Information on EC 1.7.2.4 - nitrous-oxide reductase

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

EC NUMBER
COMMENTARY hide
1.7.2.4
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RECOMMENDED NAME
GeneOntology No.
nitrous-oxide reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrogen + H2O + 2 ferricytochrome c = nitrous oxide + 2 ferrocytochrome c + 2 H+
show the reaction diagram
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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
denitrification
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Microbial metabolism in diverse environments
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nitrate reduction I (denitrification)
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nitrate reduction VII (denitrification)
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nitrifier denitrification
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Nitrogen metabolism
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SYSTEMATIC NAME
IUBMB Comments
nitrogen:cytochrome c oxidoreductase (N2O-forming)
The reaction is observed only in the direction of nitrous oxide reduction. Contains the mixed-valent dinuclear CuA species at the electron entry site of the enzyme, and the tetranuclear Cu-Z centre in the active site. In Paracoccus pantotrophus, the electron donor is cytochrome c552.
CAS REGISTRY NUMBER
COMMENTARY hide
55576-44-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 1013
UniProt
Manually annotated by BRENDA team
strain IAM1013
UniProt
Manually annotated by BRENDA team
NCIMB 11015, X-ray scattering data, 20 A resolution
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-
Manually annotated by BRENDA team
strain ATCC 8750
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-
Manually annotated by BRENDA team
strain USDA110
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-
Manually annotated by BRENDA team
strain DSM 15892T
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Manually annotated by BRENDA team
strain SN611
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-
Manually annotated by BRENDA team
strain SN611
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-
Manually annotated by BRENDA team
strain DSM 15936T
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-
Manually annotated by BRENDA team
A3151
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-
Manually annotated by BRENDA team
LMD 82.5
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-
Manually annotated by BRENDA team
strain P2
-
-
Manually annotated by BRENDA team
strain CCUG 2519
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-
Manually annotated by BRENDA team
precursor; strain C7R12
SwissProt
Manually annotated by BRENDA team
strain ED3
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-
Manually annotated by BRENDA team
strain G59
-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
strain 8A55
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Manually annotated by BRENDA team
strain 8A55
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-
Manually annotated by BRENDA team
strains N22, Kb1
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Manually annotated by BRENDA team
strain PW5
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-
Manually annotated by BRENDA team
strain PW5
-
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Manually annotated by BRENDA team
stain S1
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Manually annotated by BRENDA team
stain S1
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-
Manually annotated by BRENDA team
strain 1021; strain 50
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-
Manually annotated by BRENDA team
strain 50
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Manually annotated by BRENDA team
ATCC 29591
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Manually annotated by BRENDA team
DSM 807
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-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
N2O + 2 Fe2+ + 2 H+
N2 + H2O + 2 Fe3+
show the reaction diagram
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-
-
-
?
N2O + H2O + benzyl viologen cation radical
N2 + reduced benzyl viologen
show the reaction diagram
N2O + reduced acceptor
N2 + H2O + acceptor
show the reaction diagram
N2O + reduced benzyl viologen
N2 + H2O + benzyl viologen
show the reaction diagram
N2O + reduced benzyl viologen
N2 + oxidized benzyl viologen
show the reaction diagram
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-
-
-
?
N2O + reduced cytochrome c
N2 + H2O + cytochrome c
show the reaction diagram
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-
-
-
?
N2O + reduced cytochrome c550
N2 + H2O + cytochrome c550
show the reaction diagram
N2O + reduced methyl viologen
N2 + H2O + methyl viologen
show the reaction diagram
N2O + reduced methyl viologen
N2 + H2O + oxidized methyl viologen
show the reaction diagram
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-
-
-
?
N2O + reduced methyl viologen
N2 + oxidized methyl viologen
show the reaction diagram
N2O + reduced pseudoazurin
N2 + H2O + pseudoazurin
show the reaction diagram
N2O + reduced pseudoazurin
N2 + oxidized pseudoazurin
show the reaction diagram
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blue copper electron-transfer protein pseudoazurin is capable of mediating electron transfer to the nitrous oxide reductase. Pseudoazurin binds near copper site CuA, with parameters consistent with the formation of a transient, weakly-bound complex. Pseudoazurin a strong candidate for the physiological electron donor to th enzyme
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-
?
nitrogen + H2O + acceptor
nitrous oxide + reduced acceptor
show the reaction diagram
nitrous oxide + reduced acceptor
nitrogen + H2O + acceptor
show the reaction diagram
nitrous oxide + reduced methyl viologen
nitrogen + H2O + methyl viologen
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
N2O + reduced acceptor
N2 + H2O + acceptor
show the reaction diagram
N2O + reduced pseudoazurin
N2 + oxidized pseudoazurin
show the reaction diagram
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blue copper electron-transfer protein pseudoazurin is capable of mediating electron transfer to the nitrous oxide reductase. Pseudoazurin binds near copper site CuA, with parameters consistent with the formation of a transient, weakly-bound complex. Pseudoazurin a strong candidate for the physiological electron donor to th enzyme
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?
nitrogen + H2O + acceptor
nitrous oxide + reduced acceptor
show the reaction diagram
P94127
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-
?
nitrous oxide + reduced acceptor
nitrogen + H2O + acceptor
show the reaction diagram
nitrous oxide + reduced methyl viologen
nitrogen + H2O + methyl viologen
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cupredoxin
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a copper enzyme with cupredoxin containing blue T1 copper and red T2 copper. Blue and red copper centers form initially before they are pH-dependently transformed into purple CuA center, lower pH resulting in fewer trapped T1 and T2 coppers and faster transition. Structure overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
copper
Fe
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1 atom per mol
Ni
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0.76 atoms per mol
Zn
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2 atoms per mol
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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53% after 30 min at 0.5 mM
Acetylene
EGTA
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36% at 0.5 mM after 30 min
methylisonitrile
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N,N,N',N'-tetramethyl-p-phenylenediamine
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76% inhibition at 0.1 mM
Na2S
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temporarily inhibition; together with sulfide no inhibition
Ni2+
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50% inhibition at 0.1 mM
rotenone
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88% inhibition at 0.025 mM
S2O42-
Zn2+
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complete inhibition at 0.1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NO2-
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increases the synthesis of N2O-reductase
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.004
benzyl viologen
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-
0.0043
benzyl viologen cation radical
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pH 7.1, 25C
0.033
Fe2+
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pH 7.6, 25C
0.0024 - 0.032
N2O
0.0009 - 0.0138
reduced benzyl viologen
0.00095
reduced methyl viologen
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0.0288
reduced pseudoazurin
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pH 7.1, temperature not specified in the publication
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additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8
N2O
Pyrobaculum aerophilum
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pH 7.6, 25C
162.9
reduced benzyl viologen
Achromobacter cycloclastes
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pH 7.1, temperature not specified in the publication
89.3
reduced pseudoazurin
Achromobacter cycloclastes
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pH 7.1, temperature not specified in the publication
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
242
Fe2+
Pyrobaculum aerophilum
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pH 7.6, 25C
25
250
N2O
Pyrobaculum aerophilum
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pH 7.6, 25C
1327
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.028 - 0.035
Acetylene
0.0035
CO
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non competitive
0.000025 - 0.000045
KCN
0.00035 - 0.00064
NaN3
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.4
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fully oxidized enzyme isolated from aerobic growth conditions, pH 7.6, temperature not specified in the publication
1
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ascorbate-reduced phenanzine methosulphate electrode assay
1.6
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anaerobic form pre-reduced with ascorbate
1.97
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wild type enzyme
2.8
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anaerobic form pre-reduced with dithionite
3.3
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photoreduced methyl viologen spectrophotometric assay
3.52
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single mutant nirX
3.63
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double mutant nosXnirX
3.7
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dithionite-reduced methyl viologen electrode assay
5.5
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wild-type
8.5
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aerobic form pre-oxidized with ferricyanide
8.8
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aerobic form pre-reduced with dithionite
23
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blue-coloured form
27
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pH 7.1, 25C
29
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HdN2OR form A, i.e. reduced form
45
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HdN2OR form A, i.e. oxidized form
55
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purple coloured form
61.7
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after preincubation in 2 mM reduced benzyl viologen
78
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anaerobic form
88
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fully reduced enzyme isolated from aerobic growth conditions, pH 7.6, temperature not specified in the publication
92.4
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fully reduced enzyme isolated from aerobic growth conditions, pH 7.6, temperature not specified in the publication
122
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anaerobic form
124
purified enzyme, pH 8.0, 25C
453
enzyme purified from transgenic Nicotiana tabacum, pH 7.1, 37C
additional information
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Vmax N2O 0.2 - 0.5 micromol/min/mg, pH 7.1, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5 - 9
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with reduced benzyl viologen as electron donor
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.7 - 10.6
perturbations of the protein conformation induced by pH variations, although the principal secondary structure elements are largely unaltered
5.7 - 9.4
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pH profile with two maxima, high complexity of pH dependence, overview
6 - 9.2
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half maximum activity values at pH 6 and pH 9.2
7 - 9.5
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approx. 50% of maximal activity at pH 7.5, approx. 75% of maximal activity at pH 9.5
9 - 10
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activity is maximal after incubation at high pH values
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 85
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activity is maximal after exposure to high temperatures
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
49600
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gel fitlration
85000
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gel filtration
89000
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gel filtration
95000
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gel filtration
105000
115000
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gel filtration
118000
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gel filtration
120000
130000
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non denaturating PAGE
134000
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gel filtration
144000
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gel filtration
160000
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gel filtration
162000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 71670, deduced from nucleotide sequence
homodimer
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the purple form of enzyme, in which the copper centre is in the oxidized [2Cu2+:2Cu+] redox state, is redox active, although it is still catalytically non-competent, as its specific activity is lower than that of the activated fully reduced enzyme and comparable with that of the enzyme with the copper centre in either the [1Cu2+:3Cu+] redox state or in the redox inactive state
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method
2.4 A resolution, preliminary study
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1.6 A resolution
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building of two models of the active site reveals two distinct mechanisms. In the first model, N2O binds to the fully reduced tetranuclear Cu4S core in a bent my-(1,3)-O,N bridging fashion between the CuI and CuIV centres and subsequently extrudes N2 while generating the corresponding bridged my-oxo species. In the second model, substrate N2O binds loosely to one of the coppers of the tetranuclear Cu4S core in a terminal fashion, i.e., using only the oxygen atom. Loss of N2 generates the same my-oxo copper core. The free energies of activation predicted for these two alternative pathways are close to one another and do not provide decisive support for one over the other
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modeling of structure. The residues contributing to the semiocclusion of the T1 copper site are Trp355, Met389, and Met297. There is a negatively charged residue in the neighborhood of the T1 site, Glu296
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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85% inactivation after 20 h
70
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most of activity lost
85
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half-life 5.5 h
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
an enzyme concentration of at least 0.01 mg per ml is necessary to avoid loss of activity due to dilution
-
turnover dependent activation, nitrite and fluoride accelerate this process
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turnover dependent inactivation, promoted by zinc ions at 0.01 mM
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, retained most of its activity after aerobic storage for 2 months without any additives
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4C, under aerobic conditions, 100 h stable
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4C, under aerobic conditions, 5 mM EDTA, 3 h stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a pink form is isolated when the purification is done aerobically, a purple form when the purification is under anaerobic conditions
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ammonium sulfate, DEAE-Sephadex A-50, Sephacryl S-200
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anaerobical purification of N2OR under an Ar atmosphere by three different steps of anion exchange chromatography, followed by cytochrome c affinity chromatography and ultrafiltration
DEAE cellulose column chromatography, hydroxyapatite column chromatography, Mono Q10 anion-exchange column chromatography, and Sephadex S-200 gel filtration
DEAE-FF ion exchange chromatography and Resource Q column chromatography
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HiTrap Q Sepharose anion exchange chromatography
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isolation of the enzyme in the purple form, in which the copper centre is in the oxidized [2Cu2+:2Cu+] redox state and is redox active. Isolation of this form in the presence of oxygen from a microaerobic culture in the presence of nitrate and also from a strictly anaerobic culture
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of anaerobically grown cells
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partial purification under anaerobic conditions
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purification aerobically at 4C
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purification under anaerobic conditions
recombinant metal-free apo protein
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two forms a purple and a blue form
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of the nos gene cluster
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expressed in Escherichia coli
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expression as metal-free apo protein
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expression in Escherichia coli
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expression in Escherichia coli HB101, no activity
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expression in Nicotiana tabacum
the gene encoding the enzyme in the nos cluster is cloned from a genomic library
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C165G
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Cys165 is not available for Cu cooordination; retaines catalytic activity
C622D
-
no activity, distorted CuA-centre
E296Q
-
mutation near T1 copper site, similar biochemical and spectroscopic properties to those of the wild type
M297A
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mutation near T1 copper site, similar biochemical and spectroscopic properties to those of the wild type
M389A
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mutation near T1 copper site, similar biochemical and spectroscopic properties to those of the wild type
W355A
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mutation near T1 copper site, similar biochemical and spectroscopic properties to those of the wild type
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
degradation
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