The Crystal Structure of Cupriavidus necator Nitrate Reductase in Oxidized and Partially Reduced States
Abstract
The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C(4)mim]Cl(-). In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.
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The crystal structures of NapA from Cupriavidus necator (PDB ID: 3ML1), E. coli (PDB ID: 2NYA), R. sphaeroides (PDB ID: 1OGY), and D. desulfuricans (PDB ID: 2NAP) have been reported.
“The crystal structures of NapA from several species have been reported: Cupriavidus necator (PDB ID: 3ML1),121 E. coli (PDB ID: 2NYA),96 R. sphaeroides (PDB ID: 1OGY),186 and D. desulfuricans (PDB ID: 2NAP).119”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [121]
The Rhodobacter sphaeroides and Cupriavidus necator NapAB heterodimer structures have been determined using X-ray crystallography.
“The Rhodobacter sphaeroides and Cupriavidus necator NapAB heterodimer structures have been determined using X-ray crystallography.121,186”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [121]
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- Authors
- Coelho, C., González, P. J., Moura, J. G., Moura, I., Trincão, J., & João Romão, M.
- Affiliations
- Universidade Nova de Lisboa (PT)
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2011-05
- Volume
- 408
- Issue
- 5
- Pages
- 932-948
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W1985556019
- Cited by
- 97
- References
- 48
- Citations by year
- 2026: 3 · 2025: 8 · 2024: 5 · 2023: 7 · 2022: 4 · 2021: 3 · 2020: 10 · 2019: 6 · 2018: 5 · 2017: 4 · 2016: 4 · 2015: 10 · 2014: 9 · 2013: 8 · 2012: 8
- FWCI
- 3.73
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- European Synchrotron Radiation Facility
- Fundação para a Ciência e a Tecnologia
- European Regional Development Fund
- 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, Cupriavidus necator, molybdenum enzymes, [4Fe-4S] cluster, electron transfer, sodium dithionite, ionic liquids, redox potential, crystal structure
- MeSH terms
- Amino Acid Sequence, Cysteine, Dithionite, Electron Spin Resonance Spectroscopy, Molecular Sequence Data, Molybdenum, Protein Conformation, Crystallography, X-Ray, Periplasm, Cupriavidus necator, Catalytic Domain, Nitrate Reductase, Ionic Liquids
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