Major Mo(V) EPR Signature of Rhodobacter sphaeroides Periplasmic Nitrate Reductase Arising from a Dead-End Species That Activates upon Reduction. Relation to Other Molybdoenzymes from the DMSO Reductase Family
Abstract
Enzymes of the DMSO reductase family use a mononuclear Mo-bis(molybdopterin) cofactor (MoCo) to catalyze a variety of oxo-transfer reactions. Much functional information on nitrate reductase, one of the most studied members of this family, has been gained from EPR spectroscopy, but this technique is not always conclusive because the signature of the MoCo is heterogeneous, and which signals correspond to active species is still unsure. We used site-directed mutagenesis, EPR and protein film voltammetry to demonstrate that the MoCo in R. sphaeroides periplasmic nitrate reductase (NapAB) is subject to an irreversible reductive activation process whose kinetics we precisely define. This activation quantitatively correlates with the disappearance of the so-called "Mo(V) high-g" EPR signal, but this reductive process is too slow to be part of the normal catalytic cycle. Therefore, in NapAB, this most intense and most commonly observed signature of the MoCo arises from a dead-end, inactive state that gives a catalytically competent species only after reduction. This activation proceeds, even without substrate, according to a reduction followed by an irreversible nonredox step, both of which are pH independent. An apparently similar process occurs in other nitrate reductases (both assimilatory and membrane bound), and this also recalls the redox cycling procedure, which activates periplasmic DMSO reductases and simplifies their spectroscopic signatures. Hence we propose that heterogeneity at the active site and reductive activation are common properties of enzymes from the DMSO reductase family. Regarding NapAB, the fact that we could detect no Mo EPR signal upon reoxidizing the fully reduced enzyme suggests that the catalytically active form of the Mo(V) is thermodynamically unstable, as is the case for other enzymes of the DMSO reductase family. Our original approach, which combines spectroscopy and protein film voltammetry, proves useful for discriminating the forms of the active site on the basis of their catalytic properties. This could be applied to other enzymes for which the question arises as to the catalytic relevance of certain long-lived, spectroscopically characterized species.
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Detailed work with R. sphaeroides demonstrated that the 'high-g' species is catalytically relevant.
“Detailed work with R. sphaeroides demonstrated that the ‘high-g’ species is catalytically relevant.247”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [247]
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
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-citationCrystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods
Dias, J. M., Than, M. E., Humm, A., Huber, R., Bourenkov, G. P., Bartunik, H. D., Bursakov, S., Calvete, J., Caldeira, J., Carneiro, C., et al. (1999) · Structure · doi:10.1016/s0969-2126(99)80010-0 · Cited by 286 · see citing works
Metalloenzymes of the denitrification pathway
Tavares, P., Pereira, A. S., Moura, J. J. G., & Moura, I. (2006) · Journal of Inorganic Biochemistry · doi:10.1016/j.jinorgbio.2006.09.003 · Cited by 228 · 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-citationLi, H., Temple, C. A., Rajagopalan, K. V., & Schindelin, H. (2000) · Journal of the American Chemical Society · doi:10.1021/ja000643e · Cited by 139 · see citing works
Structure of the Molybdenum Site of Dimethyl Sulfoxide Reductase
George, G. N., Hilton, J. C., Temple, C. A., Prince, R. C., & Rajagopalan, K. V. (1999) · Journal of the American Chemical Society · doi:10.1021/ja982843k · 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
Li, H., Temple, K. L., Rajagopalan, K. V., & Schindelin, H. (2000) · Worldwide Protein Data Bank · doi:10.2210/pdb1eu1/pdb · Cited by 104 · see citing works
Butler, 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
X-ray Absorption Spectroscopy of Dimethyl Sulfoxide Reductase from Rhodobacter sphaeroides
George, G. N., Hilton, J. C., & Rajagopalan, K. V. (1996) · Journal of the American Chemical Society · doi:10.1021/ja953317l · Cited by 94 · see citing works
Hoke, K. R., Cobb, N. J., Armstrong, F. A., & Hille, R. (2004) · Biochemistry · doi:10.1021/bi0357154 · Cited by 87 · see citing works
Bastian, N. R., Kay, C., Barber, M. J., & Rajagopalan, K. V. (1991) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)52399-1 · Cited by 86 · see citing works
Bennett, B., Benson, N., McEwan, A. G., & Bray, R. C. (1994) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1994.00321.x · Cited by 86 · see citing works
Tuning a Nitrate Reductase for Function
Jepson, B. J. N., Anderson, L. J., Rubio, L. M., Taylor, C. J., Butler, C. S., Flores, E., Antonia Herrero, B., Butt, J. N., & Richardson, D. J. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m402669200 · Cited by 79 · 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
Frangioni, 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-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
Purification and characterization of the assimilatory nitrate reductase of Azotobacter vinelandii
Gangeswaran, R., Lowe, D., & Eady, R. R. (1993) · Biochemical Journal · doi:10.1042/bj2890335 · Cited by 55 · see citing works
Bursakov, S. A., Liu, M. Y., Payne, W. J., LeGall, J., Moura, I., & Moura, J. J. G. (1995) · Anaerobe · doi:10.1016/s1075-9964(95)80444-7 · Cited by 52 · 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-citationBertrand, 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
Structure of the Molybdenum Site of Escherichia coli Trimethylamine N-Oxide Reductase
Zhang, L., Nelson, K. J., Rajagopalan, K. V., et al. (2007) · Inorganic Chemistry · doi:10.1021/ic701956f · Cited by 42 · 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
Redox characteristics of the tungsten DMSO reductase of Rhodobacter capsulatus
Hagedoorn, P., Hagen, W. R., Stewart, L. J., et al. (2003) · FEBS Letters · doi:10.1016/s0014-5793(03)01359-0 · Cited by 37 · see citing works
Reductive activation of nitrate reductases
Field, S. J., Thornton, N. P., Anderson, L. J., Gates, A. J., Reilly, A., Jepson, B. J. N., Richardson, D. J., George, S. J., Cheesman, M. R., & Butt, J. N. (2005) · Dalton Transactions · doi:10.1039/b505530j · Cited by 37 · see citing works
BUTLER, C. S., CHARNOCK, J. M., GARNER, C. D., THOMSON, A. J., FERGUSON, S. J., BERKS, B. C., & RICHARDSON, D. J. (2000) · Biochemical Journal · doi:10.1042/bj3520859 · Cited by 32 · 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-citationButler, C. S., Fairhurst, S. A., Ferguson, S. J., et al. (2002) · Biochemical Journal · doi:10.1042/bj3630817 · Cited by 26 · see citing works
Bell, A. F., He, X. N., Ridge, J. P., et al. (2000) · Biochemistry · doi:10.1021/bi002065k · Cited by 22 · see citing works
Guigliarelli, B., Bertrand, P., Moré, C., et al. (1985) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/0005-2728(85)90216-6 · Cited by 22 · see citing works
Self-citationSears, H. J., Bennett, B., Spiro, S., THOMSON, A. J., & Richardson, D. J. (1995) · Biochemical Journal · doi:10.1042/bj3100311 · Cited by 21 · see citing works
Butler, C. S., Ferguson, S. J., Berks, B. C., THOMSON, A. J., Cheesman, M. R., & Richardson, D. J. (2001) · FEBS Letters · doi:10.1016/s0014-5793(01)02577-7 · Cited by 13 · see citing works
Frangioni, B., & Guigliarelli, B. (2006) · Cited by 1 · see citing works
Self-citation
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Sparacino-Watkins, C., Stolz, J. F., & Basu, P. (2014) · Chem. Soc. Rev. · doi:10.1039/c3cs60249d · Cited by 426 · see citing works
Biaso, F., Burlat, B., & Guigliarelli, B. (2012) · Inorganic Chemistry · doi:10.1021/ic201533p · Cited by 40 · see citing works
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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-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
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- Authors
- Fourmond, V., Burlat, B., Dementin, S., Arnoux, P., Sabaty, M., Boiry, S., Guigliarelli, B., Bertrand, P., Pignol, D., & Léger, C.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Corresponding author
- Léger, Christophe
- ORCID
- Venue
- The Journal of Physical Chemistry B
- Publisher
- American Chemical Society (ACS)
- Published
- 2008-11-11
- Volume
- 112
- Issue
- 48
- Pages
- 15478-15486
- ISSN
- 1520-5207 (ISSN-L)
- 1520-6106
- OpenAlex
- W2009225565
- Cited by
- 51
- References
- 43
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 1 · 2022: 5 · 2021: 2 · 2019: 5 · 2018: 2 · 2017: 3 · 2016: 5 · 2015: 2 · 2014: 5 · 2013: 8 · 2012: 2
- FWCI
- 1.51
- 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
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Keywords
- periplasmic nitrate reductase, Rhodobacter sphaeroides, molybdenum enzymes, molybdenum cofactor, reductive activation, protein film voltammetry, active site heterogeneity, redox cycling, enzyme activation kinetics
- MeSH terms
- Adsorption, Algorithms, Electrochemistry, Electron Spin Resonance Spectroscopy, Iron-Sulfur Proteins, Microwaves, Molybdenum, Mutation, Oxidation-Reduction, Oxidoreductases, Potentiometry, Rhodobacter sphaeroides, Nitrate Reductase
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