., & Armstrong, F. A. (2004) · Biochemistry · doi:10.1021/bi035869j · Cited by 90 · see citing works
Heffron, K., Léger, C., Rothery, R. A., Weiner, J. H., & Armstrong, F. A. (2001) · Biochemistry · doi:10.1021/bi002452u · Cited by 80 · see citing works
Self-citationCorrecting for Electrocatalyst Desorption and Inactivation in Chronoamperometry Experiments
Fourmond, V., Lautier, T., Baffert, C., Leroux, F., Liebgott, P.-P., Dementin, S., Rousset, M., Arnoux, P., Pignol, D., Meynial‐Salles, I., et al. (2009) · Analytical Chemistry · doi:10.1021/ac8025702 · Cited by 71 · see citing works
Self-citationFrangioni, B., Arnoux, P., Sabaty, M., Pignol, D., Bertrand, P., Guigliarelli, B., & Léger, C. (2004) · Journal of the American Chemical Society · doi:10.1021/ja0384072 · Cited by 70 · see citing works
Self-citationA needle in a haystack: The active site of the membrane‐bound complex cytochrome c nitrite reductase
Almeida, M. G., Silveira, C. M., Guigliarelli, B., Bertrand, P., Moura, J. J. G., Moura, I., & Léger, C. (2006) · FEBS Letters · doi:10.1016/j.febslet.2006.12.023 · Cited by 66 · see citing works
Self-citationLéger, C., Lederer, F., Guigliarelli, B., & Bertrand, P. (2005) · Journal of the American Chemical Society · doi:10.1021/ja055275z · Cited by 61 · see citing works
Self-citationBennett, B., Berks, B. C., Ferguson, S. J., Thomson, A. J., & Richardson, D. J. (1994) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1994.00789.x · Cited by 58 · see citing works
GOLDING, R. M., & Tennant, W. C. (1973) · Molecular Physics · doi:10.1080/00268977300101001 · Cited by 55 · see citing works
Detection and interpretation of redox potential optima in the catalytic activity of enzymes
Elliott, S. J., Léger, C., Pershad, H. R., Hirst, J., Heffron, K., Ginet, N., Blasco, F., Rothery, R. A., Weiner, J. H., & Armstrong, F. A. (2002) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/s0005-2728(02)00254-2 · Cited by 52 · see citing works
Self-citationReductive activation and redox properties of hydrogenase from Desulfovibrio gigas
Fernández, V. M., Aguirre, R., & Hatchikian, E. C. (1984) · Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology · doi:10.1016/0167-4838(84)90324-8 · Cited by 51 · see citing works
Fourmond, V., Burlat, B., Dementin, S., Arnoux, P., Sabaty, M., Boiry, S., Guigliarelli, B., Bertrand, P., Pignol, D., & Léger, C. (2008) · The Journal of Physical Chemistry B · doi:10.1021/jp807092y · Cited by 51 · see citing works
Self-citationBarker, C. D., Reda, T., & Hirst, J. (2007) · Biochemistry · doi:10.1021/bi061988y · Cited by 48 · see citing works
Pershad, H. R., Hirst, J., Cochran, B. C., Ackrell, B. A. C., & Armstrong, F. A. (1999) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/s0005-2728(99)00066-3 · Cited by 47 · see citing works
Bertrand, P., Frangioni, B., Dementin, S., Sabaty, M., Arnoux, P., Guigliarelli, B., Pignol, D., & Léger, C. (2007) · The Journal of Physical Chemistry B · doi:10.1021/jp074340j · Cited by 45 · see citing works
Self-citationGates, A. J., Richardson, D. J., & Butt, J. N. (2007) · Biochemical Journal · doi:10.1042/bj20071088 · Cited by 44 · see citing works
Hirst, J., Ackrell, B. A. C., & Armstrong, F. A. (1997) · Journal of the American Chemical Society · doi:10.1021/ja9631413 · Cited by 40 · see citing works
Mechanistic Studies of Rhodobacter sphaeroides Me2SO Reductase
Cobb, N. J., Conrads, T. P., & Hille, R. (2005) · Journal of Biological Chemistry · doi:10.1074/jbc.m412050200 · Cited by 38 · see citing works
Adams, B., SMITH, A. T., Bailey, S., McEwan, A. G., & Bray, R. C. (1999) · Biochemistry · doi:10.1021/bi9902034 · Cited by 38 · see citing works
Ackrell, B. A. C., Armstrong, F. A., Cochran, B. C., Sucheta, A., & Yu, T. (1993) · FEBS Letters · doi:10.1016/0014-5793(93)81768-u · Cited by 36 · see citing works
Reassessing the Strategies for Trapping Catalytic Intermediates during Nitrate Reductase Turnover
Fourmond, V., Sabaty, M., Arnoux, P., Bertrand, P., Pignol, D., & Léger, C. (2010) · The Journal of Physical Chemistry B · doi:10.1021/jp911443y · Cited by 33 · see citing works
Self-citationDementin, S., Arnoux, P., Frangioni, B., Grosse, S., Léger, C., Burlat, B., Guigliarelli, B., Sabaty, M., & Pignol, D. (2007) · Biochemistry · doi:10.1021/bi700928m · Cited by 31 · see citing works
Self-citationRapson, T. D., Kappler, U., & Bernhardt, P. V. (2008) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2008.06.005 · Cited by 26 · see citing works
Dynamic electrochemical experiments on hydrogenases
Armstrong, F. A. (2009) · Photosynthesis Research · doi:10.1007/s11120-009-9428-0 · Cited by 24 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
The mononuclear molybdenum enzymes
Hille, R., Hall, J., & Basu, P. (2014) · Chemical Reviews · doi:10.1021/cr400443z · Cited by 1,738 · see citing works
Fundamentals, Applications, and Future Directions of Bioelectrocatalysis
Chen, H., Simoska, O., Lim, K., Grattieri, M., Yuan, M., Dong, F., Lee, Y. S., Beaver, K., Weliwatte, N. S., Gaffney, E. M., et al. (2020) · Chemical Reviews · doi:10.1021/acs.chemrev.0c00472 · Cited by 500 · see citing works
Nitrate and periplasmic nitrate reductases
Sparacino-Watkins, C., Stolz, J. F., & Basu, P. (2014) · Chem. Soc. Rev. · doi:10.1039/c3cs60249d · Cited by 426 · see citing works
The prokaryotic Mo/W-bisPGD enzymes family: A catalytic workhorse in bioenergetic
Grimaldi, S., Schoepp‐Cothenet, B., Ceccaldi, P., Guigliarelli, B., & Magalon, A. (2013) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2013.01.011 · Cited by 159 · see citing works
Self-citationGonzález, P. J., Rivas, M. G., Mota, C., Brondino, C. D., Moura, I., & Moura, J. J. G. (2012) · Coordination Chemistry Reviews · doi:10.1016/j.ccr.2012.05.020 · Cited by 44 · see citing works
Kolaj‐Robin, O., O’Kane, S. R., Nitschke, W., Léger, C., Baymann, F., & Soulimane, T. (2010) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2010.10.009 · Cited by 42 · see citing works
Self-citationMarangon, J., Paes de Sousa, P. M., Moura, I., Brondino, C. D., Moura, J. J. G., & González, P. J. (2012) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2012.04.011 · Cited by 40 · see citing works
Biaso, F., Burlat, B., & Guigliarelli, B. (2012) · Inorganic Chemistry · doi:10.1021/ic201533p · Cited by 40 · see citing works
Self-citationJacques, J. G. J., Fourmond, V., Arnoux, P., Sabaty, M., Étienne, É., Grosse, S., Biaso, F., Bertrand, P., Pignol, D., Léger, C., et al. (2013) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2013.10.013 · Cited by 35 · see citing works
Self-citationGates, A. J., Kemp, G. L., To, C. Y., Mann, J., Marritt, S. J., Mayes, A. G., Richardson, D. J., & Butt, J. N. (2011) · Physical Chemistry Chemical Physics · doi:10.1039/c0cp02887h · Cited by 33 · see citing works
Reassessing the Strategies for Trapping Catalytic Intermediates during Nitrate Reductase Turnover
Fourmond, V., Sabaty, M., Arnoux, P., Bertrand, P., Pignol, D., & Léger, C. (2010) · The Journal of Physical Chemistry B · doi:10.1021/jp911443y · Cited by 33 · see citing works
Self-citationCracknell, J. A., & Blanford, C. F. (2012) · Chemical Science · doi:10.1039/c2sc00632d · Cited by 31 · see citing works
Reductive activation of E. coli respiratory nitrate reductase
Ceccaldi, P., Rendon, J., Léger, C., Toci, R., Guigliarelli, B., Magalon, A., Grimaldi, S., & Fourmond, V. (2015) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2015.06.007 · Cited by 28 · see citing works
Self-citationProtein Electrochemistry: Questions and Answers
Fourmond, V., & Léger, C. (2016) · Advances in biochemical engineering, biotechnology · doi:10.1007/10_2015_5016 · Cited by 26 · see citing works
Self-citationKinetics of substrate inhibition of periplasmic nitrate reductase
Jacques, J. G. J., Burlat, B., Arnoux, P., Sabaty, M., Guigliarelli, B., Léger, C., Pignol, D., & Fourmond, V. (2014) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/j.bbabio.2014.05.357 · Cited by 23 · see citing works
Self-citationMediated Electrochemistry of Nitrate Reductase from Arabidopsis thaliana
Kalimuthu, P., Fischer-Schrader, K., Schwarz, G., & Bernhardt, P. V. (2013) · The Journal of Physical Chemistry B · doi:10.1021/jp404076w · Cited by 22 · see citing works
Mintmier, B., McGarry, J. M., Bain, D. J., & Basu, P. (2020) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-020-01833-9 · Cited by 14 · see citing works
Wang, T.-H., Chen, Y.-H., Huang, J.-Y., Liu, K., Ke, S., & Chu, H. (2011) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2011.07.007 · Cited by 13 · see citing works
An Introduction to Electrochemical Methods for the Functional Analysis of Metalloproteins
Léger, C. (2013) · Elsevier eBooks · doi:10.1016/b978-0-444-56351-4.00008-7 · Cited by 11 · see citing works
Self-citationSrivastava, A. P., Allen, J. P., Vaccaro, B. J., Hirasawa, M., Alkul, S., Johnson, M. K., & Knaff, D. B. (2015) · Biochemistry · doi:10.1021/acs.biochem.5b00511 · Cited by 10 · see citing works
An introduction to electrochemical methods for the functional analysis of metalloproteins
Fourmond, V., & Léger, C. (2019) · Elsevier eBooks · doi:10.1016/b978-0-444-64225-7.00009-2 · Cited by 10 · see citing works
Self-citationMalagnini, M., Aldinio-Colbachini, A., Opdam, L. V., Giuliantonio, A. D., Fasano, A., Fourmond, V., & Léger, C. (2025) · Bioelectrochemistry · doi:10.1016/j.bioelechem.2025.108967 · Cited by 9 · see citing works
Self-citationCoelho, C., Marangon, J., Rodrigues, D., Moura, J. J. G., Romão, M. J., de Sousa, P. M. P., & dos Santos, M. M. C. (2013) · Journal of Electroanalytical Chemistry · doi:10.1016/j.jelechem.2013.01.030 · Cited by 9 · see citing works
Direct Electrochemistry of Molybdenum and Tungsten Enzymes
Fourmond, V. (2017) · Elsevier eBooks · doi:10.1016/b978-0-12-409547-2.13354-2 · Cited by 5 · see citing works
Self-citationJacob-Villedieu, A., Katipamula, S., Marchand, A., Reuillard, B., Artero, V., Fourmond, V., Faure, B., Simaan, A. J., & Léger, C. (2025) · Journal of the American Chemical Society · doi:10.1021/jacs.5c11615 · Cited by 4 · see citing works
Self-citation
- Authors
- Fourmond, V., Burlat, B., Dementin, S., Sabaty, M., Arnoux, P., Étienne, É., Guigliarelli, B., Bertrand, P., Pignol, D., & Léger, C.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Léger, Christophe
- ORCID
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2010-03-01
- Volume
- 49
- Issue
- 11
- Pages
- 2424-2432
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W1988833413
- Cited by
- 28
- References
- 43
- Citations by year
- 2025: 2 · 2020: 2 · 2019: 1 · 2018: 1 · 2017: 1 · 2016: 1 · 2015: 3 · 2014: 3 · 2013: 6 · 2012: 4
- FWCI
- 2.09
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- HAL AMU (submittedVersion)
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Physical Sciences › Environmental Science › Environmental Engineering › Microbial Fuel Cells and Bioremediation
- Keywords
- nitrate reductase, Rhodobacter sphaeroides, protein film voltammetry, paraquat, driving factors, enzyme mutants, active site residues, overpotential, redox relay, spectrophotometric enzyme assay
- MeSH terms
- Electrochemistry, Kinetics, Models, Molecular, Mutation, Rhodobacter sphaeroides, Solutions, Spectrum Analysis, Periplasm, Catalytic Domain, Mutant Proteins, Nitrate Reductase, Biocatalysis
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
