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Enzyme Nomenclature
Information on EC 1.7.2.2 - nitrite reductase (cytochrome; ammonia-forming)
Word Map on EC 1.7.2.2
- 1.7.2.2
-
six-electron
- desulfovibrio
-
voltammetry
- succinogenes
-
wolinella
-
dissimilatory
-
ammonification
- desulfuricans
- nitratireducens
-
thioalkalivibrio
- flavorubredoxin
- menaquinol
- deleyianum
- flavohemoglobins
- hildenborough
-
pentahaem
- sulfurospirillum
-
cxxch
- analysis
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
1.7.2.2
-
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RECOMMENDED NAME
GeneOntology No.
nitrite reductase (cytochrome; ammonia-forming)
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
NH3 + 2 H2O + 6 ferricytochrome c = nitrite + 6 ferrocytochrome c + 7 H+
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
reduction
-
-
-
-
reduction
-
TvNiR catalyzes the reduction of nitrite and hydroxylamine to ammonium ions without the release of any intermediates.
reduction
-
the enzyme possesses also peroxidase activity; TvNiR catalyzes the reduction of nitrite and hydroxylamine to ammonium ions without the release of any intermediates.
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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.
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CAS REGISTRY NUMBER
COMMENTARY
37256-41-0
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
physiological function
-
periplasmic multiheme c-type cytochromes cytochrome c peroxidase and cytochrome c nitrite reductase NrfA mediate resistance to hydrogen peroxide. NrfA and a cytoplasmic flavodiiron protein Fdp are key components of nitric oxide detoxification. In addition, NrfA mediates resistance to hydroxylamine stress
physiological function
-
periplasmic multiheme c-type cytochromes cytochrome c peroxidase and cytochrome c nitrite reductase NrfA mediate resistance to hydrogen peroxide. NrfA and a cytoplasmic flavodiiron protein Fdp are key components of nitric oxide detoxification. In addition, NrfA mediates resistance to hydroxylamine stress
-
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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
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome
-
-
-
-
?
hydroxylammonium + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
-
-
-
?
nitrite + ferrocytochrome c + 6 H+
NH3 + ferricytochrome c + 2 H2O
-
-
-
-
?
nitrite + reduced + acceptor
NH3 + oxidized acceptor + H2O
-
-
-
?
nitrite + reduced anthraquinone-2-sulfonate + H+
NH3 + H2O + oxidized anthraquinone-2-sulfonate
sulfite + reduced methyl viologen
H2S + oxidized methyl viologen + H2O
-
-
-
-
?
sulfite + reduced methyl viologen + H+
H2S + oxidized methyl viologen + H2O
-
-
-
-
?
ammonia + H2O + oxidized cytochrome C552
nitrite + reduced cytochrome C552
-
-
-
-
?
ammonia + H2O + oxidized cytochrome C552
nitrite + reduced cytochrome C552
-
-
-
-
?
hydroxylamine + benzyl viologen reduced
? + benzyl viologen oxidized
-
-
-
?
hydroxylamine + benzyl viologen reduced
? + benzyl viologen oxidized
-
-
-
?
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
-
-
-
?
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
10 mM hydroxylamine
-
-
ir
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
-
-
-
?
hydroxylamine + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
10 mM hydroxylamine
-
-
ir
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
-
-
-
-
?
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
-
-
-
-
?
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
-
mechanism
-
-
?
nitrite + benzyl viologen reduced + H+
NH4+ + benzyl viologen oxidized + H2O
-
-
-
?
nitrite + benzyl viologen reduced + H+
NH4+ + benzyl viologen oxidized + H2O
-
-
-
?
nitrite + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
-
-
-
?
nitrite + ferrocytochrome c
NH3 + ferricytochrome c + H2O
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
the periplasmic cytochrome c nitrite reductase, Nrf, system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. NO is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin, flavohaemoglobin and cytochrome c nitrite reductase: Determination of a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, overview
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
ccNiR catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
nitrite is the preferred substrate. ccNiR is encoded by gene nrfA performing nitrite reduction with formate. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme
-
-
?
nitrite + reduced anthraquinone-2-sulfonate + H+
NH3 + H2O + oxidized anthraquinone-2-sulfonate
about 95% conversion
-
-
?
nitrite + reduced anthraquinone-2-sulfonate + H+
NH3 + H2O + oxidized anthraquinone-2-sulfonate
about 95% conversion
-
-
?
nitrite + reduced diquat + H+
NH3 + H2O + oxidized diquat
100% conversion
-
-
?
nitrite + reduced diquat + H+
NH3 + H2O + oxidized diquat
100% conversion
-
-
?
nitrite + reduced indigo carmine + H+
NH3 + H2O + oxidized indigo carmine
75% conversion
-
-
?
nitrite + reduced indigo carmine + H+
NH3 + H2O + oxidized indigo carmine
75% conversion
-
-
?
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
-
nitrite and inhibitor CO both bind to the catalytic heme
-
-
?
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
-
-
-
-
?
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
-
-
-
?
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
-
-
-
-
?
nitrite + reduced methyl viologen
NH3 + H2O + oxidized methyl viologen
Q5F2I3
-
-
-
?
nitrite + reduced methyl viologen
NH3 + oxidized methyl viologen + H2O
-
-
-
-
?
nitrite + reduced methyl viologen
NH3 + oxidized methyl viologen + H2O
-
-
-
-
?
nitrite + reduced methyl viologen + H+
NH3 + H2O + oxidized methyl viologen
about 98% conversion
-
-
?
nitrite + reduced methyl viologen + H+
NH3 + H2O + oxidized methyl viologen
about 98% conversion
-
-
?
nitrite + reduced phenosafranine + H+
NH3 + H2O + oxidized phenosafranine
about 102% conversion
-
-
?
nitrite + reduced phenosafranine + H+
NH3 + H2O + oxidized phenosafranine
about 102% conversion
-
-
?
NO + 5 ferrocytochrome c + 5 H+
NH3 + H2O + 5 ferricytochrome c
-
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
-
-
?
NO + 5 ferrocytochrome c + 5 H+
NH3 + H2O + 5 ferricytochrome c
-
-
-
-
?
sulfite + 6 ferrocytochrome c + 6 H+
H2S + 3 H2O + 6 ferricytochrome c
-
-
-
-
?
sulfite + 6 ferrocytochrome c + 6 H+
H2S + 3 H2O + 6 ferricytochrome c
-
sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family, binding mode of sulfite to the catalytic heme center of ccNiR, overview
-
-
?
additional information
?
-
-
active site structure determination and analysis, proton transfer is coupled to electron transfer at the active site, overview
-
-
-
additional information
?
-
-
NrfA from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium, it might also participate in NO radical detoxification, detoxifying exogenously generated NO radical encountered during invasion of a human host, overview
-
-
-
additional information
?
-
-
the enzyme shows a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO radical reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO radical reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine
-
-
-
additional information
?
-
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. Important function of the Q263-calcium ion pair increasing substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand, active site structures of native and Q263 mutant NrfA enzymes, overview
-
-
-
additional information
?
-
-
no nitrate or sulfite reductase activity
-
-
-
additional information
?
-
-
not accepted electron donors: NADH, NADPH, ubiquinol-1, menadiol, formate, D-lactate, sn-glycerol-3-phosphate
-
-
-
additional information
?
-
-
no nitrate or sulfite reductase activity
-
-
-
additional information
?
-
-
not accepted electron donors: NADH, NADPH, ubiquinol-1, menadiol, formate, D-lactate, sn-glycerol-3-phosphate
-
-
-
additional information
?
-
protein film voltammetry shows that all detected electron transfer steps are one-electron in nature. Enzyme displays substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover
-
-
-
additional information
?
-
-
no reaction with chloride, bromate and nitrate
-
-
-
additional information
?
-
-
the enzyme possesses also peroxidase activity
-
-
-
additional information
?
-
-
no reaction with chloride, bromate and nitrate
-
-
-
additional information
?
-
-
the enzyme possesses also peroxidase activity
-
-
-
additional information
?
-
-
the enzyme catalyzes the last step in the metabolic pathway of nitrate dissimilation
-
-
-
additional information
?
-
-
ccNiR also reduces sulfite to sulfide linking the biogeochemical cycles of nitrogen and of sulfur
-
-
-
additional information
?
-
-
simulation of the kinetics of the reduction process reveals that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers or in the form of three consecutive elementary steps involving reduction, proton-coupled electron transfers and protonation. Recharging through two proton-coupled electron transfers is a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO radical intermediate. The radical transfer role for the active-site Tyr218, cannot be confirmed. The highly conserved calcium located in the direct proximity of the active site plays an important role in the substrate conversion through the facilitation of the proton transfer steps
-
-
-
additional information
?
-
-
simulation of the kinetics of the reduction process reveals that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers or in the form of three consecutive elementary steps involving reduction, proton-coupled electron transfers and protonation. Recharging through two proton-coupled electron transfers is a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO radical intermediate. The radical transfer role for the active-site Tyr218, cannot be confirmed. The highly conserved calcium located in the direct proximity of the active site plays an important role in the substrate conversion through the facilitation of the proton transfer steps
-
-
-
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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
nitrite + 6 ferrocytochrome c + 7 H+
NH3 + 2 H2O + 6 ferricytochrome c
-
-
-
-
?
NO + 5 ferrocytochrome c + 5 H+
NH3 + H2O + 5 ferricytochrome c
-
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
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
P0ABK9
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
P0ABK9
the periplasmic cytochrome c nitrite reductase, Nrf, system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. NO is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
protection against NO may involve reductive detoxification in low-oxygen environments, and three enzymes, flavorubredoxin, flavohaemoglobin and cytochrome c nitrite reductase: Determination of a combined role for NorV and NrfA in NO detoxification under anaerobic conditions, overview
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
-
-
-
?
nitrite + ferrocytochrome c + H+
NH3 + H2O + ferricytochrome c
-
ccNiR catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification
-
-
?
additional information
?
-
-
NrfA from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium, it might also participate in NO radical detoxification, detoxifying exogenously generated NO radical encountered during invasion of a human host, overview
-
-
-
additional information
?
-
-
the enzyme catalyzes the last step in the metabolic pathway of nitrate dissimilation
-
-
-
additional information
?
-
-
ccNiR also reduces sulfite to sulfide linking the biogeochemical cycles of nitrogen and of sulfur
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
heme
-
crystal structure of penta-heme NrfA. Four of the NrfA hemes have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand
heme
-
10 heme centres range in reduction potential from -30 to 320 mV. The heme oxidation state has a profound effect on the interactions with substrate molecules, nitrite and hydroxylamine
heme
-
NrfA contains five hemes and NrfH four hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+ 150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. The four NrfH hemes are all in a low-spin state
heme
-
contains 14 hemes; the NrfA2NrfH complex houses 14 hemes, closely-spaced hemes in this enzyme with magnetic interactions between them
methyl viologen
Q5F2I3
used as the electron donor, reduced with dithionite, artificial cofactor
methyl viologen
-
used as the electron donor, reduced with dithionite, artificial cofactor
methyl viologen
artificial cofactor, zinc-reduced or dithionite-reduced
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ca2+
Calcium
Fe2+
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
Iron
Ca2+
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. An important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand
Calcium
-
a conserved calcium ion (calcium I) with octahedral coordination is present near the active site. A second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The main role of this calcium may not be electrostatic but structural
Calcium
-
the highly conserved calcium located in the direct proximity of the active site plays an important role in the substrate conversion through the facilitation of the proton transfer steps
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
CN-
-
cyanide is a potent inhibitor of NrfA nitrite and hydroxylamine reductase activities
NO
-
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
Zn2+
EDTA protects NrfA from zinc inhibition, zinc inhibition kinetics in presence or absence of EDTA, overview
azide
-
rapid and reversible. Tighter binding of azide when the active site is oxidized than when it is reduced
CO
-
CO and nitrite both bind to the catalytic heme, bimolecular inhibition kinetics, overview; competitive inhibitor
cyanide
-
two sites for cyanide binding and inhibition, higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase
additional information
-
inhibition in the absence of Ca2+ is noted at potentials where heme 4 and/or 5 is reduced, this may implicate Ca2+ association with the propionate of heme 4 as being essential for maintaining steady-state activity from the fully reduced enzyme-substrate complex
-
additional information
-
sulfate, chloride and ammonium are ineffective inhibitors of cytochrome c nitrite reductase
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
16.4
hydroxylamine
-
in 0.1 M K+-phosphate buffer, at pH 7.0 and 30°C
0.033
nitrite
pH 7.0, 25°C, wild-type enzyme with methyl viologen as reductant
0.413
nitrite
pH 7.0, 25°C, mutant Q263E with methyl viologen as reductant
16.7
nitrite
-
in 0.1 M K+-phosphate buffer, at pH 7.0 and 30°C
additional information
additional information
-
kinetics, overview
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1064
reduced diquat
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
738
reduced methyl viologen
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
79
reduced phenosafranine
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
1195
nitrite
Thioalkalivibrio nitratireducens
-
modified enzyme that contains an additional covalent bond between residues Tyr303 and Gln360, pH 7.0, 20°C
0.023
sulfite
Thioalkalivibrio nitratireducens
-
modified enzyme that contains an additional covalent bond between residues Tyr303 and Gln360, pH 7.0, 20°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.1664
reduced diquat
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
23417
0.049
reduced methyl viologen
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
381
0.029
reduced phenosafranine
Desulfovibrio desulfuricans
Q8VNU2
apparent value, in 0.2 M phosphate buffer pH 7.6, at 37°C
23227
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
quantitation of steady-state NrfA reduction of NO radical, nitrite, and hydroxylamine
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
7
-
the sulfite reductase activity of NiR has a maximum value at neutral pH
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7 - 9
-
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
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 25
-
NrfA activity increases when the temperature is raised from 4°C to 25°C
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
in the perplasm NrfA forms a stable, membrane-associated complex with its electron donor NrfH
-
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Desulfovibrio vulgaris (strain Hildenborough / ATCC 29579 / DSM 644 / NCIMB 8303)
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)
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)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
18000
19000
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
22000
55000
61000
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
64000
-
6 * 64000, calculated from sequence analysis and spectral properties
66000
245000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
x * 61000 (NrfA) + x * 19000 (NrfH), heterooligomeric complexhas an average 2NrfA:1NrfH composition
dimer
hexamer
monomer
oligomer
hexamer
-
6 * 64000, calculated from sequence analysis and spectral properties
hexamer
Q5F2I3
determination of the oligomeric composition of NiR by small-angle X-ray scattering, overview
hexamer
-
6 * 64000, calculated from sequence analysis and spectral properties
-
oligomer
-
ccNiR 's threedimensional structure shows the characteristically close packing of heme cofactors into parallel and perpendicular motifs
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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
-
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
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
-
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
-
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
-
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
-
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
-
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
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
-
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
80
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
80
Q5F2I3
the half-life is 2.2 h, enzyme inactivation is described by a two-step scheme, about 20% residual activity of after 25 h
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
30°C, potassium phosphate buffer at pH 7.0, 24 h, no loss of activity
-
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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 wild-type and mutant enzymes from Escherichia coli strain JCB4083a by ammonium sulfate fractionation and anion exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene nrfA, expression of wild-type and mutant enzymes in Escherichia coli strain JCB4083a
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
-
mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
K100I
-
mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
K100L
-
mutagenesis of the hem-binding motif CWSCK results in almost complete loss of formate-dependent nitrite reduction
Y218F
-
active site mutant, that shows almost complete loss of nitrite reductase activity, while sulfite reduction remains unaffected
additional information
additional information
-
anaerobically grown Escherichia coli nrf mutants are more sensitive to NO radicals than the parent strain
additional information
-
effects of all eight possible combinations of norV, hmpA and nrfA single, double and triple mutations, overview. The norV nrfA mutant cannot grow after NO addition
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
development of a conductometric biosensor for nitrite detection using cytochrome c nitrite reductase immobilized on a planar interdigitated electrode. Under the optimum conditions at room temperature, the biosensor shows a fast response to nitrite with a linear range of 0.2–120 microM, a sensitivity of 0.194 microS per microM NO2?, and a detection limit of 0.05 microM. The biosensor also shows satisfactory reproducibility. When stored in potassium phosphate buffer at 4 ?C, the biosensor shows good stability over 1 month
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LINKS TO OTHER DATABASES (specific for EC-Number 1.7.2.2)
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