Reassessing the Strategies for Trapping Catalytic Intermediates during Nitrate Reductase Turnover
Abstract
We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert on a time scale that is much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in substrate inhibition and in an uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies in which attempts were made to trap catalytic intermediates of Nap in experiments carried out under turnover conditions at very high substrate concentration.
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Under reducing conditions and excess nitrate (high nitrate loading), the inactive form of Nap accumulates, causing inhibition of the enzyme.
“Furthermore, under reducing conditions and in the presence of excess nitrate (high nitrate loading), the inactive form accumulates, causing inhibition of the enzyme.205”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [205]
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 [205]
Cites
The mononuclear molybdenum enzymes
Hille, R., Hall, J., & Basu, P. (2014) · Chemical Reviews · doi:10.1021/cr400443z · Cited by 1,738 · see citing works
The Mononuclear Molybdenum Enzymes
Hille, R. (1996) · Chemical Reviews · doi:10.1021/cr950061t · Cited by 1,501 · see citing works
Molybdenum cofactors, enzymes and pathways
Schwarz, G., Mendel, R. R., & Ribbe, M. W. (2009) · Nature · doi:10.1038/nature08302 · Cited by 872 · 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
Climbing Nitrogenase: Toward a Mechanism of Enzymatic Nitrogen Fixation
Hoffman, B. M., Dean, D. R., & Seefeldt, L. C. (2009) · Accounts of Chemical Research · doi:10.1021/ar8002128 · Cited by 375 · 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-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
Roldán, 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
Jepson, B. J. N., Mohan, S., Clarke, T. A., Gates, A. J., Cole, J. A., Butler, C. S., Butt, J. N., Hemmings, A. M., & Richardson, D. J. (2007) · Journal of Biological Chemistry · doi:10.1074/jbc.m607353200 · Cited by 120 · see citing works
Najmudin, S., González, P. J., Trincão, J., Coelho, C., Mukhopadhyay, A., Cerqueira, N. M. F. S. A., Romão, C. C., Moura, I., Moura, J. J. G., Brondino, C. D., et al. (2008) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-008-0359-6 · Cited by 116 · see citing works
SOAS: 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-citationButler, C. S., Charnock, J. M., Bennett, B., Sears, H. J., Reilly, A. J., Ferguson, S. J., Garner, C. D., Lowe, D. J., Thomson, A. J., Berks, B. C., et al. (1999) · Biochemistry · doi:10.1021/bi990402n · Cited by 99 · 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
Wijma, H. J., Jeuken, L. J. C., Verbeet, M. P., et al. (2006) · Journal of Biological Chemistry · doi:10.1074/jbc.m601610200 · Cited by 80 · see citing works
Bray, R. C., Adams, B., SMITH, A. T., et al. (2000) · Biochemistry · doi:10.1021/bi0000521 · Cited by 77 · see citing works
Correcting 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-citationTrapping an Intermediate of Dinitrogen (N2) Reduction on Nitrogenase
Barney, B. M., Lukoyanov, D. A., Igarashi, R. Y., et al. (2009) · Biochemistry · doi:10.1021/bi901092z · Cited by 64 · see citing works
The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase
Cerqueira, N. M. F. S. A., Gonzalez, P. J., Brondino, C. D., Romão, M. J., Romão, C. C., Moura, I., & Moura, J. J. G. (2009) · Journal of Computational Chemistry · doi:10.1002/jcc.21280 · Cited by 56 · 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-citationFourmond, 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-citationProtein Film Voltammetry of Copper-Containing Nitrite Reductase Reveals Reversible Inactivation
Wijma, H. J., Jeuken, L. J. C., Verbeet, M. P., et al. (2007) · Journal of the American Chemical Society · doi:10.1021/ja071274q · Cited by 49 · 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-citationGonzález, P. J., Rivas, M. G., Brondino, C. D., Bursakov, S. A., Moura, I., & Moura, J. J. G. (2006) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-006-0110-0 · Cited by 45 · see citing works
Gates, A. J., Richardson, D. J., & Butt, J. N. (2007) · Biochemical Journal · doi:10.1042/bj20071088 · Cited by 44 · see citing works
Bursakov, S. A., Carneiro, C., Almendra, M. J., Duarte, R. O., Caldeira, J., Moura, I., & Moura, J. J. G. (1997) · Biochemical and Biophysical Research Communications · doi:10.1006/bbrc.1997.7560 · Cited by 42 · see citing works
Jones, A. K., Camba, R., Reid, G. A., et al. (2000) · Journal of the American Chemical Society · doi:10.1021/ja000848n · Cited by 39 · see citing works
Hofmann, M. (2009) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-009-0545-1 · Cited by 35 · see citing works
Butler, C. S., Fairhurst, S. A., Ferguson, S. J., THOMSON, A. J., Berks, B. C., RICHARDSON, D. J., & LOWE, D. J. (2002) · Biochemical Journal · doi:10.1042/0264-6021:3630817 · Cited by 32 · see citing works
Dementin, 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-citationHofmann, M. (2007) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-007-0271-5 · Cited by 29 · see citing works
Sabaty, M., Schwintner, C., Cahors, S., Richaud, P., & Verméglio, A. (1999) · Journal of Bacteriology · doi:10.1128/jb.181.19.6028-6032.1999 · Cited by 29 · see citing works
Self-citationFourmond, V., Burlat, B., Dementin, S., Sabaty, M., Arnoux, P., Étienne, É., Guigliarelli, B., Bertrand, P., Pignol, D., & Léger, C. (2010) · Biochemistry · doi:10.1021/bi902140e · Cited by 28 · 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
Butler, C. S., Fairhurst, S. A., Ferguson, S. J., et al. (2002) · Biochemical Journal · doi:10.1042/bj3630817 · Cited by 26 · see citing works
Enzymatic Reduction of Nitrate to Nitrite: Insight from Density Functional Calculations
Xie, H., & Cao, Z. (2009) · Organometallics · doi:10.1021/om9008197 · Cited by 20 · see citing works
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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
Fourmond, V., Burlat, B., Dementin, S., Sabaty, M., Arnoux, P., Étienne, É., Guigliarelli, B., Bertrand, P., Pignol, D., & Léger, C. (2010) · Biochemistry · doi:10.1021/bi902140e · Cited by 28 · see citing works
Self-citation
- Authors
- Fourmond, V., Sabaty, M., Arnoux, P., Bertrand, P., Pignol, D., & Léger, C.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Léger, Christophe
- Venue
- The Journal of Physical Chemistry B
- Publisher
- American Chemical Society (ACS)
- Published
- 2010-02-17
- Volume
- 114
- Issue
- 9
- Pages
- 3341-3347
- ISSN
- 1520-5207 (ISSN-L)
- 1520-6106
- OpenAlex
- W1982847921
- Cited by
- 33
- References
- 38
- Citations by year
- 2026: 1 · 2025: 2 · 2022: 1 · 2021: 1 · 2020: 3 · 2019: 2 · 2017: 3 · 2016: 3 · 2015: 1 · 2014: 2 · 2013: 7 · 2012: 2
- FWCI
- 2.09
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Physical Chemistry B (publishedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion)
- Funders
- Agence Nationale de la Recherche
- Division of Chemistry
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Environmental Science › Environmental Engineering › Microbial Fuel Cells and Bioremediation
- Physical Sciences › Engineering › Electrical and Electronic Engineering › Electrochemical sensors and biosensors
- Keywords
- periplasmic nitrate reductase, NAP, nitrate reduction, catalytic intermediates, enzyme inactivity, substrate inhibition, hysteresis, active site flexibility, enzyme kinetics, protein film voltammetry
- MeSH terms
- Kinetics, Molybdenum, Nitrates, Oxidation-Reduction, Periplasm, Catalytic Domain, Nitrate Reductase, Biocatalysis, Electrochemical Techniques
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