Information on EC 1.7.2.1 - nitrite reductase (NO-forming)

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

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EC NUMBER
COMMENTARY hide
1.7.2.1
-
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RECOMMENDED NAME
GeneOntology No.
nitrite reductase (NO-forming)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ammonia oxidation II (anaerobic)
-
-
Microbial metabolism in diverse environments
-
-
nitrate reduction I (denitrification)
-
-
nitrate reduction VII (denitrification)
-
-
nitrifier denitrification
-
-
nitrite-dependent anaerobic methane oxidation
-
-
Nitrogen metabolism
-
-
denitrification
-
-
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SYSTEMATIC NAME
IUBMB Comments
nitric-oxide:ferricytochrome-c oxidoreductase
The reaction is catalysed by two types of enzymes, found in the perimplasm of denitrifying bacteria. One type comprises proteins containing multiple copper centres, the other a heme protein, cytochrome cd1. Acceptors include c-type cytochromes such as cytochrome c-550 or cytochrome c-551 from Paracoccus denitrificans or Pseudomonas aeruginosa, and small blue copper proteins such as azurin and pseudoazurin. Cytochrome cd1 also has oxidase and hydroxylamine reductase activities. May also catalyse the reaction of hydroxylamine reductase (EC 1.7.99.1) since this is a well-known activity of cytochrome cd1.
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CAS REGISTRY NUMBER
COMMENTARY hide
9027-00-3
-
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
NCIB 11015
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain TK-6
SwissProt
Manually annotated by BRENDA team
strain TK-6
SwissProt
Manually annotated by BRENDA team
formerly Aquaspirillum magnetotacticum
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
wild-type strain MC58, gene nitrite reductase aniA
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain 5276
-
-
Manually annotated by BRENDA team
DSM 50135
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
deletion of cytochrome cd1-type nitrite reductase NirS gene or gene NirN results in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. Nitrite reduction is completely abolished in the DELTAnirS mutant. NirN is required for full reductase activity of NirS by maintaining a proper form of d1 heme for holo-cytochrome cd1 assembly
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ferrocytochrome c + O2
ferricytochrome c + H2O
show the reaction diagram
ferrocytochrome c-551 + NO2-
NO + ferricytochrome c-551
show the reaction diagram
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
show the reaction diagram
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
hydroxylamine + reduced pseudoazurin
NH3 + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
N,N-dimethyl-p-phenylenediamine + oxidized benzyl viologen
?
show the reaction diagram
-
-
-
-
?
NH2OH + NaNO2
N2O + H2O
show the reaction diagram
-
-
-
?
NH2OH + reduced cytochrome c550
NH3 + H2O + oxidized cytochrome c550
show the reaction diagram
-
additional electron donor: horse heart cytochrome c
-
?
nitrite + dithionite
NO + reduced dithionite
show the reaction diagram
-
type 1 copper of the fully loaded protein is reduced both directly by dithionite and indirectly by the type 2 copper site via intramolecular electron transfer
-
-
?
nitrite + electron donor
NO + oxidized electron donor
show the reaction diagram
-
-
-
-
?
nitrite + electron donor
NO + oxidized electron donor + H2O
show the reaction diagram
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
nitrite + ferrocytochrome c(gamma)
NO + H2O + ferricytochrome c(gamma)
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c2
NO + H2O + ferricytochrome c2
show the reaction diagram
nitrite + ferrocytochrome c550
NO + ferricytochrome c550
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c550
NO + oxidized ferricytochrome c550
show the reaction diagram
nitrite + H2O + reduced cytochrome cd1
nitric oxide + H+ + cytochrome cd1
show the reaction diagram
-
anaerobic assay conditions
-
-
?
nitrite + H2O + reduced pseudoazurin
nitric oxide + H+ + pseudoazurin
show the reaction diagram
nitrite + methyl viologen
NO + oxidized methyl viologen + H2O
show the reaction diagram
-
-
-
?
nitrite + reduced azurin
NO + H2O + oxidized azurin
show the reaction diagram
nitrite + reduced azurin
NO + oxidized azurin
show the reaction diagram
-
azurin purified from Pseudomonas chlororaphis
-
-
?
nitrite + reduced azurin I
NO + azurin I
show the reaction diagram
-
-
-
?
nitrite + reduced azurin I
NO + oxidized azurin I
show the reaction diagram
nitrite + reduced benzyl viologen
nitric oxide + oxidized benzyl viologen
show the reaction diagram
nitrite + reduced benzyl viologen
NO + H2O + oxidized benzyl viologen
show the reaction diagram
nitrite + reduced benzyl viologen
NO + oxidized benzyl viologen
show the reaction diagram
nitrite + reduced electron donor
NO + H2O + oxidized electron donor
show the reaction diagram
nitrite + reduced hydroquinone
nitric oxide + H2O + hydroquinone
show the reaction diagram
nitrite + reduced methyl viologen
NO + oxidized methyl viologen
show the reaction diagram
-
random sequential mechanism
-
-
?
nitrite + reduced methyl viologen
NO + oxidized methyl viologen + H2O
show the reaction diagram
nitrite + reduced pseudoazurin
NO + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
NO2 + reduced methyl viologen
NO + oxidized methylviologen
show the reaction diagram
-
-
-
?
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
NO2- + morpholine
N-nitrosomorpholine
show the reaction diagram
-
in the presence of diethyldithiocarbamic acid ethylester, nitrosation through the production of NO or NO+-like species
-
?
NO2- + Na2S2O4
NO + Na2S2O3
show the reaction diagram
-
pysiological electron donor is unknown, no activity with methyl viologen, phenazine methosulfate or N,N,N',N',-tetramethyl-p-phenylenediamine
-
?
NO2- + reduced ascorbate
NO + oxidized ascorbate
show the reaction diagram
NO2- + reduced azurin
NO + oxidized azurin
show the reaction diagram
-
putative physiological electron donor
-
?
NO2- + reduced cytochrome c550
NO + oxidized cytochrome c550
show the reaction diagram
-
unambiguously identified as physiological electron donor
-
?
NO2- + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
O2 + ferrocytochrome c
H2O + ferricytochrome c
show the reaction diagram
O2 + reduced pseudoazurin
H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
O2-. + H+
H2O2 + O2
show the reaction diagram
-
purified enzyme shows superoxide dismutase activity, approx. one-third that of pure superoxide dismutase
-
-
reduced azurin + O2
oxidized azurin + H2O
show the reaction diagram
reduced tetramethyl-4-phenylenediamine + NO2
oxidized tetrametyl-4-phenylenediamine + NO
show the reaction diagram
-
no reaction with horse ferrocytochrome c, Neurospora europaea ferrocytochrome c-552, Magnetospirillum magnetotacticum ferrocytochrome c-550 and Pseudomonas aeruginosa cytochrome c-551
-
?
reduced tetramethyl-4-phenylenediamine + O2
oxidized tetrametyl-4-phenylenediamine + H2O
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
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
show the reaction diagram
-
-
-
-
nitrite + electron donor
NO + oxidized electron donor
show the reaction diagram
-
-
-
-
?
nitrite + electron donor
NO + oxidized electron donor + H2O
show the reaction diagram
-
mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. In the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
nitrite + ferrocytochrome c2
NO + H2O + ferricytochrome c2
show the reaction diagram
-
there is likely an unidentified electron donor, in addition to c2 that transfers electrons to nitrite reductase
-
-
?
nitrite + ferrocytochrome c550
NO + ferricytochrome c550
show the reaction diagram
-
-
-
-
?
nitrite + reduced azurin I
NO + oxidized azurin I
show the reaction diagram
-
coordinate synthesis of azurin I and copper nitrite reductase in Alcaligenes xylosoxidans during denitrification
-
-
?
nitrite + reduced electron donor
NO + H2O + oxidized electron donor
show the reaction diagram
nitrite + reduced pseudoazurin
NO + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
NO2- + reduced cytochrome c550
NO + oxidized cytochrome c550
show the reaction diagram
-
unambiguously identified as physiological electron donor
-
?
NO2- + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
unambiguously identified as physiological electron donor
-
?
reduced azurin + O2
oxidized azurin + H2O
show the reaction diagram
additional information
?
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome
-
cytochrome cd1
-
heme c
heme d
-
Heme d1
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
-
copper-containing dissimilatory nitrite reductase, catalytic type 2 copper, binding site structure, analysis of binding structure and interaction with inhibitors, overview
LiCl
-
2 M, enzyme is activated by high salt concentrations
NaCl
optimum salt concentration: 2 M
NaNO3
-
2 M, enzyme is activated by high salt concentrations
NH4Cl
-
2 M, enzyme is activated by high salt concentrations
Zn
-
the recombinant enzyme, expressed in Pseudomonas putida, contains c-heme but no d1-heme. Reconstitution of this protein with Zn-protoporphyrin IX in the place of the d1-heme. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem. Reduction of the d1-heme is associated with a substantial reorganization of the coordination of the metal
Zn2+
-
present on surface of each monomer, crystallization data
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetate
-
weak, mixed-type inhibition, inhibition mode, overview
azide
-
binding mode, overview
CO
20% residual activity
diethyldithiocarbamate
formate
-
weak mixed-type inhibition, inhibition mode, overview
methylhydrazine
-
irreversible
NaN3
-
1 mM, 60% inhibition
NH2OH
-
1 mM, 50% inhibition
nitrate
-
weak inhibition, inhibition mode, overview
Nitrous oxide
-
binding mode, overview
Oxidized cytochrome c
-
-
-
phenylhydrazine
-
irreversible
Urea
-
4 M, 60% inhibition
additional information
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NapC
-
c-type cytochrome with 4 bis-histidinyl coordinated hemes capable of reducing and hence activating oxidized cytochrome cd1 nitrite reductase
-
Subtilisin
-
species of 48000 Da which contains the d1 but not the c heme
-
additional information
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00084 - 0.049
azurin
0.0305 - 0.046
cytochrome c
0.00177 - 0.0075
cytochrome c551
-
0.0018 - 0.0075
Ferrocytochrome c-551
-
0.43 - 2.5
hydroxylamine
0.434 - 2.5
NH2OH
0.00008 - 416
nitrite
0.00147 - 0.8
NO2-
0.027 - 1
O2
0.067 - 0.16
pseudoazurin
-
0.41
reduced methyl viologen
pH and temperature not specified in the publication
additional information
additional information
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7
Abz-VAA
Pseudomonas aeruginosa
-
25°C, pH 6.9, recombinant enzyme
-
1 - 3.2
azurin
1.33
cytochrome c
Pseudomonas aeruginosa
-
succinylated monomeric enzyme
0.583 - 2.8
cytochrome c551
-
0.025 - 0.043
Ferrocytochrome c-551
-
5
ferrocytochrome c2
Rhodobacter sphaeroides
-
-
0.1
ferrocytochrome V(gamma)
Rhodobacter sphaeroides
-
-
-
0.08 - 7.7
hydroxylamine
0.08 - 6.4
NH2OH
0.08 - 1478
nitrite
38 - 125
NO
0.08 - 144
NO2-
0.11 - 6.4
O2
additional information
additional information
Achromobacter cycloclastes
-
-
-
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2
azide
-
recombinant enzyme, pH 6.5, 25°C
0.0049
oxidized azurin
-
-
-
0.001 - 0.002
oxidized cytochrome b551
-
-
-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.048
-
nitrite reduction
0.33
-
electron donor azurin
2.1
-
nitrite reduction, elctron donor methyl viologen
3.32
-
reduction of N,N,N',N'-tetramethyl-4-phenyldiamine
10.8
-
recombinant enzyme, without activation, enzyme has only type 1 copper centers
22.7
-
enzyme is activated 2.5-4.5fold by freezing at -20°C for 6 h and subsequent thawing
40
-
if reduced nitrite is measured
45.2
-
native recombinant enzyme, azurin
80 - 90
-
if oxidized benzyl viologen is measured
82
-
mutant N90S, pH 7.1
107
-
native recombinant enzyme, dithionite
117
-
after storage at -20°C for 25 h and thawing in a water bath at 20°C
123
-
mutant H254F, pH 7.1
130
-
electron donor benzyl viologen
167.7
-
recombinant enzyme, after activation with CuSO4
168
-
native recombinant enzyme, methyl viologen
240
-
wild-type, pH 7.1
303
pH 6.5, 35°C
960
-
in the presence of 2 M NaCl
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6.5
-
assay at, pH dependence, overview
5.1 - 5.4
-
hydroxylamine reduction, electron donor pseudoazurin, pre-reduced cytochrome cd1; NH2OH reduction, electron donor pseudoazurin
5.4
-
hydroxylamine reduction, electron donor cytochrome c550, pre-reduced cytochrome cd1; NH2OH reduction, electron donor cytochrome c550
5.6
-
reaction with 0.5 mM nitrite and pseudoazurin as electron donor
5.7
-
nitrite reduction, electron donor cytochrome c550; nitrite reduction, electron donor cytochrome c550, pre-reduced cytochrome cd1
5.8
-
nitrite reduction, electron donor pseudoazurin; nitrite reduction, electron donor pseudoazurin, pre-reduced cytochrome cd1
6.2
-
O2 reduction, electron donor cytochrome c550; O2 reduction, electron donor cytochrome c550, pre-reduced cytochrome cd1
6.3
-
O2 reduction, electron donor pseudoazurin; O2 reduction, electron donor pseudoazurin, pre-reduced cytochrome cd1
6.5
optimum below pH 6.5
6.5 - 7
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9 - 6.2
-
pH 4.9: about 55% of maximal ativity, pH 6.2: about 65% of maximal activity, reaction with 0.5 mM nitrite and pseudoazurin as electron donor
5.2 - 6.5
-
pH 5.2: about 60% of maximal activity, pH 6.5: about 55% of maximal activity, reaction with 5 mM nitrite and pseudoazurin as electron donor
additional information
almost no activity at pH 9.0
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60 - 80
60°C: about 60% of maximal activity, 80°C: about 90% of maximal activity
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4 - 6.2
-
isoelectric focusing
9.3
isoelectric focusing
additional information
-
storage at -20°C for several months leads to an increase in the number of isoelectrophoretic forms. All preparations have two primary bands, one with a pI of 6.97 and the other of 7.02. Both bands possess significant cytochrome oxidase activity after elution from the gels. When each of the primary bands is eluted and again subject to isoelectric focusing under the same conditions as before, each band interconverts into two bands with pIs of 6.97 and 7.02
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
-
Manually annotated by BRENDA team
-
MLTC-1
Manually annotated by BRENDA team
additional information
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
the first 65 amino acid residues of theN-terminal domain constitute a mitochondrial targeting signal, sequence analysis
Manually annotated by BRENDA team
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Geobacillus kaustophilus (strain HTA426)
Geobacillus kaustophilus (strain HTA426)
Pseudoalteromonas haloplanktis (strain TAC 125)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Ralstonia pickettii (strain 12J)
Ralstonia pickettii (strain 12J)
Ralstonia pickettii (strain 12J)
Ralstonia pickettii (strain 12J)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
Rhodobacter sphaeroides (strain ATCC 17025 / ATH 2.4.3)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36500
-
3 * 36500, calculated from sequence, crystal structure
37700
-
3 * 37700, SDS-PAGE
39800
3 * 39800, SDS-PAGE, intracellular enzyme
41800
-
2 * 41800, SDS-PAGE
44300
3 * 44300, SDS-PAGE, extracellular enzyme
46000
-
3 * 46000, SDS-PAGE
53705
x * 53705, calculated, x * 60000, SDS-PAGE
54000
-
2 * 54000, SDS-PAGE
60204
-
2 * 60204, deduced from amino acid sequence
61500
1 * 61500, SDS-PAGE
63022
-
2 * 63022, electrospray mass spectroscopy
64000
-
2 * 64000, SDS-PAGE
66000
-
2 * 66000, SDS-PAGE
69000
-
gel filtration
70000
-
gel filtration
80000
-
gel filtration, gradient PAGE
83300
-
gel filtration
100000
-
gel filtration
103000
105000
-
sedimentation equlibrium
107000
-
gel filtration
113000
-
gel filtration
127000
-
gel filtration
130000
-
gel filtration
131000
-
gel filtration
133000
-
gel filtration
167000
-
gel filtration
400000
-
gel filtration
900000
gel filtration, aggregate
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 53705, calculated, x * 60000, SDS-PAGE
hexamer
monomer
octamer
-
8 * 50000, SDS-PAGE
trimer
additional information
-
enzyme NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. NirS also directly interacts with NirF, a membrane anchored lipoprotein
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor haem d1 into NirS
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
atomic resolution structures of four forms of the green Cu-nitrite reductase: structure of the resting state of the enzyme at 0.9 A, structure of then nitrite-soaked complex at 1.10 A resolution, structure of the endogenously bound NO complex at 1.12-A resolution, structure of endogenously bound nitrite and NO in the same crystal at 1.15-A resolution
-
mutant H254F loaded with either Cu2+ or Zn2+, to 1.5 A and 1.85 A, respectively. Both structures are essentially identical. Structure of mutant N90S, to 1.6 A resolution. In both the native and mutant N90S structures, a surface Zn ion is present in each monomer, bridging the two monomers through the coordinating residues His165 and Asp167 of one monomer and Glu195 of the adjacent monomer. This Zn site is similar to that described previously in several NiR structures
-
sitting-drop vapour diffusion method. Crystal structures of M144L and M144Q at 1.9 A, crystal structure of native enzyme at 1.04 A, structure of mutant enzyme C130A at 1.35 A
-
use of crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the type 1 Cu centre, but that the type 2 Cu centre does not photoreduce directly over a typical crystallographic data collection time. Internal electron transfer between the type 1 Cu and type 2 Cu centres does not occur, and the type 2 Cu centre remains oxidized
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crystals are grown at 19°C by hanging drop vapour diffusion using a reservoir of 100 mM sodium acetate, pH 4.7, 6%-10% polyethylene glycol 4000 and 1-5 mM cupric chloride, each drop is made from an equal volume of reservoir and a 15 mg/ml protein stock solution buffered in 10 mM Tris pH 7.0, crystals of mutants diffract to 1.8 A, nitrite-soaked oxidized crystals are obtained by placing crystals in reservoir solution supplemented with 5 mM sodium nitrite
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hanging-drop vapor-diffusion method, mutant enzyme M150G
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M150G crystals are grown at room temperature by hanging drop vapor diffusion method
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purified recombinant enzyme, free or in complex with small molecule inhibitors, hanging drop vapor diffusion method, room temperature, 25 mg/ml protein in 20 mM Tris-HCl, pH 7.0, is mixed with an equal volume of reservoir containing 6-10% PEG 4000, 100 mM sodium acetate, pH 4.0, addition of 20 mM of ligands 20 mM of azide, formate, or nitrate, X-ray diffraction structure determination and analysis at 1.5-1.8 A resolution
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hanging drop vapour diffusion, 0.002 ml protein solution containing 20 mg/ml protein in 20 mM Tris-Hcl, pH 7.5 are mixed with reservoir solution containing 18% polyethylene glycol 4000 and 100 mM Tris-HCl, pH 8.9 at 20°C, crystals of wild-type HdNIR and C260A mutant diffract to 2.35 A and 3.5 A, respectively
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in complex with its electron-donor protein pseudoazurin
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crystal structure
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crystals of Y25S mutant protein are grown from a solution containing 10-20 mg/ml protein in the presence of 2.2-2.4 M ammonium sulfate and 50 mM potassium phosphate, pH 7.0, crystals diffract to 1.4 A
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molecular modeling of myglobin mutant L29H/F43Y with or without nitrite shows the necessary structural features of native cytochrome cd1 nitrite reductase and that the protein can provide comparable interactions with nitrite as in native nitrite reductase
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recombinant soluble domain, residues 483-913, to 2.4 A
to 1.95 A resolution. The naturally fused type of Cu-nitrite reductase tethering a cytochrome c at the C-terminus folds as a unique trimeric domain-swapped structure and has a self-sufficient electron flow system. The C-terminal cytochrome c domain is located at the surface of the type 1 copper site in the N-terminal domain from the adjacent subunit. The heme-to-Cu distance of 10.6 A is comparable to the transient electron transfer complex of normal Cu-nitrite reductase with cytochrome c. The cytochrome c-Cu-nitrite reductase domain interaction is highly transient. An electron is directly transferred from the partner to the type 1 copper
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2.5 A resolution
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crystals of the H327A mutant are obtained by vapor diffusion technique by mixing in a 1:1 ratio the protein and a reservoir solution containing 4.0% polyethylene glycol 5000 monomethyl ether, 0.1 M sodium acetate, pH 5.5. The space group is 4(3)22 with cell dimensions 70.5 x 70.5 x 281 A. Crystals of the H369A mutant are obtained by mixing in a 1.1 ratio the protein and a reservoir solution containing 11.5% polyethylene glycol 6000, 0.2 M imidazole/malate, pH 6.5. The space group is P4(1)2(1)2 with cell dimensions 94.7 x 94.7 x 159.9 A; H327A mutant enzymes: vapour diffusion technique, mixing of the enzyme and a reservoir solution containing 4% polyethylene glycol 5000 monomethyl ether, 100 mM sodium acetate pH 5.5 in a 1/1 ratio, H369A mutant enzyme: 11.5% polyethylene glycol 6000, 200 mM imidazole/malate pH 6.5, x-ray structure of both mutants
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crystals of the H327A mutants are obtained by vapour diffusion technique. Crystals of H369A are obtained by mixing equal volumes of a reservoir solution containing 11.5% PEG 6000, 0.2 M imidazole/malate, pH 6.5, and of protein, in presence or not of 50 mM potassium nitrite and 50 mM sodium ascorbate. Crystals belong to space group P4(1)2(1)2 with cell dimensions a = b = 94.7 A, c = 159.9 A. The three-dimensional structures of NIR mutant H327A, and H369A in complex with NO solved by multiple wave-length anomalous dispersion, using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60° rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d1-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d1-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure
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structure of the reduced enzyme both in the unbound form and with the physiological product, NO, bound at the d1 heme active site
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the structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa is solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha as the starting model
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vapour diffusion at 20°C, in presence of 10% polyethylene glycol 4000, 50 mM Tris-HCl, pH 8.7, 400 mM NaCl, at a protein concentration of 14 mg/ml. The crystals are dark green elongated tetragonal prisms of dimensions 1.5 mm * 0.2 mm * 0.2 mm for the largest ones. These crystals are tetragonal with space group P4(1)(3)2(1)2 and cell dimensions a = b = 128.2 A, c = 172.6 A. They diffract at least up to 2.8 A
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structures of copper(I)-nitrite complexes with sterically hindered tris(4-imidazolyl)carbinols such as tris(1-methyl-2-ethyl-4-imidazolyl)carbinol, tris(1-methyl-2-isopropyl-4-imidazolyl)carbinol, or tris(1-pyrazolyl)methanes such as tris(3,5-dimethyl-1-pyrazolyl)methane or tris(3,5-diethyl-1-pyrazolyl)methane, and tris(3,5-diisopropyl-1-pyrazolyl)methane reveal mononoclear ny1-N-bound nitrite complexes with a distorted tetrahedral geometry
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homology modeling based on PDB entry 1BKW. Model demonstrates that the enzyme can be folded into two cupredoxin domains of the well-known CuNIR structure and implies that the conserved residues H122, D125, H127, H158, C159, H168, M173,H272, and H321 form the type 1 and type 2 Cu sites
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
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activity is not affected after treatment for 10 min, first step in purification
80
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native and purified enzyme retain 50% activity after 20 min exposure
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
urea, 4 M, 9% of initial activity, 6 M, no dissociation into subunits but irreversible inactivation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, 4 weeks
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-20°C, more than 1 year, no loss of activity
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-20°C, storage for several months leads to an increase in the number of isoelectrophoretic forms
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0°C, 3 weeks, no loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate, CM-Sephadex, DEAE-Sephadex A-50, Sephacryl S-200, Sephadex G-50, Resource Q, Poros PE/M
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butyl-toyopearl, Sepharose CL-6B, Sephacryl S-300, octyl-sepharose, hydroxyapatite
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CM-cellulose, Sephadex G-150, hydroxyapatite
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DEAE-Biogel A, TSK DEAE-5PW
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DEAE-Sepharose, Poros HP2, Superdex 200
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DEAE-Toyopearl, CM-cellulose, Sephacryl S-200
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f. sp. denitrificans
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Mono S, alkyl-superose, Sephacryl S-300, Mono Q, Mono S
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monomeric enzyme prepared by controlled succinylation of the native dimer; recombinant enzyme, contains only the c heme
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purified NiR exhibits a blue color spectrum showing maximum absorbance at 594 nm
recombinant enzyme
recombinant soluble domain
recombinant wild-type and D92E and D92N mutant enzyme
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recombinant wild-type and H139A mutant enzyme
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recombinant wild-type and Y10F mutant enzyme
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recombinant wild-type enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography, the tag is removed by thrombin, followed by anion exchange chromatography
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recombinant wild-type, H327A and H369A mutant enzyme
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soluble fraction, DEAE-cellulose, Source 15Q, Sephadex 75
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
D92E and D92N mutant enzyme
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epression in Pseudomonas putida
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expression in Escherichia coli
expression in Haloferax volcanii, intracellular enzyme
expression in Pseudomonas putida
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expression in Pseudomonas putida PAW340
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expression in Pseudomonas stutzeri
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expression of the soluble domain, residues 483-913, in Escherichia coli
expression of the wild-type enzyme in Escherichia coli strain BL21 (DE3)
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expression of wild-type and Y10F mutant enzyme in Pseudomonas putida
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expression of wild-type, D129A, D129N, H287A, I289A and I289V mutant enzymes in Escherichia coli
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expression of wild-type, H327A and H369A mutant enzyme in Pseudomonas putida
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gene aniA, DNA and amino acid sequence determination and analysis, expression and phylogenetic analysis, recombinant expression of wild-type and mutant enzymes in Escherichia coli
H139A mutant enzyme
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
W144L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
W144L/Y203L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
Y203L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
W144L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
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W144L/Y203L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
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Y203L
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visible absorption and EPR spectrum is similar to that of wild-type AcNIR. The redox potentials of the mutant is also nearly equal to that of wild-type. Although the enzymatic activities of the mutants are also the same as that of wild-type enzyme, the intermolecular electron transfer rate constants from pseudoazurin to mutant AcNIRs is 3-4fold less than that from pseudoazurin to wild-type AcNIR using electrochemical methods
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A191E
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slight increase in electron transfer rate constant
A191E/G198E
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3fold increase in electron transfer rate constant
A83D
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slight increase in electron transfer rate constant
A83D/A191E
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3fold increase in electron transfer rate constant
A83D/A191E/G198E
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4.7fold increase in electron transfer rate constant
A83D/G198E
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3fold increase in electron transfer rate constant
C130A
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inactive mutant enzyme, the loss of activity in this mutant is due to the absence of T1Cu and loss of the Cu–Cys130Sg bond rather than any change to the protein structure in this region
D92E
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mutation in type 2 Cu center, very low activity with artificial electron donors methyl viologen and sodium dithionite, 20-30% of wild-type activity with physiological electron donor azurin I
D92N
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mutation in type 2 Cu center, very low activity with artificial electron donors methyl viologen and sodium dithionite, 60-70% of wild-type activity with physiological electron donor azurin I
G198E
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2.6fold increase in electron transfer rate constant
H139A
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mutation in type 1 Cu center, very low activity with the artificial electron donor methyl viologen, no activity with the physiological electron donor azurin I
H254F
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full catalytic activity despite disruption of the primary proton channel. No change in apparent Km value for nitrite
M144L
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change in activity in the mutant is related to the perturbation of the finely poised redox potentials of the T1Cu sites of azurin and AxNiR
M144Q
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change in activity in the mutant is related to the perturbation of the finely poised redox potentials of the T1Cu sites of azurin and AxNiR
N90S
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disruption of H-bonding in the high-pH proton channel results in an 70% decrease in specific activity. No change in apparent Km value for nitrite
I257A
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3.7% of wild-type activity
I257G
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2.5% of wild-type activity
I257L
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26% of wild-type activity
I257M
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4% of wild-type activity
I257T
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1.4% of wild-type activity
I257V
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125% of wild-type activity
M150H
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mutant enzyme shows very low catalytic activity
I257A
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3.7% of wild-type activity
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I257G
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2.5% of wild-type activity
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I257L
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26% of wild-type activity
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I257M
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4% of wild-type activity
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I257V
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125% of wild-type activity
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M150G
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mutation increases the reorganization energy by 0.3 eV (30 kJ/mol), binding of the nearby Met62 to the type-1 Cu site lowers the reorganization energy back to approximately the wild-type value
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M150T
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mutation increases the reorganization energy by 0.3 eV (30 kJ/mol)
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C114A
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lacks the type I copper ion in the N-terminal domain, shows catalytic activity
C260A
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lacks the type I copper ion in the C-terminal domain, no nitrite-reduction activity
C114A
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lacks the type I copper ion in the N-terminal domain, shows catalytic activity
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C260A
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lacks the type I copper ion in the C-terminal domain, no nitrite-reduction activity
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H280L
a naturally occuring, enzyme-inactivating mutation in the disease-associated strain i1332, a 9-residues insertion located close to the type I Cu-site and mutation of the catalytic histidine at position 280
M106H
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inactive protein, the unusual highly cooperative and strongly hysteretic redox titration of the wild-type is lost in the mutant protein
Y25S
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unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, O2, and hydroxylamine without a reduuctive activation step
D129A
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low activity
D129N
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low activity
H287A
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very low activity
I289A
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activity comparable to wild-type
I289V
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activity comparable to wild-type
M182T
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activity comparable to wild-type
additional information
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replacement of the long 15-residue type 1 copper-binding loop of nitrite reductase with that from Paracoccus versutus cupredoxin amicyanin. The sizable loop contraction does not have a significant effect on the structures of both the type 1 and type 2 CuII sites. The crystal structure of the variant with ZnII at both the type 1 and type 2 sites shows a coordination geometry of the type 2 site that is almost identical to that found in the wild-type protein. In the type 1 centre, the positions of most of the coordinating residues are altered with the largest difference observed for the coordinating His residue in the centre of the mutated loop. This ligand moves away from the active site, which results in a more open metal centre with a coordinating water molecule. The reduction potential of the type i centre is reduced by 200 mV. The resulting unfavourable driving force for electron transfer between the two copper sites, and an increased reorganisation energy for the type 1 centre, contribute to the loop variant having very little nitrite reductase activity
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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study on use of stabilized hemoglobin as alternative to red cells. Cross-linked hemoglobin bis-tetramers with good oxygen delivery potential have 3fold enhanced nitrite reductase activity, compared to native protein and cross-linked tetramers. Conjugation of four polyethylene glocol chains to the bis-tetramer at each beta-Cys-93 produces a material with additionallly 2.5fold increased nitrite reductase activity while retaining cooperativity
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LINKS TO OTHER DATABASES (specific for EC-Number 1.7.2.1)
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ExPASy
KEGG
MetaCyc
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NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
SYSTERS
Protein Mutant Database
InterPro (database of protein families, domains and functional sites)