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Nitrate reductases are molybdoenzymes that reduce nitrate (NO
) to nitrite (NO
). This reaction is critical for the production of protein in most crop plants, as nitrate is the predominant source of nitrogen in fertilized soils.[2]

nitrate reductase
Nitrate reductase.png
structure of nitrate reductase A from E. coli[1]
EC number1.7.99.4
CAS number37256-45-4
IntEnzIntEnz view
ExPASyNiceZyme view
MetaCycmetabolic pathway
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Molybdopterin oxidoreductase (nitrate reductase alpha subunit)
OPM superfamily3
OPM protein1kqf
4Fe-4S dicluster domain
(nitrate reductase beta subunit)
Nitrate reductase gamma subunit
OPM superfamily3
OPM protein1q16
Nitrate reductase delta subunit
Nitrate reductase cytochrome c-type subunit (NapB)
Periplasmic nitrate reductase protein NapE



Eukaryotic nitrate reductases are part of the sulfite oxidase family of molybdoenzymes. They transfer electrons from NADH or NADPH to nitrate.


Prokaryotic nitrate reductases belong to the DMSO reductase family of molybdoenzymes and have been classified into three groups, assimilatory nitrate reductases (Nas), respiratory nitrate reductase (Nar), and periplasmic nitrate reductases (Nap).[3] The active site of these enzymes is a Mo ion that is bound to the four thiolate functions of two pterin molecules. The coordination sphere of the Mo is completed by one amino-acid side chain and oxygen and/or sulfur ligands. The exact environment of the Mo ion in certain of these enzymes (oxygen versus sulfur as a sixth molybdenum ligand) is still debated. The Mo is covalently attached to the protein by a cysteine ligand in Nap, and an aspartate in Nar.[4]


Prokaryotic nitrate reductases have two major types, transmembrane nitrate reductases and periplasmic nitrate reductases. The transmembrane nitrate reductase (NAR) does proton translocation and can contribute to the generation of ATP by the proton motive force. The periplasmic nitrate reductase (NAP) does not do proton translocation and does not contribute to the proton motive force.[5]

The transmembrane respiratory nitrate reductase[6] is composed of three subunits; an 1 alpha, 1 beta and 2 gamma. It is the second nitrate reductase enzyme which it can substitute for the NRA enzyme in Escherichia coli allowing it to use nitrate as an electron acceptor during anaerobic respiration.[7] A transmembrane nitrate reductase that can function as a proton pump (similar to the case of anaerobic respiration) has been discovered in a diatom Thalassiosira weissflogii.[8]

The nitrate reductase of higher plants, algae, and fungi is a homodimeric cytosolic protein with five conserved domains in each monomer: 1) an Mo-MPT domain that contains the single molybdopterin cofactor, 2) a dimer interface domain, 3) a cytochrome b domain, and 4) an NADH domain that combines with 5) an FAD  domain to form the cytochrome b reductase fragment.[9] There exists a GPI-anchored variant that is found on the outer face of the plasma membrane. Its exact function is still not clear.[10]


In prokaryotic periplasmic nitrate reductase, a nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the MoV species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with MoVI ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308.

The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism is that of molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of MoV species. MoVI intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the MoVI species allow water molecule dissociation and the concomitant enzymatic turnover.[11]

Similar to the prokaryotic nitrate reduction mechanism, in eukaryotic nitrate reductase, an oxygen in nitrate binds to Mo in the (IV) oxidation state, displacing a hydroxide ion. Then the Mo d-orbital electrons flip over, creating a double bond between Mo (VI) and that oxygen, ejecting nitrite. The Mo(VI) double bond to oxygen is reduced via electrons from NAD(P)H passed through the intramolecular transport chain.[12]


Nitrate reductase (NR) is regulated at the transcriptional and translational levels induced by light, nitrate, and possibly a negative feedback mechanism. First, nitrate assimilation is initiated by the uptake of nitrate from the root system, reduced to nitrite by nitrate reductase, and then nitrite is reduced to ammonia by nitrite reductase. Ammonia then goes into the GS-GOGAT pathway to be incorporated into amino acids.[13] When the plant is under stress, instead of reducing nitrate via NR to be incorporated into amino acids, the nitrate is reduced to nitric oxide which can have many damaging effects on the plant. Thus, the importance of regulating nitrate reductase activity is to limit the amount of nitric oxide being produced.

Inactivation of nitrate reductaseEdit

The inactivation of nitrate reductase has many steps and many different signals that aid in the inactivation of the enzyme. Specifically in spinach, the very first step of nitrate reductase inactivation is the phosphorylation of NR on the 543-serine residue. The very last step of nitrate reductase inactivation is the binding of the 14-3-3 adapter protein, which is initiated by the presence of Mg2+ and Ca2+.[14] Higher plants and some algae post-translationally regulate NR by phosphorylation of serine residues and subsequent binding of a 14-3-3 protein.[15]

Anoxic conditionsEdit

Studies were done measuring the nitrate uptake and nitrate reductase activity in anoxic conditions to see if there was a difference in activity level and tolerance to anoxia. These studies found that nitrate reductase, in anoxic conditions improves the plants tolerance to being less aerated.[14] This increased activity of nitrate reductase was also related to a increase in nitrite release in the roots. The results of this study showed that the dramatic increase in nitrate reductase in anoxic conditions can be directly attributed to the anoxic conditions inducing the dissociation of 14-3-3 protein from NR and the dephosphorylation of the nitrate reductase.[14]


Nitrate reductase activity can be used as a biochemical tool for predicting grain yield and grain protein production.[16][17]

Nitrate reductase can be used to test nitrate concentrations in biofluids.[18]

Nitrate reductase promotes amino acid production in tea leaves.[19] Under south Indian conditions, it is reported that tea plants sprayed with various micronutrients (like Zn, Mn and B) along with Mo enhanced the amino acid content of tea shoots and also the crop yield.[20]


  1. ^ PDB: 1Q16​; Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NC (September 2003). "Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A". Nature Structural Biology. 10 (9): 681–7. doi:10.1038/nsb969. PMID 12910261.
  2. ^ Marschner, Petra, ed. (2012). Marschner's mineral nutrition of higher plants (3rd ed.). Amsterdam: Elsevier/Academic Press. p. 135. ISBN 9780123849052.
  3. ^ Moreno-Vivián, Conrado Cabello, Purificación Martínez-Luque, Manuel Blasco, Rafael Castillo, Francisco. Prokaryotic Nitrate Reduction: Molecular Properties and Functional Distinction among Bacterial Nitrate Reductases. American Society for Microbiology. OCLC 678511191.CS1 maint: multiple names: authors list (link)
  4. ^ Tavares P, Pereira AS, Moura JJ, Moura I (December 2006). "Metalloenzymes of the denitrification pathway". Journal of Inorganic Biochemistry. 100 (12): 2087–100. doi:10.1016/j.jinorgbio.2006.09.003. PMID 17070915.
  5. ^ Kuypers MM, Marchant HK, Kartal B (May 2018). "The microbial nitrogen-cycling network". Nature Reviews. Microbiology. 16 (5): 263–276. doi:10.1038/nrmicro.2018.9. PMID 29398704.
  6. ^ "ENZYME entry: EC". ENZYME Enzyme nomenclature database. Retrieved 25 April 2019.
  7. ^ Blasco F, Iobbi C, Ratouchniak J, Bonnefoy V, Chippaux M (June 1990). "Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon". Molecular & General Genetics. 222 (1): 104–11. doi:10.1007/BF00283030 (inactive 2019-08-21). PMID 2233673.
  8. ^ Jones GJ, Morel FM (May 1988). "Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase". Plant Physiology. 87 (1): 143–7. doi:10.1104/pp.87.1.143. PMC 1054714. PMID 16666090.
  9. ^ Campbell WH (June 1999). "Nitrate Reductase Structure, Function and Regulation: Bridging the Gap between Biochemistry and Physiology". Annual Review of Plant Physiology and Plant Molecular Biology. 50 (1): 277–303. doi:10.1146/annurev.arplant.50.1.277. PMID 15012211.
  10. ^ Tischner R (October 2000). "Nitrate uptake and reduction in higher and lower plants". Plant, Cell and Environment. 23 (10): 1005–1024. doi:10.1046/j.1365-3040.2000.00595.x.
  11. ^ Cerqueira NM, Gonzalez PJ, Brondino CD, Romão MJ, Romão CC, Moura I, Moura JJ (November 2009). "The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase". Journal of Computational Chemistry. 30 (15): 2466–84. doi:10.1002/jcc.21280. PMID 19360810.
  12. ^ Fischer K, Barbier GG, Hecht HJ, Mendel RR, Campbell WH, Schwarz G (April 2005). "Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site". The Plant Cell. 17 (4): 1167–79. doi:10.1105/tpc.104.029694. PMC 1087994. PMID 15772287.
  13. ^ Taiz L, Zeiger E, Moller IM, Murphy A (2014). Plant Physiology and Development (6 ed.). Massachusetts: Sinauer Associates, Inc. p. 356. ISBN 978-1-60535-353-1.
  14. ^ a b c Allègre A, Silvestre J, Morard P, Kallerhoff J, Pinelli E (December 2004). "Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia" (PDF). Journal of Experimental Botany. 55 (408): 2625–34. doi:10.1093/jxb/erh258. PMID 15475378.
  15. ^ Wang Y, Bouchard JN, Coyne KJ (September 2018). "Expression of novel nitrate reductase genes in the harmful alga, Chattonella subsalsa". Scientific Reports. 8 (1): 13417. Bibcode:2018NatSR...813417W. doi:10.1038/s41598-018-31735-5. PMC 6128913. PMID 30194416.
  16. ^ Croy LI, Hageman RH (1970). "Relationship of nitrate reductase activity to grain protein production in wheat". Crop Science. 10 (3): 280–285. doi:10.2135/cropsci1970.0011183X001000030021x.
  17. ^ Dalling MJ, Loyn RH (1977). "Level of activity of nitrate reductase at the seedling stage as a predictor of grain nitrogen yield in wheat (Triticum aestivum L.)". Australian Journal of Agricultural Research. 28 (1): 1–4. doi:10.1071/AR9770001.
  18. ^ Mori, Hisakazu (2001). "Determination of Nitrate in Biological Fluids Using Nitrate Reductase in a Flow System". Journal of Health Science. 47 (1): 65–67. doi:10.1248/jhs.47.65. ISSN 1344-9702.
  19. ^ Ruan J, Wu X, Ye Y, Härdter R (1988). "Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensis L". J. Sci. Food Agric. 76 (3): 389–396. doi:10.1002/(SICI)1097-0010(199803)76:3<389::AID-JSFA963>3.0.CO;2-X.
  20. ^ Venkatesan S (November 2005). "Impact of genotype and micronutrient applications on nitrate reductase activity of tea leaves". J. Sci. Food Agric. 85 (3): 513–516. doi:10.1002/jsfa.1986.

External linksEdit

This article incorporates text from the public domain Pfam and InterPro: IPR003816