Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum
Open accessAbstract
Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
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?
Reexamination of the D. desulfuricans NapA structure with single-wavelength anomalous diffraction experiments revealed that the sixth ligand is a sulfide, rather than an oxo.
“However, reexamination of the D. desulfuricans NapA structure with single-wavelength anomalous diffraction experiments revealed that the sixth ligand is a sulfide, rather than an oxo.120”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [120]
Studies with D. desulfuricans suggest that the 'nitrate species' is not catalytically relevant.
“Studies with D. desulfuricans suggest that the ‘nitrate species’ is not catalytically relevant.120,246”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [120]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
<i>Coot</i> : model-building tools for molecular graphics
Emsley, P., & Cowtan, K. (2004) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444904019158 · Cited by 30,571 · see citing works
The PyMOL Molecular Graphics System
DeLano, W. L. (2002) · Medical Entomology and Zoology · Cited by 18,147 · see citing works
The CCP4 suite: programs for protein crystallography
Number Collaborative Computational Project (1994) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444994003112 · Cited by 17,299 · see citing works
Refinement of Macromolecular Structures by the Maximum-Likelihood Method
Murshudov, G. N., Vagin, A. A., & Dodson, E. J. (1997) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444996012255 · Cited by 14,671 · see citing works
Jones, T. A., Zou, J.-Y., Cowan, S. W., & Kjeldgaard, M. (1991) · Acta Crystallographica Section A Foundations of Crystallography · doi:10.1107/s0108767390010224 · Cited by 12,650 · see citing works
Cell biology and molecular basis of denitrification
Zumft, W. G. (1997) · Microbiology and Molecular Biology Reviews · doi:10.1128/mmbr.61.4.533-616.1997 · Cited by 3,483 · see citing works
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
A discussion of the solution for the best rotation to relate two sets of vectors
Kabsch, W. (1978) · Acta Crystallographica Section A · doi:10.1107/s0567739478001680 · Cited by 1,151 · see citing works
Biological inorganic chemistry: structure and reactivity
(2007) · Choice Reviews Online · doi:10.5860/choice.44-6242 · Cited by 575 · see citing works
Richardson, D. J. (2000) · Microbiology · doi:10.1099/00221287-146-3-551 · Cited by 544 · see citing works
Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A
Bertero, M. G., Rothery, R. A., Palak, M., Hou, C., Lim, D., Blasco, F., Weiner, J. H., & Strynadka, N. C. J. (2003) · Nature Structural & Molecular Biology · doi:10.1038/nsb969 · Cited by 494 · see citing works
Crystal 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
Self-citationBacterial nitrate reductases: Molecular and biological aspects of nitrate reduction
González, P. J., Correia, C., Moura, I., Brondino, C. D., & Moura, J. J. G. (2006) · Journal of Inorganic Biochemistry · doi:10.1016/j.jinorgbio.2005.11.024 · Cited by 273 · see citing works
Self-citationArchitecture of NarGH Reveals a Structural Classification of Mo-bisMGD Enzymes
Jormakka, M., Richardson, D. J., Byrne, B., & Iwata, S. (2004) · Structure · doi:10.1016/j.str.2003.11.020 · Cited by 212 · see citing works
Raaijmakers, H. C. A., Macieira, S., Dias, J. M. L., Teixeira, S. C. M., Bursakov, S. A., Huber, R., Moura, J. J. G., Moura, I., & Romão, M. J. (2002) · Structure · doi:10.1016/s0969-2126(02)00826-2 · Cited by 186 · see citing works
Self-citationRaaijmakers, H. C. A., & Romão, M. J. (2006) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-006-0129-2 · Cited by 167 · see citing works
Self-citationLiu, M. C., & Peck, H. D. (1981) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)43021-9 · Cited by 156 · see citing works
Structural 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
Mo 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
Self-citationSchneider, T. R. (2002) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444901019291 · Cited by 137 · 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
Objective comparison of protein structures: error-scaled difference distance matrices
Schneider, T. R. (2000) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444900003723 · Cited by 101 · 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 the Molybdenum Site of Escherichia coli Formate Dehydrogenase
George, G. N., Colangelo, C. M., Dong, J., Scott, R. A., Khangulov, S. V., Gladyshev, V. N., & Stadtman, T. C. (1998) · Journal of the American Chemical Society · doi:10.1021/ja973004l · Cited by 79 · 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., Herrero, A., 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., Gutteridge, S., Lamy, M. T., et al. (1983) · Biochemical Journal · doi:10.1042/bj2110227 · Cited by 71 · 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
Groysman, S., & Holm, R. H. (2007) · Inorganic Chemistry · doi:10.1021/ic062441a · Cited by 56 · see citing works
A nonoctahedral dioxo molybdenum complex with a coordinated partial disulfide bond
Stiefel, E. I., Miller, K. F., Bruce, A. E., et al. (1980) · Journal of the American Chemical Society · doi:10.1021/ja00530a054 · Cited by 51 · see citing works
Brondino, C. D., Rivas, M. G., Romão, M. J., Moura, J. J. G., & Moura, I. (2006) · Accounts of Chemical Research · doi:10.1021/ar050104k · Cited by 49 · 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
Self-citationObservation of Ligand-Based Redox Chemistry at the Active Site of a Molybdenum Enzyme
George, G. N., Costa, C., Moura, J. J. G., & Moura, I. (1999) · Journal of the American Chemical Society · doi:10.1021/ja9841761 · Cited by 43 · see citing works
Self-citationBursakov, 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
Self-citationBerg, J. M., Spira, D. J., Hodgson, K. O., et al. (1984) · Inorganic Chemistry · doi:10.1021/ic00189a028 · Cited by 39 · see citing works
Young, C. G. (2007) · Journal of Inorganic Biochemistry · doi:10.1016/j.jinorgbio.2007.06.040 · Cited by 35 · see citing works
Laughlin, L. J., Eagle, A. A., George, G. N., Tiekink, E. R. T., & Young, C. G. (2007) · Inorganic Chemistry · doi:10.1021/ic061213d · Cited by 30 · see citing works
Jiang, J., & Holm, R. H. (2004) · Inorganic Chemistry · doi:10.1021/ic030301k · Cited by 29 · see citing works
Hofmann, M. (2007) · JBIC Journal of Biological Inorganic Chemistry · doi:10.1007/s00775-007-0271-5 · Cited by 29 · see citing works
Tenderholt, A. L., Szilagyi, R. K., Holm, R. H., et al. (2007) · Journal of Inorganic Biochemistry · doi:10.1016/j.jinorgbio.2007.07.011 · Cited by 28 · see citing works
Goh, L. Y., Wong, R. C. S., Yip, W.-H., et al. (1991) · Organometallics · doi:10.1021/om00050a015 · Cited by 26 · see citing works
Faller, J. W., & Ma, Y. (1989) · Organometallics · doi:10.1021/om00105a006 · Cited by 25 · see citing works
Coelho, C., González, P. J., Trincão, J., Carvalho, A. L., Najmudin, S., Hettman, T., Dieckman, S., Moura, J. J. G., Moura, I., & Romão, M. J. (2007) · Acta Crystallographica Section F Structural Biology and Crystallization Communications · doi:10.1107/s1744309107022129 · Cited by 24 · see citing works
Self-citationSugimoto, H., Sakurai, T., Miyake, H., et al. (2005) · Inorganic Chemistry · doi:10.1021/ic0509128 · Cited by 20 · see citing works
Astashkin, A. V., Klein, E. L., & Enemark, J. H. (2007) · Journal of Inorganic Biochemistry · doi:10.1016/j.jinorgbio.2007.05.015 · Cited by 18 · see citing works
Halcrow, M. A., Huffman, J. C., & Christou, G. (1994) · Inorganic Chemistry · doi:10.1021/ic00095a005 · Cited by 12 · see citing works
Rys, A. Z., Lebuis, A.-M., Shaver, A. G., et al. (1999) · Organometallics · doi:10.1021/om980813k · Cited by 11 · see citing works
Dias, J. M. L., Bursakov, S. A., Carneiro, C., Moura, J. J. G., Moura, I., & Romão, M. J. (1999) · Acta Crystallographica Section D Biological Crystallography · doi:10.1107/s0907444998014735 · Cited by 6 · see citing works
Self-citation
Flagged first, then by citations
Research works printed but not linked: 1
- Stiefel EI, Miller KF, Bruce AE, Corbin JL, Berg JM, Hodgson KO (1980) J Am Chem Soc 102(10):3624-3626
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
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
Maia, L. B., & Moura, J. J. G. (2014) · Chemical Reviews · doi:10.1021/cr400518y · Cited by 401 · see citing works
Self-citationThe molybdenum oxotransferases and related enzymes
Hille, R. (2013) · Dalton Transactions · doi:10.1039/c2dt32376a · Cited by 133 · see citing works
Coelho, C., González, P. J., Moura, J. G., Moura, I., Trincão, J., & João Romão, M. (2011) · Journal of Molecular Biology · doi:10.1016/j.jmb.2011.03.016 · Cited by 97 · see citing works
Self-citationSpectroscopic studies of molybdenum and tungsten enzymes
Pushie, M. J., & George, G. N. (2011) · Coordination Chemistry Reviews · doi:10.1016/j.ccr.2011.01.056 · Cited by 86 · 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
Self-citationPushie, M. J., Cotelesage, J. J., & George, G. N. (2014) · Metallomics · doi:10.1039/c3mt00177f · Cited by 55 · see citing works
Marangon, 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
Self-citationBiaso, F., Burlat, B., & Guigliarelli, B. (2012) · Inorganic Chemistry · doi:10.1021/ic201533p · Cited by 40 · 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
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
Simpson, P. J. L., McKinzie, A. A., & Codd, R. (2010) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2010.06.002 · Cited by 20 · see citing works
Necessity of fine tuning in Mo(iv) bis(dithiolene) complexes to warrant nitrate reduction
Majumdar, A., Pal, K., & Sarkar, S. (2009) · Dalton Transactions · doi:10.1039/b815436h · Cited by 19 · see citing works
- Authors
- 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., & Romão, M. J.
- Affiliations
- Rede de Química e Tecnologia (PT)
- Universidade Nova de Lisboa (PT)
- Universidad Nacional del Litoral (AR)
- Corresponding author
- Romão, Carlos C.
- Brondino, Carlos D.
- Romão, Maria J.
- ORCID
- Venue
- JBIC Journal of Biological Inorganic Chemistry
- Published
- 2008-03-08
- Volume
- 13
- Issue
- 5
- Pages
- 737-753
- ISSN
- 0949-8257 (ISSN-L)
- 1432-1327
- OpenAlex
- W2150426398
- Cited by
- 116
- References
- 48
- Citations by year
- 2026: 5 · 2025: 6 · 2024: 3 · 2023: 4 · 2022: 6 · 2021: 4 · 2020: 3 · 2019: 6 · 2018: 5 · 2017: 5 · 2016: 7 · 2015: 8 · 2014: 12 · 2013: 10 · 2012: 6
- FWCI
- 4.74
- Type
- journal article
- Language
- en
- Open access
- green
- hdl.handle.net
- Licence
- other-oa
- www.springer.com/tdm (tdm)
- Locations
- JBIC Journal of Biological Inorganic Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- Funders
- Consejo Nacional de Investigaciones Científicas y Técnicas
- Fundação para a Ciência e a Tecnologia (POCI2010)
- 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 › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Keywords
- periplasmic nitrate reductase, Desulfovibrio desulfuricans, molybdenum coordination, sulfur-containing ligands, disulfide bonds, [4Fe-4S] cluster, active site structure, hyperfine structure
- MeSH terms
- Binding Sites, Catalysis, Data Interpretation, Statistical, Disulfides, Electron Spin Resonance Spectroscopy, Ligands, Models, Molecular, Molybdenum, Protein Conformation, Sulfur, Crystallography, X-Ray, Desulfovibrio desulfuricans, Nitrate Reductase
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
