Information on EC 1.7.7.1 - ferredoxin-nitrite reductase

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

EC NUMBER
COMMENTARY hide
1.7.7.1
-
RECOMMENDED NAME
GeneOntology No.
ferredoxin-nitrite reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NH3 + 2 H2O + 6 oxidized ferredoxin = nitrite + 6 reduced ferredoxin + 7 H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
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redox reaction
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-
-
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reduction
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
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nitrate reduction II (assimilatory)
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nitrate reduction VI (assimilatory)
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Nitrogen metabolism
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nitrate assimilation
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SYSTEMATIC NAME
IUBMB Comments
ammonia:ferredoxin oxidoreductase
An iron protein. Contains siroheme and [4Fe-4S] clusters.
CAS REGISTRY NUMBER
COMMENTARY hide
37256-44-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
leek
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Manually annotated by BRENDA team
cyanobacterium
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Manually annotated by BRENDA team
cyanobacterium
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-
Manually annotated by BRENDA team
sunflower
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Manually annotated by BRENDA team
barley
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
french bean
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
hydroxylamine + reduced ferredoxin
ammonia + H2O + oxidized ferredoxin
show the reaction diagram
hydroxylamine can serve as an electron-accepting substrate for the enzyme and the product of hydroxylamine reduction is ammonia. Hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme
-
-
?
hydroxylamine + reduced ferredoxin
NH3 + H2O + oxidized ferredoxin
show the reaction diagram
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-
-
-
?
nitrite + 6 reduced ferredoxin
ammonia + 2 H2O + 6 oxidized ferredoxin
show the reaction diagram
nitrite + 6 reduced ferredoxin + 7 H+
NH3 + 2 H2O + 6 oxidized ferredoxin
show the reaction diagram
nitrite + FADH2
NH3 + FAD + H2O
show the reaction diagram
nitrite + NADPH + H+
NH3 + NADP+ + H2O
show the reaction diagram
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10% of maximum activity compared to reduced methyl viologen as electron donor
-
-
?
nitrite + reduced 2,6-dichlorophenolindophenol
NH3 + oxidized 2,6-dichlorophenolindophenol + H2O
show the reaction diagram
-
less than 35% of maximum activity compared to reduced methyl viologen as electron donor
-
-
?
nitrite + reduced ferredoxin
ammonia + oxidized ferredoxin
show the reaction diagram
nitrite + reduced ferredoxin
NH3 + H2O + oxidized ferredoxin
show the reaction diagram
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
show the reaction diagram
nitrite + reduced phenazine methosulphate
NH3 + oxidized phenazine methosulphate + H2O
show the reaction diagram
-
less than 35% of maximum activity compared to reduced methyl viologen as electron donor
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-
?
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
nitrite + 6 reduced ferredoxin + 7 H+
NH3 + 2 H2O + 6 oxidized ferredoxin
show the reaction diagram
nitrite + reduced ferredoxin
ammonia + oxidized ferredoxin
show the reaction diagram
nitrite + reduced ferredoxin
NH3 + H2O + oxidized ferredoxin
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
iron-sulfur:siroheme cofactor
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siroheme
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
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slightly inhibitory
4-hydroxymercuribenzoate
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8-hydroxyquinoline
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7% inhibition at 5 mM
bathophenanthroline
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slight
cyanide
hydroxylamine
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34% inhibition at 10 mM
Mersalyl
N-acetylsuccinimide
N-bromosuccinimide
N-ethylmaleimide
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47% inhibition at 1 mM
o-phenanthroline
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15% inhibition at 5 mM
p-chloromercuribenzoate
p-hydroxymercuribenzoate
Phenyl mercury acetate
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-
Phenylglyoxal
pyridoxal-5'-phosphate
75% inhibition of the enzyme with ferredoxin as electron donor after exposure to NaBH4, but no inhibition of the enzyme with methyl viologen as electron donor
SO32-
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24% inhibition at 10 mM
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbic acid
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addition of ascorbic acid to a reconstituted system composed of nitrite reductase and ferredoxin proteins leads to reduction of nitrite at a rate nearly equal to that is obtainable with the NADPH/ferredoxin-NADPH-reductase/ferredoxin-nitrite reductase system
additional information
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no activation after treatment with 100 mM NaCl
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006 - 0.1
Ferredoxin
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0.063 - 0.91
methyl viologen
0.069
NADPH
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0.01 - 8.6
nitrite
0.0077 - 0.0746
reduced ferredoxin
1.9
reduced methyl viologen
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pH 7.0, 60°C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10 - 227
nitrite
101.4 - 203.2
reduced ferredoxin
additional information
additional information
Spinacia oleracea
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turnover number decreases about 20% after treatment with N-bromosuccinimide
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
125 - 473
nitrite
168
4300 - 14200
reduced ferredoxin
265
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
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The assay mixture contains 50 mM Tris-HCl (pH 7.5), 0.5 mM NaNO2, 1 mM methylviologen, and 50 ml of extract in a final volume of 0.9 ml. The reaction is started by the addition of 100 ml of 0.12 M Na2S2O4 dissolved in 0.2 M NaHCO3 and then incubated at 30°C for 60 min
15.8
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pH 7.0, 60°C
90
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methyl viologen as electron donor
120
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ferredoxin as electron donor
135
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methyl viologen as electron donor
188
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ferredoxin as electron donor
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 8.3
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pH 6.8: about 75% of activity maximum, pH 8.3: about 70% of activity maximum
7.8 - 8.3
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appreciable amounts of activity at pH 7.8 and pH 8.3 in Tris buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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increase of activity up to 50°C
60
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when measured in the presence of 3.3 M NaCl
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25000
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1 * 63000 + 1 * 25000, SDS-PAGE
35000
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1 * 64000 + 1 * 35000, SDS-PAGE
52000
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gel filtration
54000
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gel filtration
58000
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1 * 58000 + 1 * 66000, SDS-PAGE
59650
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mass spectroscopy
60000 - 63000
62690
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amino acid composition
63700
x * 63700, calculated
64000
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1 * 64000 + 1 * 35000, SDS-PAGE
64830
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calculated from amino acid composition
68000
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gel filtration, SDS-PAGE
69000
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native PAGE
74000
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gel filtration
100000
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gel filtration
126000
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gel filtration, native enzyme
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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spinach: the native enyzme with a molecular weight of 85000 can be split into a modified form with a MW of 61000 that retains activity with the non-physiological electron-donor methyl viologen, but loses most of the ferredoxin-linked activity and an 24000 MW coupling protein fragment in which the ferredoxin-binding domain is located
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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66 kDa subunit is phosphorylated
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
modeling of structure based on spinach nitrite reductase. Arginine and lysine residues are involved in electrostatically-stabilized binding to ferredoxin
the biological unit, NrfH2NrfA4, houses 28 c-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for c-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. The midpoint redox potential of the NrfH menaquinol-interacting methionine-coordinated heme is -270 mV; the biological unit, NrfH2NrfA4, houses 28 c-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for c-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. The redox potential of the catalytic lysine-coordinated high spin heme of NrfA is -50 mV
wild-type, to 1.25 A resolution and mutants Q448K, M175E, M175G, M175K to 2.0, 1.7. 1.7, 19 A resolution, respectively. The structure provides detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups
sitting drop vapour diffusion method
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NMR-study of protein-protein interaction of ferredoxin and nitrite reductase shows three acidic regions of ferredoxin to be major sites for the interaction with the enzyme, indicating that the complex is stabilized through electrostatic interaction
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
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enzyme in crude extract is rapidly inactivated below pH 7
394413
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
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1 h, pH 8.0, 75 mM Tris-HCl, stable
45
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instable after 5 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
repetitive freezing and thawing or prolonged dialysis produces a loss of enzyme activity
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the stability of the enzyme depends on the salt concentration. The maximum stability is found in the presence of 3.6 M NaCl
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unstable in crude extracts and in solutions below pH 7
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when the enzyme is dialysed against a 50 mM phosphate buffer, pH 7.0, containing 20% glycerol, the enzyme can be stored for 2 weeks without any significant loss of activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 10% glycerol, 10 mM 2-mercaptoethanol, stable for several weeks
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-20°C, 50% glycerol, for at least 6 months
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-20°C, several months
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-24°C, 30% loss of activity within 4 months
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4°C, 0.1 M potassium phosphate buffer, pH 7.7, 1 week
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4°C, 1 week
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4°C, 200 mM Tris-HCl buffer, pH 7.5, 200 mM NaCl, 10% glycerol, for at least 1 month
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4°C, 30% loss of activity within 3 weeks
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in Escherichia coli
expressed in Escherichia coli
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expression in Escherichia coli
fusion with beta-galactosidase
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K449Q
mutation does not affect enzymatic activity
M175E
the conformation of the Gln47 and Lys91 side-chains changes significantly. Compared with the conformation in wild-type, the Gln47 side-chain rotates about 120 degrees and the amino group in the Lys91 side-chain moves toward the side-chain of residue 175
M175G
the conformation of the Gln47 and Lys91 side-chains changes significantly. Compared with the conformation in wild-type, the Gln47 side-chain rotates about 120 degrees and the amino group in the Lys91 side-chain moves toward the side-chain of residue 175
M175K
the conformation of the Gln47 and Lys91 side-chains changes significantly. Compared with the conformation in wild-type, the Gln47 side-chain rotates about 120 degrees and the amino group in the Lys91 side-chain moves toward the side-chain of residue 175
Q448K
mutation had only a negligible effect on the overall fold of the protein. In addition, the mutation did not affect the hydrogen bond interaction at the distal position of the siroheme
C514S
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almost complete loss of activity
C518S
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almost complete loss of activity
G513A
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increased Km for nitrite compared to the wild type enzyme
G513E
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loss of most of the activity
G513V
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loss of most of the activity
G519A
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68% activity of wild type enzyme
G519E
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8% activity of wild type enzyme
G519V
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9% activity of wild type enzyme
P515A
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marginal loss of activity
P515S
-
marginal loss of activity
P515T
-
marginal loss of activity
additional information
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various mutants that differ in enzymatic activity
Show AA Sequence (519 entries)
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