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Enzyme Nomenclature
Information on EC 1.7.5.1 - nitrate reductase (quinone)
Word Map on EC 1.7.5.1
- 1.7.5.1
-
reductases
-
denitrify
-
denitrification
- narghi
-
dissimilatory
-
nark
- chlorate
-
nitrate-reducing
- molybdopterin
-
molybdoenzyme
-
low-potential
-
thiosphaera
- pantotropha
-
fnr-like
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
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EC NUMBER 
COMMENTARY 
1.7.5.1
-
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RECOMMENDED NAME
GeneOntology No.
nitrate reductase (quinone)
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nitrate + a quinol = nitrite + a quinone + H2O
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
nitrite:quinone oxidoreductase
A membrane-bound enzyme which supports anaerobic respiration on nitrate under anaerobic conditions and in the presence of nitrate. Contains the bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) cofactor, iron-sulfur clusters and heme b. Escherichia coli expresses two forms NarA and NarZ, both being comprised of three subunits.
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
respiratory nitrate reductase 1 alpha chain
E1WG01
UniProt
respiratory nitrate reductase 1 alpha chain
E1WG01
UniProt
-
395164, 658071, 674425, 674613, 695649, 696060, 696079, 696200, 696201, 696212, 696375, 696520, 697207, 697691, 697693, 697708, 700254, 700372, 726366
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-
narH: P11349, narG: P09152, narI: P11350
P11349 and P09152 and P11350
SwissProt
P09152: alpha-chain, P11349: beta-chain, P0AF26: delta-chain
P09152 and P11349 and P0AF26
SwissProt
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
-
the cytochrome c containing nitrate reductase plays a role in electron transport for denitrification in Thermus thermophilus, narC mutants are defective in anaerobic growth with nitrite, NO and N2O and present decreased NADH oxidation coupled to these electron acceptors
physiological function
E1WG01
under nitrate-rich conditions, the nar and nap genes encoding a membrane-bound form and a periplasmic form of nitrate reductase, as well as NO-regulated genes encoding flavohaemoglobin, flavorubredoxin and hybrid cluster protein are induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state is released as N2O when nitrite has accumulated to millimolar levels. In a narG mutant lacking membrane-bound nitrate reductase, the steady-state rate of N2O production was about 30-fold lower than that of the wild-type. A combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N2O production, and this can account for up to 20% of the nitrate catabolized
physiological function
-
Streptomyces coelicolor has a high capacity for nitrate reduction both during aerobic growth and when the bacterium is incubated anaerobically. During aerobic growth in liquid medium the bacterium is able to reduce 50 mM nitrate stoichiometrically to nitrite. A mutant lacking all three NarGHJI operons fails to reduce nitrate
physiological function
-
mutants deficient in all three nitrate reductases narGHI, narXYZ, napFDAGHCB are capable of sustaining 48% of protoporphyrinogen IX oxidases activity and 65% of wild-type activity, respectively
physiological function
-
under nitrate-rich conditions, the nar and nap genes encoding a membrane-bound form and a periplasmic form of nitrate reductase, as well as NO-regulated genes encoding flavohaemoglobin, flavorubredoxin and hybrid cluster protein are induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state is released as N2O when nitrite has accumulated to millimolar levels. In a narG mutant lacking membrane-bound nitrate reductase, the steady-state rate of N2O production was about 30-fold lower than that of the wild-type. A combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N2O production, and this can account for up to 20% of the nitrate catabolized
-
physiological function
-
Streptomyces coelicolor has a high capacity for nitrate reduction both during aerobic growth and when the bacterium is incubated anaerobically. During aerobic growth in liquid medium the bacterium is able to reduce 50 mM nitrate stoichiometrically to nitrite. A mutant lacking all three NarGHJI operons fails to reduce nitrate
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
nitrate + 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol
nitrite + 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone + H2O
-
i.e. decylubiquinol
-
-
?
nitrate + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
nitrite + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone + H2O
nitrate + 5-hydroxy-2-methyl-1,4-naphthoquinol
nitrite + 5-hydroxy-2-methyl-1,4-naphthoquinone + H2O
-
-
-
-
?
nitrate + 5-hydroxy-2-methyl-naphthalene-1,4-diol
nitrite + 5-hydroxy-2-methyl-naphthalene-1,4-dione + H2O
nitrate + menaquinol
nitrite + menaquinone + H2O
-
only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. In vivo quinol interacts directly with the gamma subunit that is lost during solubilization
-
-
?
nitrate + quinol
nitrite + quinone
-
NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. The semiquinone is located within the quinol oxidation site QD
-
-
?
nitrate + reduced methyl viologen
nitrite + oxidized ethyl viologen + H2O
-
-
-
-
?
nitrate + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
nitrite + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone + H2O
-
-
-
-
?
nitrate + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
nitrite + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone + H2O
-
-
-
?
nitrate + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
nitrite + 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone + H2O
-
-
-
?
nitrate + 2-methyl-1,4-naphthoquinol
nitrite + 2-methyl-1,4-naphthoquinone + H2O
-
i.e menadiol
-
-
?
nitrate + 2-methyl-1,4-naphthoquinol
nitrite + 2-methyl-1,4-naphthoquinone + H2O
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i.e. menadiol
-
-
?
nitrate + 2-methyl-1,4-naphthoquinol
nitrite + 2-methyl-1,4-naphthoquinone + H2O
-
i.e. menadiol. As the reduction of nitrate to nitrite requires two electrons, there must necessarily be two successive bindings of quinone, with transfer of one electron to the hemes, then to the [Fe-S] cluster, to be finally accumulated at the level of the molybdenum cofactor to be able to undertake the catalytic reaction. There are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol. A two-pathway electron transfer model for nitrate reductase A is proposed
-
-
?
nitrate + 2-methyl-1,4-naphthoquinol
nitrite + 2-methyl-1,4-naphthoquinone + H2O
-
i.e. menadiol. Electrons from menadiol oxidation are transferred initially to heme bL
-
-
?
nitrate + 5-hydroxy-1,4-naphthoquinol
nitrite + 5-hydroxy-1,4-naphthoquinone + H2O
-
i.e. juglone
-
-
?
nitrate + 5-hydroxy-1,4-naphthoquinol
nitrite + 5-hydroxy-1,4-naphthoquinone + H2O
-
i.e. reduced form of juglone
-
-
?
nitrate + 5-hydroxy-2-methyl-naphthalene-1,4-diol
nitrite + 5-hydroxy-2-methyl-naphthalene-1,4-dione + H2O
-
i.e plumbagin
-
-
?
nitrate + 5-hydroxy-2-methyl-naphthalene-1,4-diol
nitrite + 5-hydroxy-2-methyl-naphthalene-1,4-dione + H2O
-
i.e. reduced form of plumbagin
-
-
?
nitrate + 5-hydroxy-2-methyl-naphthalene-1,4-diol
nitrite + 5-hydroxy-2-methyl-naphthalene-1,4-dione + H2O
-
i.e. reduced form of plumbagin
i.e. plumbagin
-
?
nitrate + duroquinol
nitrite + duroquinone + H2O
-
if quinols are used as the electron donor the enzyme operates by a two-site, enzyme-substitution mechanism
-
-
?
nitrate + quinol
nitrite + quinone + H2O
P09152 and P11349 and P0AF26
-
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
first enzyme involved in respiratory denitrification in prokaryotes
-
-
-
nitrate + quinol
nitrite + quinone + H2O
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in order to use nitrate as an electron acceptor, Escherichia coli synthesises three distinct enzymes: a membrane-bound enzyme (nitrate reductase A, NarGHI) encoded by the narGHJI operon and a soluble periplasmic nitrate reductase (NapAB, EC 1.9.6.1) encoded by the napFDAGHBC operon. A second membrane-bound nitrate reductase (nitrate reductase Z, NarZYV) encoded by the NarZYWV operon is biochemically similar to NarGHI. Whereas NarGHI synthesis is induced by nitrate under anaerobic conditions, NarZYV is expressed at a cryptic level and may assist Escherichia coli in transition from aerobic to anaerobic respiration (physiological role of this isoenzyme at the onset of the stationary growth phase in rich media). NapAB is mainly expressed in the presence of low concentrations of nitrate under both aerobic and anaerobic conditions, and its expression is suppressed at high nitrate concentrations. Conversely, NarGHI is maximally expressed when nitrate concentration is elevated, and under these conditions becomes the predominant enzyme in Escherichia coli. Thus, NapAB (Ec 1.9.6.1) and NarGHI seem to function in different ranges of nitrate concentration in a complementary way to support anaerobic respiration on nitrate under anaerobic conditions and in the presence of nitrate
-
-
?
nitrate + quinol
nitrite + quinone + H2O
P09152 and P11349 and P0AF26
model of electron transfer in the nitrate reductase: electrons are provided by quinones to the NarI subunit and subsequently transferred to NarH, which eventually delivers them to the molybdenum cofactor where nitrate reduction takes place
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
nitrate reductase A reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
under anaerobic conditions in the presence of nitrate, Escherichia coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A, NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. The arrangement, coordination scheme and unique environment of the redox-active prosthetic groups is revealed
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
the enzyme is essential for the fungal denitrification. The fungal formate dehydrogenase can supply electrons via quinol/quinone pool to nitrate reductase A
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
the membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH
-
-
?
nitrate + reduced benzyl viologen
nitrite + benzyl viologen
-
-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
-
-
-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
P11349 and P09152 and P11350
-
-
-
?
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
-
when reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, Theorell-Chance mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme
-
-
?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
-
-
-
-
?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
-
when reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, Theorell-Chance mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme
-
-
?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
-
catalysis under substrate-limiting conditions clearly occurs via two pathways with distinct kinetic properties reversibly linked by a redox event. This redox event may be integral to the catalytic cycle of the active site or occur at a center, remote from the description of active-site chemistry, which serves to switch NarGH between two catalytically competent forms
-
-
?
nitrate + tetramethyl-p-benzoquinol
nitrite + tetramethyl-p-benzoquinone + H2O
-
i.e. duroquinol
-
-
?
nitrate + tetramethyl-p-benzoquinol
nitrite + tetramethyl-p-benzoquinone + H2O
P11349 and P09152 and P11350
i.e. duroquinol
-
-
?
nitrate + tetramethyl-p-benzoquinol
nitrite + tetramethyl-p-benzoquinone + H2O
-
i.e. duroquinol. As the reduction of nitrate to nitrite requires two electrons, there must necessarily be two successive bindings of quinone, with transfer of one electron to the hemes, then to the [Fe-S] cluster, to be finally accumulated at the level of the molybdenum cofactor to be able to undertake the catalytic reaction. There are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol. A two-pathway electron transfer model for nitrate reductase A is proposed
-
-
?
nitrate + ubiquinol
nitrite + ubiquinone + H2O
-
if quinols are used as the electron donor the enzyme operates by a two-site, enzyme-substitution mechanism
-
-
?
additional information
?
-
-
Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). The two enzymes are encoded by distinct operons located within two different loci on the Escherichia coli chromosome. The narGHJI operon, encoding nitrate reductase A, is located in the chlC locus at 27 min, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system. The narZYWV operon, encoding nitrate reductase Z, is located in the chlZ locus located at 32.5 min, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWV operon and the narU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in Escherichia coli
-
-
-
additional information
?
-
-
nitrate reductase Z expression is regulated in a manner opposite to that of nitrate reductase A. The narGHJZ operon is aerobically repressed, strongly induced by nitrate and positively regulated by the fnr gene product. The expression of narZ is anaerobically repressed, induced weakly, if at all, by nitrate and negatively regulated by the fnr gene product. The opposing regulation of these two enzymes suggests that a function of nitrate reductase Z may be to catalyse the immediate flow of electrons to nitrate during an aerobic/anaerobic transition when the bacterium is grown in the presence of nitrate
-
-
-
additional information
?
-
-
NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. The NRZ operon is controlled mainly at the level of transcription and is induced 10fold at the onset of stationary phase in rich media. Expression of NRZ nitrate reductase is highly growth phase dependent and is controlled by the alternative vegetative sigma factor RpoS. RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions
-
-
-
additional information
?
-
-
bromate and chlorate are substrates of the enzyme
-
-
-
additional information
?
-
-
the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction
-
-
-
additional information
?
-
-
nitrate reductase Z is synthesized in small amounts, the expression of its structural genes does not seem to be induced by nitrate, repressed by oxygen or activated by the product of the fnr gene. The nitrate reductase Z in mutant LCB79/pLCB14 couples formate oxidation with nitrate reduction probably via quinones and type-b cytochromes
-
-
-
additional information
?
-
-
nitrate reductase Z is able to use both nitrate and chlorate as substrate
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
nitrate + quinol
nitrite + quinone + H2O
-
first enzyme involved in respiratory denitrification in prokaryotes
-
-
-
nitrate + quinol
nitrite + quinone + H2O
-
in order to use nitrate as an electron acceptor, Escherichia coli synthesises three distinct enzymes: a membrane-bound enzyme (nitrate reductase A, NarGHI) encoded by the narGHJI operon and a soluble periplasmic nitrate reductase (NapAB, EC 1.9.6.1) encoded by the napFDAGHBC operon. A second membrane-bound nitrate reductase (nitrate reductase Z, NarZYV) encoded by the NarZYWV operon is biochemically similar to NarGHI. Whereas NarGHI synthesis is induced by nitrate under anaerobic conditions, NarZYV is expressed at a cryptic level and may assist Escherichia coli in transition from aerobic to anaerobic respiration (physiological role of this isoenzyme at the onset of the stationary growth phase in rich media). NapAB is mainly expressed in the presence of low concentrations of nitrate under both aerobic and anaerobic conditions, and its expression is suppressed at high nitrate concentrations. Conversely, NarGHI is maximally expressed when nitrate concentration is elevated, and under these conditions becomes the predominant enzyme in Escherichia coli. Thus, NapAB (Ec 1.9.6.1) and NarGHI seem to function in different ranges of nitrate concentration in a complementary way to support anaerobic respiration on nitrate under anaerobic conditions and in the presence of nitrate
-
-
?
nitrate + quinol
nitrite + quinone + H2O
P09152 and P11349 and P0AF26
model of electron transfer in the nitrate reductase: electrons are provided by quinones to the NarI subunit and subsequently transferred to NarH, which eventually delivers them to the molybdenum cofactor where nitrate reduction takes place
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
nitrate reductase A reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
under anaerobic conditions in the presence of nitrate, Escherichia coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A, NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. The arrangement, coordination scheme and unique environment of the redox-active prosthetic groups is revealed
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
the enzyme is essential for the fungal denitrification. The fungal formate dehydrogenase can supply electrons via quinol/quinone pool to nitrate reductase A
-
-
?
nitrate + quinol
nitrite + quinone + H2O
-
the membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH
-
-
?
additional information
?
-
-
Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). The two enzymes are encoded by distinct operons located within two different loci on the Escherichia coli chromosome. The narGHJI operon, encoding nitrate reductase A, is located in the chlC locus at 27 min, along with several functionally related genes: narK, encoding a nitrate/nitrite antiporter, and the narXL operon, encoding a nitrate-activated, two component regulatory system. The narZYWV operon, encoding nitrate reductase Z, is located in the chlZ locus located at 32.5 min, a region which includes a narK homologue, narU, but no apparent homologue to the narXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both the narGHJI operon and the narK gene are positively regulated by two transacting factors Fnr and NarL-phosphate, activated respectively by anaerobiosis and nitrate, while the narZYWV operon and the narU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase in Escherichia coli
-
-
-
additional information
?
-
-
nitrate reductase Z expression is regulated in a manner opposite to that of nitrate reductase A. The narGHJZ operon is aerobically repressed, strongly induced by nitrate and positively regulated by the fnr gene product. The expression of narZ is anaerobically repressed, induced weakly, if at all, by nitrate and negatively regulated by the fnr gene product. The opposing regulation of these two enzymes suggests that a function of nitrate reductase Z may be to catalyse the immediate flow of electrons to nitrate during an aerobic/anaerobic transition when the bacterium is grown in the presence of nitrate
-
-
-
additional information
?
-
-
NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. The NRZ operon is controlled mainly at the level of transcription and is induced 10fold at the onset of stationary phase in rich media. Expression of NRZ nitrate reductase is highly growth phase dependent and is controlled by the alternative vegetative sigma factor RpoS. RpoS-mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress-associated conditions
-
-
-
additional information
?
-
-
nitrate reductase Z is synthesized in small amounts, the expression of its structural genes does not seem to be induced by nitrate, repressed by oxygen or activated by the product of the fnr gene. The nitrate reductase Z in mutant LCB79/pLCB14 couples formate oxidation with nitrate reduction probably via quinones and type-b cytochromes
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cytochrome bD
-
NarI is strongly associated with heme bD, Lys86 is required for its stabilization
-
cytochrome bH
-
both heme bL and heme bH are crucial components in the electron-transfer pathway from the subunit NarI through subunit NarH to the catalytic subunit NarG. Without heme bL electrons cannot be transferred from menaquinol to heme bH. On the other hand, in the absence of heme bH, electrons cannot be transferred from the reduced heme bL to the catalytic dimer NarGH. A complex of menadione radical anion associated with the enzyme, is formed during the process of heme reduction by menadiol
-
cytochrome bL
-
both heme bL and heme bH are crucial components in the electron-transfer pathway from the subunit NarI through subunit NarH to the catalytic subunit NarG. Without heme bL electrons cannot be transferred from menaquinol to heme bH. On the other hand, in the absence of heme bH, electrons cannot be transferred from the reduced heme bL to the catalytic dimer NarGH. A complex of menadione radical anion associated with the enzyme, is formed during the process of heme reduction by menadiol
-
heme b
-
the anchor subunit NarI contains two b-type hemes. Electron transfer out of NarI is mediated by two hemes, one of relatively low midpoint potential Em (heme bL), and one of relatively high Em (heme bH)
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
-
the enzyme possesses a molybdopterin guanine dinucleotide active center. Two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
-
evidence for the presence of interactions between the molybdenum cofactor (Moco) biosynthetic machinery and aponitrate reductase A. The final stages of molybdenum cofactor biosynthesis occurs on a complex made up by MogA, MoeA, MobA, and MobB, which is also in charge with the delivery of the mature cofactor onto the aponitrate reductase A in a NarJ-assisted process
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
-
the molybdo-bis(molybdopterin guanine dinucleotide)-binding subunit NarG is organized in four domains around the molybdo-bis(molybdopterin guanine dinucleotide) cofactor
bis(molybdopterin guanine dinucleotide)molybdenum cofactor
-
structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) cofactor in the catalytic mechanism; the enzyme uses a molybdo-bis(molybdopterin guanine dinucleotide) cofactor for catalytic mechanism
cytochrome
-
NarI is strongly associated with heme bD, Lys86 is required for its stabilization
-
cytochrome
-
although the spectral studies of nitrate reductase Z reveals the presence of a b-type cytochrome subunit (1.5 mol/molecule of 230000 Da), none can be detected in the SDS-PAGE
-
cytochrome
-
the spectrophotometric studies indicate that reduction of the cytochrome hemes varies according to the analogue of quinone used, and in no cases is it complete
-
cytochrome b
-
the enzyme contains two b-type hemes in the gamma subunit. The two b-type centres are functional parts of the enzyme
-
cytochrome b
-
partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the subunits of the enzyme. The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction
-
cytochrome c
-
the isolated preparation contains heme c in a sub-stoichiometric amount with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes
cytochrome c
-
NarC contains a periplasmic cytochrome c, which is required for membrane attachment and maturation of the NarG catalytic subunit of the enzyme
heme
-
the reduction of NarGHI hemes by menaquinol, the reduction exhibits four phases, a transient species associated with the enzyme is kinetically correlated to the second reduction of the hemes
heme
-
the spectrophotometric studies indicate that reduction of the cytochrome hemes varies according to the analogue of quinone used, and in no cases is it complete
heme
-
the transmembrane subunit NarI coordinates two low-spin hemes, heme bP and heme bD, which mediate electron transfer from the Q-site to the [Fe-S] clusters in NarH
additional information
-
molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI
-
additional information
-
the semiquinone is located within the quinol oxidation site QD
-
additional information
-
the transmembrane subunit NarI provides the quinol binding and oxidation site (Q-site)
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe
Mo
Fe
-
the catalytic subunit of Escherichia coli nitrate reductase A contains a [4Fe-4S] cluster with a high-spin ground state
Fe
-
complete coordination of the four Fe-S centers of the beta-subunit from Escherichia coli nitrate reductase
Fe
-
coordination model for the four [Fe-S] centres of the electron-transfer subunit NarH, coordination scheme of the [Fe-S] clusters, functional role of [Fe-S] centres
Fe
-
the 230000 Da complex contains 13 atoms iron and 12 atoms labile sulfur/molecules
Fe
-
the four iron-sulfur centers of nitrate reductase A belong to two classes with markedly different redox potentials. The high-potential group comprises a [3Fe-4S] and a [4Fe-4S] cluster whose midpoint potentials are +60 mV and +80 mV, respectively. Although these centers are magnetically isolated, they are coupled by a significant anticooperative redox interaction of about 50 mV. The [4Fe-4S]1+ center occurs in two different conformations as shown by its composite EPR spectrum. The low-potential group contains two [4Fe-4S] clusters with more typical redox potentials (-200 mV and -400 mV). In the fully reduced state, the three [4Fe-4S]1+ centers are magnetically coupled. The iron-sulfur centers nitrate reductase Z and nitrate reductase A, exhibit essentially the same characteristics, except that the midpoint potentials of the high-potential centers of nitrate reductase Z appear negatively shifted by about 100 mV. A correspondence between the high-potential iron-sulfur clusters of the two enzymes can be proposed
Fe
P09152 and P11349 and P0AF26
cysteine arrangements typical of iron-sulfur centers are found in the NarH polypeptide. This suggests that the latter is an electron transfer unit of the nitrate reductase complex
Fe
-
domain I of subunit NarG holds the [4Fe-4S] cluster FS0. The coordination scheme of FS0 is: His50, Cys54, Cys58, Cys93. NarH contains three [4Fe-4S] clusters, FS1, FS2, FS3 and one [3Fe-4S] cluster, FS4; domain I of the catalytic subunit NadG holds the [4Fe-4S] cluster FS0
Mo
-
the enzyme contains a molybdenum cofactor. The enzyme-specific chaperone NarJ coordinates assembly and molybdenum cofactor acquisition of the heterotrimeric enzyme during the biogenesis process
Mo
-
the enzyme uses a molybdo-bis(molybdopterin guanine dinucleotide) cofactor for catalytic mechanism
Mo
-
the enzyme possesses a molybdopterin guanine dinucleotide active center. Two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
diethyl dicarbonate
-
the quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate
additional information
-
p-chloromercuribenzoate (0.5 mM) or 2-heptyl-4-hydroxyquinolin N-oxide (1 mM) are almost without effect on the purified enzyme tested with reduced viologen as electron donor
-
2-n-heptyl-4-hydroxyquinoline N-oxide
-
the quinol-dependent, but not the viologen dye dependent, activity is inhibited reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide
2-n-heptyl-4-hydroxyquinoline N-oxide
-
has significant effects on the reduction kinetics of NarGHIH56R and NarGHIH205Y
2-n-heptyl-4-hydroxyquinoline N-oxide
-
quinol-binding-site inhibitor, elicits a much stronger inhibitory effect than stigmatellin on the reoxidation of the hemes
Stigmatellin
-
has significant effects on the reduction kinetics of NarGHIH56R and NarGHIH205Y
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00008
2-n-heptyl-4-hydroxyquinoline N-oxide
Escherichia coli
-
pH 7, substrate: 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
the transmembrane subunit NarI anchors narGH to the cytoplasmic side of the membrane
-
-
the enzyme-specific chaperone NarJ controls the quality of the enzyme addressed to the membrane via binding to the N-terminal tail of the iron-sulfur containing subunit NarG
-
nitrate reductase A (encoded by narGHJI operon) and nitrate reductase Z (encoded by narZYWv operon) are membrane-bound
-
the membrane-intrinsic subunit NarI anchors NarGH to the membrane through a predominantly hydrophobic interface with both subunits
-
alpha and beta polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. The gamma polypeptide is the only subunit which is embedded in the membrane
-
the membrane-anchored protein directs electrons from quinol oxidation at the membrane anchor, NarI, to the site of nitrate reduction in the membrane extrinsic [Fe-S] cluster and Mo-bis-MGD containing dimer, NarGH
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
24000
-
x * 129000 (alpha) + x * 66000 (beta) + x * 24000 (gamma), SDS-PAGE
25500
P09152 and P11349 and P0AF26
x * 138700 + x * 57700, x * 26500, x * 25500, the narGHJI operon that encodes the nitrate reductase encodes four polypeptides NarG (138700 Da), NarH (57700 Da), NarJ (26500 Da) and NarI (25500 Da), calculated from sequence
26500
P09152 and P11349 and P0AF26
x * 138700 + x * 57700, x * 26500, x * 25500, the narGHJI operon that encodes the nitrate reductase encodes four polypeptides NarG (138700 Da), NarH (57700 Da), NarJ (26500 Da) and NarI (25500 Da), calculated from sequence
57700
P09152 and P11349 and P0AF26
x * 138700 + x * 57700, x * 26500, x * 25500, the narGHJI operon that encodes the nitrate reductase encodes four polypeptides NarG (138700 Da), NarH (57700 Da), NarJ (26500 Da) and NarI (25500 Da), calculated from sequence
60000
-
1 * 150000 (alphaz) + 1 * 60000 (betaz) + a b-type cytochrome subunit, SDS-PAGE
66000
-
x * 129000 (alpha) + x * 66000 (beta) + x * 24000 (gamma), SDS-PAGE
129000
-
x * 129000 (alpha) + x * 66000 (beta) + x * 24000 (gamma), SDS-PAGE
138700
P09152 and P11349 and P0AF26
x * 138700 + x * 57700, x * 26500, x * 25500, the narGHJI operon that encodes the nitrate reductase encodes four polypeptides NarG (138700 Da), NarH (57700 Da), NarJ (26500 Da) and NarI (25500 Da), calculated from sequence
150000
-
1 * 150000 (alphaz) + 1 * 60000 (betaz) + a b-type cytochrome subunit, SDS-PAGE
223900
-
multiple isomorphous replacement and anaomalous scattering (MIRAS), crystallographic data
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotrimer
-
electron transfer can occur from the menaquinol binding site in NarI to the molybdo-bis(molybdopterin guanine dinucleotide) active site in NarG, where nitrate is reduced to nitrite
?
-
1 * 150000 (alphaz) + 1 * 60000 (betaz) + a b-type cytochrome subunit, SDS-PAGE
?
P09152 and P11349 and P0AF26
x * 138700 + x * 57700, x * 26500, x * 25500, the narGHJI operon that encodes the nitrate reductase encodes four polypeptides NarG (138700 Da), NarH (57700 Da), NarJ (26500 Da) and NarI (25500 Da), calculated from sequence
?
-
alpha and beta polypeptides are essentially peripheral polypeptides that are attached to the membrane by protein-protein interactions, presumably via the gamma polypeptide
?
-
x * 129000 (alpha) + x * 66000 (beta) + x * 24000 (gamma), SDS-PAGE
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of NarGHI at 1.9 A resolution, crystals of native and selenomethionine-substituted NarGHI are obtained by vapor diffusion with sitting drops; sitting-drop vapor diffusion method, crystals of native and selenomethionine-substituted NarGHI, crystal structure at 1.9 A resolution
-
the crystal structure of Escherichia coli nitrate reductase A in complex with pentachlorophenol is determined to 2.0 A of resolution
-
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freezing in liquid nitrogen may be repeated up to six times, with a reduction of 20% of the activity
-
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is remarkably resistant to air inactivation since only 2-5% of the activity is lost after a 1 h treatment
-
697691
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, the purified preparation can be stored up to three days without inactivation
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C196A
-
mutation results in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzyme
C227A
-
mutation results in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzyme
C263A
-
mutant retains significant nitrate reductase activity. EPR analysis shows that the highest redox potential [4Fe-4S] cluster (center 1) is selectively removed by the C263A mutation
C26A
-
mutant retains significant nitrate reductase activity. Mutation likely eliminates the lowest potential [4Fe-4S] cluster (center 4)
H187Y
H205Y
-
mutant without heme bH but with heme bL, a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. A transient species, likely to be associated with a semiquinone radical anion, is generated not only on reduction of the wild-type enzyme but also on reduction of NarGHIH56R and NarGHIH205Y. Compared to the wild type, no significant heme reoxidation is observed for NarGHIH56R and NarGHIH205Y. This result indicates that a single mutation removing heme bH blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH
H49S
the mutant lacks catalytic activity and the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor but retains the GDP moieties
H56R
-
mutant without heme bH but with heme bL, a smaller and slower heme reduction compared to that of the wild-type enzyme is observed. A transient species, likely to be associated with a semiquinone radical anion, is generated not only on reduction of the wild-type enzyme but also on reduction of NarGHIH56R and NarGHIH205Y. Compared to the wild type, no significant heme reoxidation is observed for NarGHIH56R and NarGHIH205Y. This result indicates that a single mutation removing heme bH blocks the electron-transfer pathway from the subunit NarI to the catalytic dimer NarGH
H56Y
-
a semiquinone is detected in the mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme; mutant lacks the distal heme bD, a EPR signal of the semiquinone is observed
H66Y
K86A
R94S
the mutant shows a concomitant decrease in enzyme turnover to about 30% of the wild type
H49S
-
the mutant lacks catalytic activity and the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor but retains the GDP moieties
-
additional information
-
mutant enzyme lacking the highest-potential [4Fe-4S] cluster is devoid of menadione activity, but still retains duroquinone activity
H187Y
-
mutant lacking heme bL but having heme bH, the heme reduction by menadiol is abolished
H187Y
-
mutant lacking the distal heme bD, no EPR signal of the semiquinone is observed; mutant lacks the distal heme bD, no EPR signal of the semiquinone is observed
H66Y
-
mutant lacking heme bL but having heme bH, the heme reduction by menadiol is abolished
H66Y
-
mutant lacking the distal heme bD, no EPR signal of the semiquinone is observed; mutant lacks the distal heme bD, no EPR signal of the semiquinone is observed
K86A
-
mutant has a lower plumbagin:nitrate oxidoreductase activity than the wild-type enzyme, 10/s compared with 68/s, respectively
K86A
-
mutation dramatically reduces the rate of oxidation of both menaquinol and ubiquinol analogues; the mutation close to heme bD leads to the loss of the EPR signal of the semiquinone, although both hemes are present, the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues
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LINKS TO OTHER DATABASES (specific for EC-Number 1.7.5.1)
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