Information on EC 1.7.2.2 - nitrite reductase (cytochrome; ammonia-forming)

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

EC NUMBER
COMMENTARY hide
1.7.2.2
-
RECOMMENDED NAME
GeneOntology No.
nitrite reductase (cytochrome; ammonia-forming)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NH3 + 2 H2O + 6 ferricytochrome c = nitrite + 6 ferrocytochrome c + 7 H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
formate to nitrite electron transfer
-
-
nitrate reduction IV (dissimilatory)
-
-
Nitrogen metabolism
-
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Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
ammonia:ferricytochrome-c oxidoreductase
Found as a multiheme cytochrome in many bacteria. The enzyme from Escherichia coli contains five hemes c and requires Ca2+. It also reduces nitric oxide and hydroxylamine to ammonia, and sulfite to sulfide.
CAS REGISTRY NUMBER
COMMENTARY hide
37256-41-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
formerly Achromobacter fischeri
-
-
Manually annotated by BRENDA team
several strains
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
serovar typhimurium
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
-
comparison of reaction mechanisms of nitrogenase, EC 1.18.6.1, and multiheme cytochrome c nitrite reductase, ccNIR, EC 1.7.2.2, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ammonia + H2O + oxidized cytochrome C552
nitrite + reduced cytochrome C552
show the reaction diagram
hydroxylamine + benzyl viologen reduced
? + benzyl viologen oxidized
show the reaction diagram
hydroxylamine + ferricytochrome c
nitrite + ferrocytochrome c
show the reaction diagram
hydroxylamine + ferrocytochrome c
? + ferricytochrome c
show the reaction diagram
-
-
-
-
?
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome
show the reaction diagram
-
-
-
-
?
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome c + H2O
show the reaction diagram
hydroxylamine + ferrocytochrome c
NH3 + H2O + ferricytochrome c
show the reaction diagram
hydroxylamine + reduced benzyl viologen
? + oxidized benzyl viologen
show the reaction diagram
hydroxylamine + reduced methyl viologen
?
show the reaction diagram
-
low activity
-
-
?
hydroxylammonium + ferrocytochrome c
NH3 + ferricytochrome c + H2O
show the reaction diagram
-
-
-
-
?
nitric oxide + ferrocytochrome c
NH3 + ferricytochrome c + H2O
show the reaction diagram
-
-
-
-
?
nitrite + 4 ferrocytochrome c + 5 H+
hydroxylamine + H2O + 4 ferricytochrome c
show the reaction diagram
-
cf. EC 1.7.2.6
-
-
?
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
show the reaction diagram
nitrite + acceptor
NH3 + oxidized acceptor + H2O
show the reaction diagram
-
-
-
-
?
nitrite + benzyl viologen reduced + H+
NH4+ + benzyl viologen oxidized + H2O
show the reaction diagram
nitrite + FADH2
NH4+ + FAD
show the reaction diagram
nitrite + ferrocytochrome c
NH3 + ferricytochrome c + H2O
show the reaction diagram
nitrite + ferrocytochrome c + 6 H+
NH3 + ferricytochrome c + 2 H2O
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
show the reaction diagram
nitrite + FMNH2
NH4+ + FMN
show the reaction diagram
-
-
-
?
nitrite + reduced + acceptor
NH3 + oxidized acceptor + H2O
show the reaction diagram
-
-
-
?
nitrite + reduced anthraquinone-2-sulfonate + H+
NH3 + H2O + oxidized anthraquinone-2-sulfonate
show the reaction diagram
nitrite + reduced diquat + H+
NH3 + H2O + oxidized diquat
show the reaction diagram
nitrite + reduced indigo carmine + H+
NH3 + H2O + oxidized indigo carmine
show the reaction diagram
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
show the reaction diagram
nitrite + reduced methyl viologen
NH3 + oxidized methyl viologen + H2O
show the reaction diagram
nitrite + reduced methyl viologen + H+
NH3 + H2O + oxidized methyl viologen
show the reaction diagram
nitrite + reduced phenosafranine + H+
NH3 + H2O + oxidized phenosafranine
show the reaction diagram
NO + 5 ferrocytochrome c + 5 H+
NH3 + H2O + 5 ferricytochrome c
show the reaction diagram
NO radical + reduced methyl viologen
?
show the reaction diagram
-
-
-
-
?
sulfite + 6 ferrocytochrome c + 6 H+
H2S + 3 H2O + 6 ferricytochrome c
show the reaction diagram
sulfite + ?
sulfide + ?
show the reaction diagram
sulfite + reduced methyl viologen
H2S + oxidized methyl viologen + H2O
show the reaction diagram
-
-
-
-
?
sulfite + reduced methyl viologen + H+
H2S + oxidized methyl viologen + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
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
hydroxylamine + ferricytochrome c
nitrite + ferrocytochrome c
show the reaction diagram
hydroxylamine + ferrocytochrome c
NH3 + H2O + ferricytochrome c
show the reaction diagram
nitrite + 4 ferrocytochrome c + 5 H+
hydroxylamine + H2O + 4 ferricytochrome c
show the reaction diagram
-
cf. EC 1.7.2.6
-
-
?
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
show the reaction diagram
nitrite + acceptor
NH3 + oxidized acceptor + H2O
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
show the reaction diagram
NO + 5 ferrocytochrome c + 5 H+
NH3 + H2O + 5 ferricytochrome c
show the reaction diagram
-
important role for the enzyme in nitric oxide management in oxygen-limited environments. Nitric oxide is a key element in host defense against invasive pathogens
-
-
?
sulfite + 6 ferrocytochrome c + 6 H+
H2S + 3 H2O + 6 ferricytochrome c
show the reaction diagram
-
-
-
-
?
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c
Ferredoxin
Q5F2I3
[4Fe-4S]-ferredoxin cofactor
-
heme c
methyl viologen
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Calcium
copper
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study on reaction of copper(II)-nitrito complexes with tridentate and tetradentate, with either O,O'-bidentate or O-unidentate modes of nitrite binding to the cupric center . Differing modes of nitrite coordination to the copper(II) ion lead to differing kinetic behavior
Fe
-
8-11 mol per subunit
Fe3+
active site heme Fe(III) iron, the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview
additional information
-
no non-heme iron
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
azide
CN-
-
cyanide is a potent inhibitor of NrfA nitrite and hydroxylamine reductase activities
CuCl2
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0.01 mM,70% inhibition; 0.1 mM, 69% inhibition
CuSO4
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0.01 mM, 68% inhibition
Cyanate
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-
cyanide
KCN
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0.1 mM, 84% inhibition; 1 mM, 100%
KNO3
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1 mM, 6% inhibition
NaN3
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1 mM, 16% inhibition
nitrate
nitrite
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substrate inhibition
NO
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wild-type cultures are more sensitive to the addition of a pulse of NO when grown under fermentative conditions compared with anaerobic respiratory conditions, sensitivity of different wild-type and mutant strains, overview
phosphate
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weak inhibitor
sulfate
Thiocyanate
-
effective inhibitor
Zn2+
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EDTA protects NrfA from zinc inhibition, zinc inhibition kinetics in presence or absence of EDTA, overview
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cvardiolipin
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cardiolipin liposomes induce nitrite reductase activity of Fe(II)-cytc. The values observed can be compared with the kinetics of the NO2-mediated conversion of other ferrous heme-proteins. Cardiolipins facilitate the NO2-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom
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NrfE
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gene product necessary for the assembly of the holoenzyme
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NrfF
-
gene product necessary for the assembly of the holoenzyme
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NrfG
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gene product necessary for the assembly of the holoenzyme
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1 - 170
hydroxylamine
0.00025 - 16.7
nitrite
0.3
NO radical
-
pH 7.0, 20°C, reduction
0.0064
reduced diquat
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
0.015
reduced methyl viologen
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
0.0027
reduced phenosafranine
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
60
sulfite
-
in 0.1 M K+-phosphate buffer, at pH 7.0 and 30°C
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
197 - 3160
hydroxylamine
390
nitric oxide
-
turnover rate in intact cell
7 - 2860
nitrite
840
NO radical
-
pH 7.0, 20°C, reduction
1064
reduced diquat
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
738
reduced methyl viologen
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
79
reduced phenosafranine
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
0.023 - 0.039
sulfite
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1664
reduced diquat
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
0.049
reduced methyl viologen
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
0.029
reduced phenosafranine
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
18
nitrite
-
pH 8.3, 20°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.03
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, oxidation, pH 7.0, 37°C
0.04
below, purified recombinant MBP-tagged enzyme mutant W434Y, substrate hydroxylamine, oxidation, pH 7.0, 37°C
0.07
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, oxidation, pH 7.0, 37°C
0.1
-
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, oxidation, pH 7.0, 37°C
1
-
purified recombinant MBP-tagged wild-type enzyme, substrate nitrite, pH 7.0, 37°C
1.2
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purified recombinant MBP-tagged wild-type enzyme, substrate nitrite, pH 7.0, 37°C
2.9
-
purified recombinant MBP-tagged enzyme mutant W434Y, substrate nitrite, pH 7.0, 37°C
8
purified recombinant MBP-tagged enzyme mutant W434Y, substrate nitrite, pH 7.0, 37°C
16.7
purified recombinant MBP-tagged enzyme mutant W434Y, substrate nitrite, pH 7.0, 37°C
25
-
nitrite reduction
27
purified recombinant MBP-tagged wild-type enzyme, substrate nitrite, pH 7.0, 37°C
29
-
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, reduction, pH 7.0, 37°C
68
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, reduction, pH 7.0, 37°C
77
purified recombinant MBP-tagged enzyme mutant W434Y, substrate hydroxylamine, reduction, pH 7.0, 37°C
98
-
purified recombinant MBP-tagged enzyme mutant W434Y, substrate hydroxylamine, reduction, pH 7.0, 37°C
149
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, reduction, pH 7.0, 37°C
150
Q5F2I3
purified native enzyme
158
-
purified recombinant MBP-tagged wild-type enzyme, substrate hydroxylamine, reduction, pH 7.0, 37°C
181
purified recombinant MBP-tagged wild-type enzyme, substrate nitrite, pH 7.0, 37°C
225
purified recombinant MBP-tagged enzyme mutant W434Y, substrate hydroxylamine, reduction, pH 7.0, 37°C
682
-
nitrite
810
-
nitrite, oligomeric form
850
-
nitrite, monomeric form
970
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nitrite, oligomeric form
1050
-
nitrite, monomeric form
2690
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hydroxylamine
2930
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benzyl viologen; nitrite
additional information
-
quantitation of steady-state NrfA reduction of NO radical, nitrite, and hydroxylamine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
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the sulfite reductase activity of NiR has a maximum value at neutral pH, the sulfite reductase activity of NiR decreases with increasing pH and is absent at pH 9.0, the activity measured at pH 7.8 is 20% of the activity measured at pH 7.0
additional information
-
pH dependence of NrfA at 4-25°C, overview
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 25
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NrfA activity increases when the temperature is raised from 4°C to 25°C
10 - 60
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activity increases up to 60
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298)
Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298)
Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298)
Shewanella oneidensis (strain MR-1)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Thioalkalivibrio nitratireducens (strain DSM 14787 / UNIQEM 213 / ALEN2)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
Wolinella succinogenes (strain ATCC 29543 / DSM 1740 / LMG 7466 / NCTC 11488 / FDC 602W)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19000
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
50590
-
apoprotein, calculation from DNA sequence
53590
-
MALDI-TOF-MS
57000
-
SDS-PAGE
58000
-
native enzyme, gel filtration
61000
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
64000
-
6 * 64000, calculated from sequence analysis and spectral properties
69000
-
SDS-PAGE
72000
-
native enzyme, gel filtration
120000
-
subunit NrfA, SDS-PAGE
245000
380000 - 390000
Q5F2I3
gel filtration
760000
-
complex form, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
hexamer
homodimer
-
-
monomer
oligomer
polymer
-
x * 56000 + x * 18000, gel filtration
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method, structure determined to 2.3 A resolution
-
crystals are grown by the vapour-diffusion technique by mixing equal amounts of protein and reservoir solution, which contains about 10% (w/v) PEG 4K in 0.1 M HEPES pH 7.5 buffer
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mutant H264N, to 2.15 A resolution. Homodimeric protein, the constellation of His/His and Lys-coordinated c-hemes is indistinguishable from those of the native enzyme
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purified NrfA, hanging drop vapor diffusion method, five conditions that result in several different crystal packing motifs, e.g. 0.001 ml of protein solution containing 10 mg/ml of NrfA mixed with 0.001 ml of mother liquor containing 100 mM HEPES, pH 7.5, 20% PEG 10000 and incubated in hanging drop trays with a 1 ml well solution at 4°C, 5-14 days, other conditions, overview. X-ray diffraction structure determination and analysis at 1.7-2.5 A resolution
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purified recombinant wild-type and mutant Q263E, 10 mg/ml protein, under aerobic conditions by the vapor diffusion hanging drop method using 20% v/v PEG 10000 in 100 mM Na-HEPES, pH 7.5, 20% ethylene glycol as cryoprotectant, X-ray diffraction structure determination and analysis at 1.74 A and 2.04 A resolution, respectively, molecular replacement
vapour diffusion method. Crystals belong to space group P2(1)2(1)2(1) with apparent cell parameters of a = 81.47 A, b = 90.84 and c = 294.87 A and contain four molecules of NrfA per asymmetric unit
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crystallization of cytochrome c552, 2.2 A resolution
-
UV/Vis spectropotentiometric results yield highly reproducible values for the heme midpoint potentials, which can be assigned to specific hemes in each protomer. Addition of the strong-field ligand cyanide leads to a 70 mV positive shift of the active site's midpoint potential, the cyanide binds to the initially 5-coordinate high-spin heme and triggers a high-spin to low-spin transition. With cyanide present three of the remaining hemes give rise to distinctive and readily assignable EPR spectral changes upon reduction
-
alone or in complex with nitrite and cyanide, hanging drop vapor diffusion method, using 0.2 M trisodium citrate dihydrate, 0.1 M Tris-HCl pH 8.5 and 30% (v/v) PEG 400
-
apoenzyme and its complexes with the substrate nitrite and the inhibitor azide. The subunit of NiR consists of the N-terminal domain which has an unique fold and contains three hemes and the catalytic C-terminal domain which hosts the remaining five hemes. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, two conserved Ca2+-binding sites. In addition, the enzyme has a covalent bond between the catalytic tyrosine and the adjacent cysteine and an unusual topography of the product channels that open into the void interior space of the protein hexamer
-
diffraction data sets with increasing absorbed doses. The structures reveal gradual changes associated with the reduction of the catalytic hemes by X-rays. The conversion of the nitrite ions bound in the active sites to NO species is observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic heme and the iron ion of the heme takes place. In the sulfite complex no enzymatic reaction is detected
hanging drop vapour diffusion method
-
modified form of the enzyme that contains an additional covalent bond between residues Tyr303 and Gln360 in complex with phosphate to 1.45 A resolution, and with sulfite to 1.8 A resolution, structure of unmodified enzyme in complex with nitrite to 1.83 A resolution. Structure reveal the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site
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ccNIR as apoenzyme or with bound sulfite or nitrite to the catalytic heme center, X-ray diffraction structure determination and analysis at 1.30-1.75 A resolution
-
enzyme in complex with nitrite
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sitting drop vapour diffusion using ammonium sulfate as precipitant. Crystal growth of the NrfHA complex is strongly dependent on the presence of detergent, presumably because of the need to shield the hydrophobic transmembrane helix of NrfH from the solvent. The crystals grown belong to space group I422; Triton X-114 significant improvement in crystal size and quality
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
at an enzyme concentration of 2 mg/ml in potassium phosphate buffer pH 7.0 the enzyme is stable at 30°C for at least 24 h
50 - 70
Q5F2I3
at least 50 h, purified enzyme, stable
50
-
incubation at 50°C results in 1.5fold increase of activity
70
-
at 70°C the enzyme looses 50% of its activity in about 3 h
86
Q5F2I3
melting of the TvNiR molecule is an irreversible highly cooperative process with the transition temperature of 86°C and the transition enthalpy of about J/mol of hexamer
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
NrfASo is unstable unless heme attachment is timely accomplished
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
30°C, potassium phosphate buffer at pH 7.0, 24 h, no loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme 57.7fold by anion exchange chromatography and gel filtration
Q5F2I3
non-denaturing preparative polyacrylamide gel electrophoresis
-
Q-Sepharose HP column chromatography, Superdex S200 gel filtration and S-200 HR column chromatography
-
recombinant MBP-tagged enzyme from Wolinella succinogenes strain DSM 1740 by affinity chromatography, cleavage of the MBP-tag by TEV protease
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recombinant wild-type and mutant enzymes from Escherichia coli strain JCB4083a by ammonium sulfate fractionation and anion exchange chromatography
recombinant wild-type and mutant MBP-tagged enzymes from Wolinella succinogenes strain DSM 1740 by affinity chromatography, cleavage of the MBP-tag by TEV protease
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene haoA, recombinant expression in Wolinella succinogenes strain DSM 1740 as maltose-binding-protein fusion His6-tagged enzyme
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gene haoA, recombinant expression of wild-type and mutant enzymes in Wolinella succinogenes strain DSM 1740 as maltose-binding-protein fusion His6-tagged enzyme
gene nrfA, expression of wild-type and mutant enzymes in Escherichia coli strain JCB4083a
recombinant expression of wild-type and mutant enzymes in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H264N
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mutant is unable to catalyze nitrite reduction but able to reduce hydroxylamine. The mutant simultaneously binds nitrite and electrons at the catalytic heme
Q263E
site-directed mutagenesis, the mutation leads to introduction of a negative charge into the vicinity of the active site heme, and the mutant shows reduced activity compared to the wild-type enzyme. The high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme
K100H
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mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
K100I
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mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
K100L
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mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
H268M
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one of the EPR-silent heme's histidine axial ligands is replaced with a methionine
K119H
site-directed mutagensis, mutation of the essential lysine residue of the non-canonical HBM (CX2CK), inactive mutant. The mutant NrfA protein displays a similar pattern of rapid degradation like the wild-type without maturation
K119L
site-directed mutagensis, mutation of the essential lysine residue of the non-canonical HBM (CX2CK), inactive mutant. The mutant NrfA protein displays a similar pattern of rapid degradation like the wild-type without maturation
H277N
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mutant is unable to catalyze nitrite reduction but able to reduce hydroxylamine. The mutant simultaneously binds nitrite and electrons at the catalytic heme
Y218F
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active site mutant, that shows almost complete loss of nitrite reductase activity, while sulfite reduction remains unaffected
H277N
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mutant is unable to catalyze nitrite reduction but able to reduce hydroxylamine. The mutant simultaneously binds nitrite and electrons at the catalytic heme
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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