Information on EC 1.7.5.1 - nitrate reductase (quinone)

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.7.5.1
-
RECOMMENDED NAME
GeneOntology No.
nitrate reductase (quinone)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrate + a quinol = nitrite + a quinone + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrate reduction I (denitrification)
-
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nitrate reduction III (dissimilatory)
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nitrate reduction IX (dissimilatory)
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nitrate reduction VII (denitrification)
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nitrate reduction VIII (dissimilatory)
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nitrate reduction VIIIb (dissimilatory)
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Nitrogen metabolism
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Microbial metabolism in diverse environments
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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.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
nitrate reductase alpha subunit
UniProt
Manually annotated by BRENDA team
nitrate reductase alpha subunit
UniProt
Manually annotated by BRENDA team
DSM 50135
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-
Manually annotated by BRENDA team
nitrate reductase 1 alpha subunit
UniProt
Manually annotated by BRENDA team
respiratory nitrate reductase 1 alpha chain
E1WG01
UniProt
Manually annotated by BRENDA team
strains HB27, PRQ16, and PRQ25
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
quinone site variants Lys86 and Gly65, Q-site inhibitor HOQNO, and their effects on heme bD, overview; quinone site variants Lys86 and Gly65, Q-site inhibitor HOQNO, and their effects on heme bD, overview; quinone site variants Lys86 and Gly65, Q-site inhibitor HOQNO, and their effects on heme bD, overview
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
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
show the reaction diagram
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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
show the reaction diagram
nitrate + 2-methyl-1,4-naphthoquinol
nitrite + 2-methyl-1,4-naphthoquinone + H2O
show the reaction diagram
nitrate + 5-hydroxy-1,4-naphthoquinol
nitrite + 5-hydroxy-1,4-naphthoquinone + H2O
show the reaction diagram
nitrate + 5-hydroxy-2-methyl-1,4-naphthoquinol
nitrite + 5-hydroxy-2-methyl-1,4-naphthoquinone + H2O
show the reaction diagram
-
-
-
-
?
nitrate + 5-hydroxy-2-methyl-naphthalene-1,4-diol
nitrite + 5-hydroxy-2-methyl-naphthalene-1,4-dione + H2O
show the reaction diagram
nitrate + duroquinol
nitrite + duroquinone + H2O
show the reaction diagram
nitrate + menaquinol
nitrite + menaquinone + H2O
show the reaction diagram
-
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
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-
?
nitrate + quinol
nitrite + quinone
show the reaction diagram
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NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. The semiquinone is located within the quinol oxidation site QD
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-
?
nitrate + quinol
nitrite + quinone + H2O
show the reaction diagram
nitrate + reduced benzyl viologen
nitrite + benzyl viologen
show the reaction diagram
nitrate + reduced benzyl viologen
nitrite + oxidized benzyl viologen + H2O
show the reaction diagram
nitrate + reduced methyl viologen
nitrite + oxidized ethyl viologen + H2O
show the reaction diagram
-
-
-
-
?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen + H2O
show the reaction diagram
nitrate + tetramethyl-p-benzoquinol
nitrite + tetramethyl-p-benzoquinone + H2O
show the reaction diagram
nitrate + ubiquinol
nitrite + ubiquinone + H2O
show the reaction diagram
nitrite + a quinone + H2O
nitrate + a quinol
show the reaction diagram
-
-
-
?
nitrite + demethylmenaquinone + H2O
nitrate + demethylmenaquinol
show the reaction diagram
nitrite + menadione + H2O
nitrate + menadiol
show the reaction diagram
-
-
-
?
nitrite + methylmenaquinone + H2O
nitrate + 2-methylmenaquinol
show the reaction diagram
-
-
-
?
nitrite + methylmenaquinone + H2O
nitrate + methylmenaquinol
show the reaction diagram
-
-
-
?
nitrite + naphthoquinone + H2O
nitrate + naphthoquinol
show the reaction diagram
-
-
-
?
nitrite + ubiquinone + H2O
nitrate + ubiquinol
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
nitrate + quinol
nitrite + quinone + H2O
show the reaction diagram
nitrite + a quinone + H2O
nitrate + a quinol
show the reaction diagram
P09152, P11349, P11350
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-
-
?
nitrite + demethylmenaquinone + H2O
nitrate + demethylmenaquinol
show the reaction diagram
P09152, P11349, P11350
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-
-
?
nitrite + ubiquinone + H2O
nitrate + ubiquinol
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,4-Naphthoquinone
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2-methylnaphthalene-1,4-dione
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4Fe-4S-center
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bis(molybdopterin guanine dinucleotide)molybdenum cofactor
cytochrome
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cytochrome b
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cytochrome bD
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NarI is strongly associated with heme bD, Lys86 is required for its stabilization
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cytochrome bH
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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
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cytochrome bL
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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
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cytochrome c
demethylmenaquinone
DMKH2, endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme; DMKH2, endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme; DMKH2, endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme
flavin
heme b
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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)
menaquinone
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there are more than one menaquinol binding sites in NarGHI
molybdenum bis-molybdopterin guanine dinucleotide
molybdo-bis(pyranopterin guanine dinucleotide)
quinone
heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating His66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate; heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating His66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate; heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating His66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate
ubiquinone
[4Fe-4S] cluster
[4Fe-4S]-center
a single tetranuclear iron-sulfur [4Fe-4S] cluster, known as FS0, is bound to subunit NarG. NarH contains three [4Fe-4S] clusters (FS1-FS3) and one trinuclear iron-sulfur cluster ([3Fe-4S], FS4); a single tetranuclear iron-sulfur [4Fe-4S] cluster, known as FS0, is bound to subunit NarG. NarH contains three [4Fe-4S] clusters (FS1-FS3) and one trinuclear iron-sulfur cluster ([3Fe-4S], FS4); a single tetranuclear iron-sulfur [4Fe-4S] cluster, known as FS0, is bound to subunit NarG. NarH contains three [4Fe-4S] clusters (FS1-FS3) and one trinuclear iron-sulfur cluster ([3Fe-4S], FS4)
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Molybdenum
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-n-heptyl-4-hydroxyquinoline N-oxide
azide
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5 mM, 95-98% inhibition
cyanide
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1 mM, 95-98% inhibition
diethyl dicarbonate
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the quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate
KCN
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1 mM, complete inhibition
NaN3
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10 mM, complete inhibition
Pentachlorophenol
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mixed inhibition
Stigmatellin
additional information
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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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.057
2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
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pH 7
0.282
5-hydroxy-2-methyl-1,4-naphthoquinol
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pH 7
0.147
5-hydroxy-2-methyl-naphthalene-1,4-diol
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pH 7.0
0.5 - 3.9
nitrate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
68
2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinol
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pH 7
120
5-hydroxy-2-methyl-1,4-naphthoquinol
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pH 7
10 - 68
nitrate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.057 - 0.49
Pentachlorophenol
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00008 - 0.0015
2-n-heptyl-4-hydroxyquinoline N-oxide
0.0004
Pentachlorophenol
Escherichia coli
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pH 7.0
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
assay at; assay at; assay at
7.5
assay at; assay at; assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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NarG and NarH are cytoplasmic subunits
Manually annotated by BRENDA team
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the transmembrane subunit NarI anchors narGH to the cytoplasmic side of the membrane
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24000
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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
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1 * 150000 (alphaz) + 1 * 60000 (betaz) + a b-type cytochrome subunit, SDS-PAGE
66000
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x * 129000 (alpha) + x * 66000 (beta) + x * 24000 (gamma), SDS-PAGE
129000
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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
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1 * 150000 (alphaz) + 1 * 60000 (betaz) + a b-type cytochrome subunit, SDS-PAGE
200000
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soluble alpha/beta enzyme, gel filtration
223900
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multiple isomorphous replacement and anaomalous scattering (MIRAS), crystallographic data
230000
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alpha(Z)beta(Z) complex, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotrimer
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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
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
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highly ordered crystals of apomolybdo-NarGHI are obtained
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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 hide
LITERATURE
60
-
20 min, stable
65
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4 min, 50% loss of activity
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
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697691
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, slow freezing leads to a 30% loss of activity
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4°C, the purified preparation can be stored up to three days without inactivation
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
-
P09152 and P11349 and P0AF26
gene cluster NarGHI, overexpression in Escherichia coli strain LCB79; gene cluster NarGHI, overexpression in Escherichia coli strain LCB79; gene cluster NarGHI, overexpression in Escherichia coli strain LCB79
gene narG in the narGHI gene cluster
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
nitrate reductase A is synthesized optimally at NO3- concentrations of 10 mM or above
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C196A
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mutation results in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzyme
C227A
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mutation results in the full loss of the four Fe-S clusters and of the Mo-cofactor, leading to inactive enzyme
C263A
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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
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mutant retains significant nitrate reductase activity. Mutation likely eliminates the lowest potential [4Fe-4S] cluster (center 4)
G65A
site-directed mutageness of subunit NarI, mutant G65A is able to support growth and retains significant quinol:nitrate oxidoreductase activity; site-directed mutageness of subunit NarI, mutant G65A is able to support growth and retains significant quinol:nitrate oxidoreductase activity; site-directed mutageness of subunit NarI, mutant G65A is able to support growth and retains significant quinol:nitrate oxidoreductase activity
H205Y
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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
H49C
the mutant lacks catalytic activity
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
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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
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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
R94S
the mutant shows a concomitant decrease in enzyme turnover to about 30% of the wild type
H49C
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the mutant lacks catalytic activity
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H49S
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the mutant lacks catalytic activity and the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor but retains the GDP moieties
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R94S
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the mutant shows a concomitant decrease in enzyme turnover to about 30% of the wild type
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additional information
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mutant enzyme lacking the highest-potential [4Fe-4S] cluster is devoid of menadione activity, but still retains duroquinone activity