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

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY
1.7.2.1
-
RECOMMENDED NAME
GeneOntology No.
nitrite reductase (NO-forming)
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
proposed reaction mechanism
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
electron-transfer mechanism
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
electron transfer from c heme to d1 heme is very slow, order of seconds
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
rate constants of intermolecular electron transfer
Pseudomonas nautica 617
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
a copper protein, cytochrome c-552 or cytochrome c-553 from Pseudomonas denitrificans acts as acceptor
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
mechanism
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
ordered mechanism in which electron transfer is gated by binding of nitrite to the type 2 Cu centre
-
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
ammonia oxidation II (anaerobic)
-
intra-aerobic nitrite reduction
-
Microbial metabolism in diverse environments
-
nitrate reduction I (denitrification)
-
nitrate reduction VII (denitrification)
-
nitrifier denitrification
-
Nitrogen metabolism
-
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
AcNIR
Achromobacter cycloclastes IAM1013
-
-
-
AfNiR
Alcaligenes faecalis S-6
-
;
-
C551-O2 oxidoreductase
-
-
cd1 nitrite reductase
-
-
copper-containing dissimilatory nitrite reductase
P38501
-
copper-containing dissimilatory nitrite reductase
Alcaligenes faecalis S-6
P38501
-
-
copper-containing nitrite reductase
-
-
copper-containing nitrite reductase
P38501
-
copper-containing nitrite reductase
Alcaligenes faecalis S-6
P38501
-
-
copper-containing nitrite reductase
-
-
copper-containing nitrite reductase
Hyphomicrobium denitrificans A3151
-
-
-
cytochrome c-551:O2, NO2- oxidoreductase
-
-
-
-
cytochrome cd
-
-
-
-
cytochrome cd
-
-
cytochrome cd1 nitrite reductase
Q3LGA9
-
cytochrome cd1 nitrite reductase
Q3LGA9
-
-
cytochrome cd1 nitrite reductase
-
-
cytochrome cd1 nitrite reductase
P72181
-
cytochrome cd1 nitrite reductase
-
-
cytochrome oxidase
-
-
-
-
dissimilatory nitrite reductase cytochrome cd1
-
-
EC 1.9.3.2
-
formerly
HdNIR
Hyphomicrobium denitrificans A3151
-
-
-
hemoglobin
-
-
NiR
Alcaligenes faecalis S-6
P38501
-
-
NirK
A9XR52
-
NirK
uncultured bacterium KSU-1
-
-
-
nitrite reductase
Q9JYE1
-
nitrite reductase
P72181
-
nitrite reductase
-
-
nitrite reductase
P24040
-
nitrite reductase
Pseudomonas stutzeri ZoBell
P24040
-
-
oxidase, Pseudomonas cytochrome
-
-
-
-
PNR
Pseudomonas aeruginosa 5276
-
-
-
Pseudomonas cytochrome oxidase
-
-
-
-
Pseudomonas cytochrome oxidase
-
-
reductase, nitrite (cytochrome)
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9027-00-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Achromobacter cycloclastes IAM1013
IAM1013
-
-
Manually annotated by BRENDA team
Alcaligenes faecalis S-6
S-6
-
-
Manually annotated by BRENDA team
Alcaligenes faecalis S-6
strain S-6
UniProt
Manually annotated by BRENDA team
NCIB 11015
-
-
Manually annotated by BRENDA team
Hyphomicrobium denitrificans A3151
A3151
-
-
Manually annotated by BRENDA team
formerly Aquaspirillum magnetotacticum
-
-
Manually annotated by BRENDA team
wild-type strain MC58, gene nitrite reductase aniA
UniProt
Manually annotated by BRENDA team
formerly Micrococcus denitrificans
-
-
Manually annotated by BRENDA team
formerly Thiosphaera pantotropha
-
-
Manually annotated by BRENDA team
mutant H327A
-
-
Manually annotated by BRENDA team
strain 5276
-
-
Manually annotated by BRENDA team
Pseudomonas aeruginosa 5276
strain 5276
-
-
Manually annotated by BRENDA team
Pseudomonas nautica 617
-
-
-
Manually annotated by BRENDA team
addition of nitrate to oxygen-limited cells induces cytochrome cd1 synthesis
-
-
Manually annotated by BRENDA team
strain ZoBell
-
-
Manually annotated by BRENDA team
strain ZoBell ATCC 14405
UniProt
Manually annotated by BRENDA team
Pseudomonas stutzeri ZoBell
strain ZoBell
-
-
Manually annotated by BRENDA team
Pseudomonas stutzeri ZoBell
strain ZoBell ATCC 14405
UniProt
Manually annotated by BRENDA team
f. sp. denitrificans
-
-
Manually annotated by BRENDA team
strain 2.4.3
-
-
Manually annotated by BRENDA team
uncultured bacterium KSU-1
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
A4TUE6, -
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
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ferrocytochrome c + O2
ferricytochrome c + H2O
show the reaction diagram
-
-
-
?
ferrocytochrome c + O2
ferricytochrome c + H2O
show the reaction diagram
-
in the presence of ascorbate, N,N,N',N-tetramethyl-p-phenylenediamine and cytochrome c-553
-
?
ferrocytochrome c-551 + NO2-
NO + ferricytochrome c-551
show the reaction diagram
-
-
-
-
ferrocytochrome c-551 + NO2-
NO + ferricytochrome c-551
show the reaction diagram
-
-
-
?
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
-
-
-
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
-
-
-
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
-
-
?
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
-
-
-
?
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
-
-
?
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
also reacts with horse heart cytochrome c
-
-
ferrocytochrome c-551 + O2
H2O + ferricytochrome c-551
show the reaction diagram
-
inactive with eukaryotic cytochromes c
-
-
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
show the reaction diagram
-
-
-
-
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
show the reaction diagram
P24474
-
-
-
?
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
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
P24474
-
-
-
?
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
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
P25006
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
-
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
artificial electron donors: reduced methyl viologen, phenazine methosulfate and to a lesser extend hydroquinone, highly purified enzyme has cytochrome c oxidase activity
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
artificial electron donors: thionine, brilliant cresyl blue, methylene blue, 2,6-dichlorophenolindophenol or Pseudomonas stutzeri cytochrome c-552 and 558
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
artificial electron donor: reduced benzyl viologen
-
?
nitrite + ferrocytochrome c
nitric oxide + H2O + ferricytochrome c
show the reaction diagram
-
artificial electron donor: reduced benzyl viologen
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
Alcaligenes faecalis S-6
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
Pseudomonas stutzeri ZoBell
-
-
-
-
?
nitrite + ferrocytochrome c(gamma)
NO + H2O + ferricytochrome c(gamma)
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 + ferrocytochrome c550
NO + oxidized 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
Alcaligenes faecalis, Alcaligenes faecalis S-6
-
reduction of pseudoazurin by ascorbate
-
-
?
nitrite + methyl viologen
NO + oxidized methyl viologen + H2O
show the reaction diagram
A9XR52
-
-
-
?
nitrite + reduced azurin
NO + H2O + oxidized azurin
show the reaction diagram
Hyphomicrobium denitrificans, Hyphomicrobium denitrificans A3151
-
-
-
-
?
nitrite + reduced azurin
NO + oxidized azurin
show the reaction diagram
-
azurin purified from Pseudomonas chlororaphis
-
-
?
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 azurin I
NO + azurin I
show the reaction diagram
O68601
-
-
-
?
nitrite + reduced benzyl viologen
NO + H2O + 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 + H2O + oxidized benzyl viologen
show the reaction diagram
Achromobacter cycloclastes IAM1013
-
-
-
-
?
nitrite + reduced benzyl viologen
NO + oxidized benzyl viologen
show the reaction diagram
-
-
-
-
?
nitrite + reduced benzyl viologen
NO + oxidized benzyl viologen
show the reaction diagram
-
random sequential mechanism
-
-
?
nitrite + reduced benzyl viologen
nitric oxide + oxidized benzyl viologen
show the reaction diagram
uncultured bacterium, uncultured bacterium KSU-1
-
-
-
-
?
nitrite + reduced electron donor
NO + H2O + oxidized electron donor
show the reaction diagram
Hyphomicrobium denitrificans, Hyphomicrobium denitrificans A3151
-
-
-
-
?
nitrite + reduced hydroquinone
nitric oxide + H2O + hydroquinone
show the reaction diagram
-, Q3LGA9
-
-
-
?
nitrite + reduced hydroquinone
nitric oxide + H2O + hydroquinone
show the reaction diagram
-, Q3LGA9
-
-
-
?
nitrite + reduced methyl viologen
NO + oxidized methyl viologen + H2O
show the reaction diagram
Alcaligenes faecalis, Alcaligenes faecalis S-6
-
-
-
-
?
nitrite + reduced methyl viologen
NO + oxidized methyl viologen
show the reaction diagram
-
random sequential mechanism
-
-
?
nitrite + reduced pseudoazurin
NO + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
P38501
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
r
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
cytochrome c550 and pseudoazurin are the alternative electron mediator proteins between the cytochrome bc1 and the cytochrome cd1 type nitrite reductase
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
pseudoazurin from Achromobacter cycloclastes
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
random sequential mechanism
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
the rate-determining step in the enzyme reaction sequence is not the intermolecular electron transfer process between pseudoazurin and AcNIR, but the reduction of substrate by AcNIR in the steady-state assay system
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
Alcaligenes faecalis S-6
-
-
-
-
r
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
Achromobacter cycloclastes IAM1013
-
the rate-determining step in the enzyme reaction sequence is not the intermolecular electron transfer process between pseudoazurin and AcNIR, but the reduction of substrate by AcNIR in the steady-state assay system
-
-
?
NO2 + reduced methyl viologen
NO + oxidized methylviologen
show the reaction diagram
-
-
-
?
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
-
probably most dominant activity in vivo
-
-
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
-
role in respiration
-
-
?
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
Pseudomonas nautica 617
-
-
-
-
NO2- + reduced ascorbate
NO + oxidized ascorbate
show the reaction diagram
-
physiological electron donor is not known, 7% activity if NADH is used as artificial electron donor
-
?
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
-
-
-
?
NO2- + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
unambiguously identified as physiological electron donor
-
?
O2 + ferrocytochrome c
H2O + ferricytochrome c
show the reaction diagram
Pseudomonas stutzeri, Pseudomonas stutzeri ZoBell
-
-
-
-
?
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
-
putative physiological electron donor
-
-
?
reduced azurin + O2
oxidized azurin + H2O
show the reaction diagram
-
not known whether azurin donates electrons in vivo in parallel or sequentially to cytochrome c551
-
-
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
-
-
-
?
hydroxylamine + reduced pseudoazurin
NH3 + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells
-
-
-
additional information
?
-
-
nitrite reductase in both oxidized and reduced states forms with NO two distinct compounds at both hemes. These compounds, in addition to the oxidized and reduced enzymes, are formed during the turnover of this enzyme as functional intermediates
-
-
-
additional information
?
-
P38501
density functional theory study of nitrite and nitric oxide adducts
-
-
-
additional information
?
-
-
theoretical investigation has provided a number of insights into the reaction mechanism of copper nitrite reductase: The results presented in this paper indicate that the hydroxyl-intermediate pathway appears unlikely for the main reaction. The results also indicate that Asp-92 may play a significant structural role through hydrogen-bonding to the protonated oxygen of the nitrite substrate, and thereby elongating the N O bond, which is to be cleaved
-
-
-
additional information
?
-
Q9JYE1
a conserved and functional aniA gene is not essential for meningococcal survival
-
-
-
additional information
?
-
-
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
-
-
-
additional information
?
-
-
activity is associated with histidine/methionine coordination at heme c, and the cytochrome cd1 is activated by exposure to its physiological substrate without the necessity of passing through the reduced state
-
-
-
additional information
?
-
P24040
activity is associated with histidine/methionine coordination at heme c, and the cytochrome cd1 is activated by exposure to its physiological substrate without the necessity of passing through the reduced state, reactivity toward nitrite is also observed for oxidized cytochrome cd1 from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent FeIII heme d1 species in nitrite reduction
-
-
-
additional information
?
-
-
electrocatalytic reduction of nitrite to NO by CuMe2bpaCl2, as a model for the active site of copper containing nitrite reductase. The 77-K EPR spectrum of CuMe2bpaCl2 in the collagen matrix reveals the typical axial signals of a tetragonal Cu2+ chromophore. The redox potential is -63 mV at pH 5.5. In the presence of nitrite, an increase in the cathodic current is observed in the cyclic voltammogram of CuMe2bpaCl2 in the collagen matrix. Upon reaching -300 mV, a linear generation of NO is observed for the CuMe2bpaCl2/collagen film-coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution using the Michaelis-Menten equation, results in Vmax 3.16 nM per s and Km 1.1 mM at pH 5.5. The current increase and the reaction rate are dependent on the pH of the solution. The mechanism of nitrite reduction by the copper complex in the collagen matrix is the same mechanism as that of the enzyme in aqueous solution
-
-
-
additional information
?
-
-
study on strucuture-function relationship using 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. All of these complexes are good functional models of Cu-NiR that form NO and copper(II) acetate complexes well from reactions with acetic acid under anaerobic conditions. The copper(I) nitrite complex with the tris(1-methyl-2-ethyl-4-imidazolyl)carbinol ligand, which is similar to the highly conserved three-histidine (His)3 ligand environment in the catalytic site of Cu-NiR, has the highest Cu-NiR activity
-
-
-
additional information
?
-
Alcaligenes faecalis, Alcaligenes faecalis S-6
-
the active site residue is Ile257. The small molecules formate, acetate and nitrate mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom and interacting wit the oxidized catalytic metal ion, overview. Nitrite and the substrate mimic bind in the same asymmetric, bidentate manner
-
-
-
additional information
?
-
Pseudomonas stutzeri ZoBell
P24040
activity is associated with histidine/methionine coordination at heme c, and the cytochrome cd1 is activated by exposure to its physiological substrate without the necessity of passing through the reduced state, reactivity toward nitrite is also observed for oxidized cytochrome cd1 from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent FeIII heme d1 species in nitrite reduction
-
-
-
additional information
?
-
Pseudomonas aeruginosa 5276
-
nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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 c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
Alcaligenes faecalis S-6
-
-
-
-
?
nitrite + ferrocytochrome c
NO + H2O + ferricytochrome c
show the reaction diagram
Pseudomonas stutzeri ZoBell
-
-
-
-
?
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
Hyphomicrobium denitrificans, Hyphomicrobium denitrificans A3151
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + H2O + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
r
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
-
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
-
cytochrome c550 and pseudoazurin are the alternative electron mediator proteins between the cytochrome bc1 and the cytochrome cd1 type nitrite reductase
-
-
?
nitrite + reduced pseudoazurin
NO + oxidized pseudoazurin
show the reaction diagram
Alcaligenes faecalis S-6
-
-
-
-
r
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
-
probably most dominant activity in vivo
-
-
NO2- + ferrocytochrome c
NO + ferricytochrome c
show the reaction diagram
-
role in respiration
-
-
?
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
-
putative physiological electron donor
-
-
?
reduced azurin + O2
oxidized azurin + H2O
show the reaction diagram
-
not known whether azurin donates electrons in vivo in parallel or sequentially to cytochrome c551
-
-
ferrocytochrome c-551 + O2
ferricytochrome c-551 + H2O
show the reaction diagram
-
-
-
-
additional information
?
-
-
nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells
-
-
-
additional information
?
-
Q9JYE1
a conserved and functional aniA gene is not essential for meningococcal survival
-
-
-
additional information
?
-
-
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
-
-
-
additional information
?
-
Pseudomonas aeruginosa 5276
-
nitrite reductase from Pseudomonas aeruginosa released by antimicrobial agents and complement induces interleukin-8 production in bronchial epithelial cells
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
cytochrome
-
contains cytochrome, probably of the c and a2 types
-
cytochrome
-
cytochrome c-552 or cytochrome c-553 from Pseudomonas denitrificans acts as acceptor
-
cytochrome cd1
-
the heme d1 is not in the oxoferryl (FeIVdO) state but is low-spin FeIII weakly coupled to a radical species, binding structure, overview
-
cytochrome cd1
-
cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer. Heme c, which transfers electrons from donor proteins into the active site, has two histidine ligands in the oxidized enzyme from Paracoccus pantotrophus. During reduction of this enzyme, Tyr25 dissociates from the distal side of heme d1, and one heme c ligand is replaced by methionine
-
cytochrome cd1
P24040
cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer. Heme c, which transfers electrons from donor proteins into the active site, has two histidine ligands in the oxidized enzyme from Paracoccus pantotrophus. During reduction of this enzyme, Tyr25 dissociates from the distal side of heme d1, and one heme c ligand is replaced by methionine. Spectral analysis, overview
-
cytochrome cd1
-
the c heme is located in a predominantly alpha-helical domain of the enzyme, whereas the d1 heme resides in a beta-propeller structure
-
heme
-
heme c has bis-histidine ligation and FeIII heme d1 [d1(FeIII)] is bound by histidine and tyrosine. The cytochrome cd1 heme d1 is not in the oxoferryl (FeIVdO) state but is low-spin FeIII weakly coupled to a radical species, structure, overview
heme
-
characterization of the nitrosyl d1 heme complex by high-field-pulse electron papramagnetic spectroscopy spectra and derived 14N hyperfine and quadrupole interactions. Residue Y10 does not influence the NO ligand orientation in the reduced state in solution
heme
-
cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer
heme
P24040
heme c and heme d1, cytochromes cd1 are dimeric bacterial nitrite reductases, which contain two hemes per monomer
heme
-
two hemes in cytochrome cd1, the c heme is located in a predominantly alpha-helical domain of the enzyme, whereas the d1 heme resides in a beta-propeller structure
heme
-
nitrite reductase activitiy of cytochrome c is dependent on the pentacoordination of the heme iron
Heme c
-
c heme is the electron accepting pole of the enzyme
Heme c
Pseudomonas nautica 617
-
-
Heme c
-
electron uptake site
Heme c
-
oxidation-reduction behavior
heme d
-
oxidation-reduction behavior, formation of an oxygenated intermediate at heme d
-
Heme d1
-
reduction of the substrate occurs at the d1-heme site
Heme d1
Pseudomonas nautica 617
-
-
Heme d1
-
nitrite reduction site
Heme d1
-
the recombinant enzyme, expressed in Pseudomonas putida, contains c-heme but no d1-heme
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
copper
-
one subunit contains a type 1 copper center, the second subunit a type 2 copper center, activity of copper depleted enzyme can be restored
copper
-
copper protein
copper
-
contains 2 type 1 copper atoms per molecule but no other types of copper
copper
-
5.5 ng/mg protein; copper protein
copper
-
copper protein
copper
-
blue copper enzyme that shows little absorbance in the 460 nm range, purified recombinant nonactivated enzyme contains 1.97 mol Copper/mol enzyme, the CuSO4 activated enzyme contains 5.97 mol copper/mol enzyme i.e. 6 copper atoms per trimer, CuSO4 activation restores type 2 copper centers which are the sites of catalysis
copper
-
green copper enzyme
copper
-
blue copper enzyme, 1 type 1 and 1 type 2 Cu atoms per subunit
copper
-
green copper enzyme
copper
-
the type 1 Cu center functions as electron acceptor from the physiological electron donor azurin I, the type 2 Cu center is involved in binding and reduction of the substrate
copper
-
enzyme contains 2 type I copper and 1 type II copper ions
copper
-
3.2 copper ions per trimer, most probably 3 type 1 and 3 type 2 copper, respectively
copper
-
enzyme contains type 1 and type 2 copper centers, 6.3 copper atoms per trimer, blue copper enzyme
copper
-
copper-containing nitrite reductase
copper
-
2.6 atoms copper per subunit, 2 type 1 Cu and 0.6 type 2 Cu
copper
-
copper containing nitrite reductase
copper
A9XR52
ligands of the type 1 and 2 Cu centers are completely conserved in NiR. They are His253, Cys294, His302, and Met307 for the type 1 Cu center and His258, His293, and His456 for type 2.Asp256 and His405 residues are important for enzyme catalysis by forming a hydrogen bond network on the type 2 Cu center. The EPR spectrum is also characteristic of NirK proteins that contain both type 1 and type 2 copper centers
copper
-
copper atoms are coordinated by H122, C159, H168, and M173 for the type 1 Cu site and H127, H158, and H321 for the type 2 Cu site. The absorption spectrum of NirK displays two peaks at 598 and 449 nm characteristic for type I Cu proteins
copper
-
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
Cu2+
-
copper-containing dissimilatory nitrite reductase, catalytic type 2 copper, binding site structure, analysis of binding structure and interaction with inhibitors, overview
Iron
-
heme iron
Iron
-
heme containing enzyme
Iron
-
in heme c and heme d1
Iron
-
in hemes c and d1, the cytochrome cd1 heme d1 is not in the oxoferryl (FeIVdO) state but is low-spin FeIII weakly coupled to a radical species, FeIII heme d1 [d1(FeIII)] is bound by histidine and tyrosine, structure, overview
Iron
-
in the two hemes of cytochrome cd1
Iron
-
nitrite reductase activitiy of cytochrome c is dependent on the pentacoordination of the heme iron
KCl
-
2 M, enzyme is activated by high salt concentrations
LiCl
-
2 M, enzyme is activated by high salt concentrations
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
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
acetate
-
weak, mixed-type inhibition, inhibition mode, overview
azide
-
binding mode, overview
CN-
-
0.047 mM, 50% inhibition of nitrite reduction
CO
A9XR52
20% residual activity
diethyldithiocarbamate
-
1 mM, complete inhibition
diethyldithiocarbamate
A9XR52
1 mM, 10% residual activity
EDTA
-
1 mM, complete inhibition after preincubation for 10 min
EDTA
-
1 mM, 70% inhibition after 1 h
EDTA
A9XR52
1 mM, 60% resiudal activity
formate
-
weak mixed-type inhibition, inhibition mode, overview
KCN
-
1 mM, 72% inhibition
KCN
-
1 mM, complete inhibition
KCN
A9XR52
1 mM, 5% residual activity
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
methylhydrazine
-
irreversible
additional information
-
relatively ineffective at 1 mM: CO, bathocuproine, diethyl dithiocarbamate, o-phenanthroline and alpha,alpha'-dipyridyl
-
additional information
-
endogenous inhibitor of nitrite reductase from Pseudomonas aeruginosa: a non-blue copper-containing glycoprotein of 10000 Da that contains 1 atom of EPR-detectable type II copper
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
cytochrome cd1 is activated by exposure to its physiological substrate without the necessity of passing through the reduced state
-
additional information
P24040
cytochrome cd1 is activated by exposure to its physiological substrate without the necessity of passing through the reduced state
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00084
-
azurin
-
recombinant Y10F mutant enzyme, at pH 6.2 and 27C
0.035
-
azurin
-
recombinant wild-type and Y10F mutant enzyme, at pH 6.2 and 27C
0.0305
-
cytochrome c
-
succinylated monomeric enzyme
0.046
-
cytochrome c
-
-
0.00177
-
cytochrome c551
-
recombinant wild-type enzyme, at pH 6.2 and 27C
-
0.0018
-
cytochrome c551
-
recombinant H327A mutant enzyme, O2 reduction
-
0.002
-
cytochrome c551
-
recombinant wild-type enzyme, O2 reduction
-
0.0056
-
cytochrome c551
-
also known as cytochrome c8
-
0.0075
-
cytochrome c551
-
recombinant H369A mutant enzyme, O2 reduction
-
0.0018
-
Ferrocytochrome c-551
-
pH 6.2, 25C, mutant enzyme H327A
-
0.002
-
Ferrocytochrome c-551
-
pH 6.2, 25C, wild-type enzyme
-
0.0075
-
Ferrocytochrome c-551
-
pH 6.2, 25C, mutant enzyme H369A
-
0.43
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c
0.68
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550
1.15
-
hydroxylamine
-
25C, pH 7.0, electron donor horse heart cytochrome c
1.38
-
hydroxylamine
-
25C, pH 7.0, electron donor cytochrome c550
0.434
-
NH2OH
-
pre-reduced enzyme, electron donor horse heart cytochrome c
0.68
-
NH2OH
-
pre-reduced enzyme, electron donor cytochrome c550
1.15
-
NH2OH
-
initially oxidized enzyme, electron donor horse heart cytochrome c
1.38
-
NH2OH
-
initially oxidized enzyme, electron donor cytochrome c550
2.5
-
NH2OH
-
pre-reduced enzyme, electron donor pseudoazurin
0.00008
-
nitrite
-
25C, A83D/A191E/G198E triple mutant
0.00024
-
nitrite
-
25C, A191E/G198E double mutant
0.00025
-
nitrite
-
25C, A83D/G198E double mutant
0.0003
-
nitrite
-
25C, A83D/A191E double mutant
0.0005
-
nitrite
-
25C, G198E mutant
0.00096
-
nitrite
-
25C, A191E mutant
0.001
-
nitrite
-
25C, A83D mutant
0.0013
-
nitrite
-
25C, wild-type
0.006
-
nitrite
-
pH 6.2, 25C, wild-type enzyme
0.007
-
nitrite
-
25C, pH 7.0, electron donor cytochrome c550; 25C, pH 7.0, electron donor horse heart cytochrome c
0.011
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c
0.012
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550
0.019
-
nitrite
-
25C, pH 7.0, electron donor pseudoazurin
0.034
-
nitrite
-
wild-type, pH 7.1
0.0357
-
nitrite
-
-
0.036
-
nitrite
-
25C, pH 7.0
0.049
-
nitrite
-
25C, pH 6.0
0.05
-
nitrite
-
mutant H254F, pH 7.1; mutant N90S, pH 7.1
0.053
-
nitrite
-
25C, pH 6.5
0.063
-
nitrite
-
25C, pH 7.0, Y25S mutant enzyme
0.068
-
nitrite
-
25C, pH 7.5
0.071
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin
0.071
-
nitrite
-
25C, pH 7.0, activated wild-type enzyme
0.099
-
nitrite
-
mutant enzyme M150H
0.22
-
nitrite
-
25C, pH 8.0
0.25
-
nitrite
-
pH 6.5, 25C
0.62
-
nitrite
-
25C, pH 5.5
126
-
nitrite
-
mutant enzyme M150T
133
-
nitrite
-
mutant enzyme M150G
416
-
nitrite
-
wild-type enzyme
0.006
-
NO2-
-
recombinant wild-type enzyme
0.0073
-
NO2-
-
initially oxidized enzyme, electron donor horse heart cytochrome c
0.011
-
NO2-
-
pre-reduced enzyme, electron donor horse heart cytochrome c
0.012
-
NO2-
-
pre-reduced enzyme, electron donor cytochrome c550
0.0187
-
NO2-
-
initially oxidized enzyme, electron donor pseudoazurin
0.046
-
NO2-
-
reduced cytochrome c as electron donor
0.067
-
NO2-
-
initially oxidized enzyme, electron donor cytochrome c550
0.071
-
NO2-
-
pre-reduced enzyme, electron donor pseudoazurin
0.027
-
O2
-
cytochrome c oxidase activity
0.036
-
O2
-
wild-type enzyme
0.148
-
O2
-
pre-reduced enzyme, electron donor cytochrome c550
0.15
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550
0.16
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin
0.161
-
O2
-
pre-reduced enzyme, electron donor pseudoazurin
0.166
-
O2
-
pre-reduced enzyme, electron donor horse heart cytochrome c
0.17
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c
0.21
-
O2
-
25C, pH 7.0, electron donor cytochrome c550
0.214
-
O2
-
initially oxidized enzyme, electron donor cytochrome c550
0.47
-
O2
-
25C, pH 7.0, electron donor horse heart cytochrome c; initially oxidized enzyme, electron donor horse heart cytochrome c
1
-
O2
-
mutant enzyme H369A
0.067
-
pseudoazurin
-
25C, pH 6.0
-
0.077
-
pseudoazurin
-
25C, pH 6.5
-
0.085
-
pseudoazurin
-
25C, pH 8.0
-
0.1
-
pseudoazurin
-
25C, pH 7.0
-
0.16
-
pseudoazurin
-
25C, pH 7.5
-
2.5
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin
additional information
-
additional information
-
-
-
additional information
-
additional information
-
steady-state kinetics
-
additional information
-
additional information
-
Michaelis-Menten kinetics
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1
-
azurin
-
recombinant Y10F mutant enzyme, at pH 6.2 and 27C
1.12
-
azurin
-
recombinant wild-type enzyme, at pH 6.2 and 27C
1.33
-
cytochrome c
-
succinylated monomeric enzyme
0.583
-
cytochrome c551
-
recombinant wild-type enzyme, at pH 6.2 and 27C
-
0.6
-
cytochrome c551
-
recombinant Y10F mutant enzyme, at pH 6.2 and 27C
-
2.8
-
cytochrome c551
-
at pH 7.0
-
0.025
-
Ferrocytochrome c-551
-
pH 6.2, 25C, wild-type enzyme
-
0.032
-
Ferrocytochrome c-551
-
pH 6.2, 25C, mutant enzyme H369A
-
0.043
-
Ferrocytochrome c-551
-
pH 6.2, 25C, mutant enzyme H327A
-
5
-
ferrocytochrome c2
-
-
0.1
-
ferrocytochrome V(gamma)
-
-
-
0.08
-
hydroxylamine
-
25C, pH 7.0, electron donor cytochrome c550
0.2
-
hydroxylamine
-
25C, pH 7.0, electron donor horse heart cytochrome c
3
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c
3.2
-
hydroxylamine
-
25C, pH 7.0, wild-type, pre-reduction with dithionite
3.5
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550
6.4
-
hydroxylamine
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin
0.08
-
NH2OH
-
initially oxidized enzyme, electron donor cytochrome c550
0.2
-
NH2OH
-
initially oxidized enzyme, electron donor horse heart cytochrome c
3
-
NH2OH
-
pre-reduced enzyme, electron donor horse heart cytochrome c
3.5
-
NH2OH
-
pre-reduced enzyme, electron donor cytochrome c550
6.4
-
NH2OH
-
pre-reduced enzyme, electron donor pseudoazurin
0.08
-
nitrite
-
pH 6.2, 25C, mutant enzyme H327A; pH 6.2, 25C, mutant enzyme H369A
0.62
-
nitrite
-
pH 7.0, electron donor azurin
2.1
-
nitrite
-
25C, pH 7.0, electron donor cytochrome c550
2.4
-
nitrite
-
25C, pH 7.0, electron donor horse heart cytochrome c
2.7
-
nitrite
-
25C, pH 7.0, wild-type, without pre-reduction with dithionite
5.3
-
nitrite
-
25C, pH 7.0, electron donor pseudoazurin
8
-
nitrite
-
pH 6.2, 25C, wild-type enzyme
41
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c
67
-
nitrite
-
25C, pH 7.0, Y25S mutant enzyme
68
-
nitrite
-
25C, pH 7.0, wild-type, pre-reduction with dithionite
71
-
nitrite
-
25C, pH 8.0
74
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550
144
-
nitrite
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin
219
-
nitrite
-
25C, pH 7.5
243
-
nitrite
-
pH 7.0, electron donor benzyl viologen
320
-
nitrite
-
25C, pH 5.5
392
-
nitrite
-
25C, pH 7.0
1046
-
nitrite
-
25C, pH 6.5
1478
-
nitrite
-
25C, pH 6.0
38
-
NO
-
25C, pH 6.5
64
-
NO
-
25C, pH 7.0
109
-
NO
-
25C, pH 7.5
125
-
NO
-
25C, pH 8.0
0.08
-
NO2-
-
recombinant H327A and H369a mutant enzymes
2.1
-
NO2-
-
initially oxidized enzyme, electron donor cytochrome c550
2.4
-
NO2-
-
initially oxidized enzyme, electron donor horse heart cytochrome c
5.3
-
NO2-
-
initially oxidized enzyme, electron donor pseudoazurin
8
-
NO2-
-
recombinant wild-type enzyme
41
-
NO2-
-
pre-reduced enzyme, electron donor horse heart cytochrome c
74
-
NO2-
-
pre-reduced enzyme, electron donor cytochrome c550
80
-
NO2-
-
electron donor: reduced tetramethyl-4-phenylenediamine
106
-
NO2-
-
all-ferric nitrite-bound complex, electron donor pseudoazurin
144
-
NO2-
-
pre-reduced enzyme, electron donor pseudoazurin
0.11
-
O2
-
25C, pH 7.0, electron donor horse heart cytochrome c; initially oxidized enzyme, electron donor horse heart cytochrome c
0.17
-
O2
-
25C, pH 7.0, electron donor cytochrome c550; initially oxidized enzyme, electron donor cytochrome c550
2.8
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor horse heart cytochrome c; pre-reduced enzyme, electron donor horse heart cytochrome c
3
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor cytochrome c550; pre-reduced enzyme, electron donor cytochrome c550
3.2
-
O2
-
25C, pH 7.0, wild-type, pre-reduction with dithionite
6.2
-
O2
-
25C, pH 7.0, Y25S mutant enzyme
6.4
-
O2
-
25C, pH 7.0, pre-reduced cytochrome cd1, electron donor pseudoazurin; pre-reduced enzyme, electron donor pseudoazurin
7.7
-
hydroxylamine
-
25C, pH 7.0, Y25S mutant enzyme
additional information
-
additional information
-
-
-
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2
-
azide
-
recombinant enzyme, pH 6.5, 25C
0.0049
-
Oxidized azurin
-
-
-
0.001
0.002
oxidized cytochrome b551
-
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.048
-
Pseudomonas nautica 617
-
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
21.1
-
-
-
22.7
-
-
enzyme is activated 2.5-4.5fold by freezing at -20C for 6 h and subsequent thawing
23.4
-
-
-
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 -20C for 25 h and thawing in a water bath at 20C
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
-
A9XR52
pH 6.5, 35C
960
-
-
in the presence of 2 M NaCl
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
-
reaction with 5 mM nitrite and pseudoazurin as electron donor
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
7
-
cytochrome c oxidase activity
6.5
-
A9XR52
optimum below pH 6.5
6.5
-
P24040
-
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
P24040
almost no activity at pH 9.0
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
60
80
-, Q3LGA9
60C: about 60% of maximal activity, 80C: about 90% of maximal activity
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.4
6.2
-
isoelectric focusing
9.3
-
-, Q3LGA9
isoelectric focusing
additional information
-
-
storage at -20C 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
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
additional information
-
enzyme is released from bacteria, after exposure to different antimicrobial agents
Manually annotated by BRENDA team
additional information
Pseudomonas aeruginosa 5276
-
enzyme is released from bacteria, after exposure to different antimicrobial agents
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
enzyme is released from bacteria, after exposure to different antimicrobial agents
-
Manually annotated by BRENDA team
Pseudomonas aeruginosa 5276
-
enzyme is released from bacteria, after exposure to different antimicrobial agents
-
-
Manually annotated by BRENDA team
-
equally distributed between the soluble- and the membrane fraction
Manually annotated by BRENDA team
A9XR52
the first 65 amino acid residues of theN-terminal domain constitute a mitochondrial targeting signal, sequence analysis
Manually annotated by BRENDA team
-
75% of enzyme activity found in the soluble fraction
-
Manually annotated by BRENDA team
Pseudomonas nautica 617
-
-
-
Manually annotated by BRENDA team
Alcaligenes faecalis S-6
-
-
-
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Alcaligenes xylosoxydans xylosoxydans
Pseudoalteromonas haloplanktis (strain TAC 125)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
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)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
69000
-
-
gel filtration
70000
-
-
gel filtration
75000
-
-, Q3LGA9
gel filtration
79000
-
-
-
80000
-
-
gel filtration, gradient PAGE
83300
-
-
gel filtration
100000
-
-
gel filtration
103000
-
-
sedimentation equilibrium
105000
-
-
sedimentation equlibrium
107000
-
-
gel filtration
113000
-
-
gel filtration
127000
-
-
gel filtration
130000
-
-
gel filtration
131000
-
Pseudomonas nautica 617
-
gel filtration
133000
-
-
gel filtration
167000
-
-
gel filtration
400000
-
-
gel filtration
900000
-
A9XR52
gel filtration, aggregate
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
A9XR52
x * 53705, calculated, x * 60000, SDS-PAGE
dimer
-
2 * 66000, SDS-PAGE
dimer
-
2 * 60000, SDS-PAGE
dimer
-
2 * 60204, deduced from amino acid sequence
dimer
-
2 * 54000, SDS-PAGE
dimer
Pseudomonas nautica 617
-
2 * 60000, SDS-PAGE
dimer
-
2 * 39000-42000, enzyme composed of 2 nonidentical subunits: one contains Cu2+ type 1, the second subunit contains Cu2+ type 2, SDS-PAGE
dimer
-
2 * 37000, SDS-PAGE
dimer
-
2 * 41800, SDS-PAGE
dimer
-
2 * 64000, SDS-PAGE
dimer
-
2 * 63022, electrospray mass spectroscopy
dimer
-
2 * 60000
dimer
Pseudomonas stutzeri ZoBell
-
; 2 * 60000
-
hexamer
-
6 * 50000
hexamer
Hyphomicrobium denitrificans A3151
-
6 * 50000
-
monomer
-, Q3LGA9
1 * 61500, SDS-PAGE
octamer
-
8 * 50000, SDS-PAGE
trimer
-
recombinant enzyme
trimer
-
3 * 37000, calculated from sequence, crystal structure
trimer
-
3 * 36500, calculated from sequence, crystal structure
trimer
-
3 * 46000, SDS-PAGE
trimer
-
3 * 37700, SDS-PAGE
monomer
-
1 * 61500, SDS-PAGE
-
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
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
crystals are grown at 19C 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
-
hanging-drop vapor-diffusion method, mutant enzyme M150G
-
M150G crystals are grown at room temperature by hanging drop vapor diffusion method
-
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
-
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 20C, crystals of wild-type HdNIR and C260A mutant diffract to 2.35 A and 3.5 A, respectively
-
in complex with its electron-donor protein pseudoazurin
-
crystal structure
-
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
-
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
-
recombinant soluble domain, residues 483-913, to 2.4 A
Q6ABN0
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
-
2.5 A resolution
-
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
-
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
-
structure of the reduced enzyme both in the unbound form and with the physiological product, NO, bound at the d1 heme active site
-
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
-
vapour diffusion at 20C, 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
-
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
-
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
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
70
-
-
activity is not affected after treatment for 10 min, first step in purification
80
-
-
native and purified enzyme retain 50% activity after 20 min exposure
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
urea, 4 M, 9% of initial activity, 6 M, no dissociation into subunits but irreversible inactivation
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
0C, 3 weeks, no loss of activity
-
-20C, storage for several months leads to an increase in the number of isoelectrophoretic forms
-
-20C, more than 1 year, no loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme
-
recombinant wild-type and D92E and D92N mutant enzyme
-
recombinant wild-type and H139A mutant enzyme
-
recombinant enzyme
-
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
-
CM-cellulose, Sephadex G-150, hydroxyapatite
-
Mono S, alkyl-superose, Sephacryl S-300, Mono Q, Mono S
-
purified NiR exhibits a blue color spectrum showing maximum absorbance at 594 nm
A9XR52
butyl-toyopearl, Sepharose CL-6B, Sephacryl S-300, octyl-sepharose, hydroxyapatite
-
ammonium sulfate, CM-Sephadex, DEAE-Sephadex A-50, Sephacryl S-200, Sephadex G-50, Resource Q, Poros PE/M
-
recombinant enzyme
-
DEAE-Toyopearl, CM-cellulose, Sephacryl S-200
-
DEAE-Sepharose, Poros HP2, Superdex 200
-
recombinant soluble domain
Q6ABN0
monomeric enzyme prepared by controlled succinylation of the native dimer; recombinant enzyme, contains only the c heme
-
recombinant enzyme
-
recombinant wild-type and Y10F mutant enzyme
-
recombinant wild-type, H327A and H369A mutant enzyme
-
soluble fraction, DEAE-cellulose, Source 15Q, Sephadex 75
-
DEAE-Biogel A, TSK DEAE-5PW
Pseudomonas nautica 617
-
f. sp. denitrificans
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
D92E and D92N mutant enzyme
-
expression in Escherichia coli
-
H139A mutant enzyme
-
expression of the wild-type enzyme in Escherichia coli strain BL21 (DE3)
-
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
Q9JYE1
expression of the soluble domain, residues 483-913, in Escherichia coli
Q6ABN0
epression in Pseudomonas putida
-
expression in Escherichia coli
-
expression in Pseudomonas putida
-
expression in Pseudomonas putida PAW340
-
expression in Pseudomonas stutzeri
-
expression of wild-type and Y10F mutant enzyme in Pseudomonas putida
-
expression of wild-type, H327A and H369A mutant enzyme in Pseudomonas putida
-
expression of wild-type, D129A, D129N, H287A, I289A and I289V mutant enzymes in Escherichia coli
-
expression in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
W144L
-
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
-
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
-
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
Achromobacter cycloclastes IAM1013
-
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
Achromobacter cycloclastes IAM1013
-
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
Achromobacter cycloclastes IAM1013
-
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
-
A191E
-
slight increase in electron transfer rate constant
A191E/G198E
-
3fold increase in electron transfer rate constant
A83D
-
slight increase in electron transfer rate constant
A83D/A191E
-
3fold increase in electron transfer rate constant
A83D/A191E/G198E
-
4.7fold increase in electron transfer rate constant
A83D/G198E
-
3fold increase in electron transfer rate constant
C130A
-
inactive mutant enzyme, the loss of activity in this mutant is due to the absence of T1Cu and loss of the CuCys130Sg bond rather than any change to the protein structure in this region
D92E
-
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
-
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
-
2.6fold increase in electron transfer rate constant
H139A
-
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
-
full catalytic activity despite disruption of the primary proton channel. No change in apparent Km value for nitrite
M144L
-
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
-
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
-
3.7% of wild-type activity
I257G
-
2.5% of wild-type activity
I257L
-
26% of wild-type activity
I257M
-
4% of wild-type activity
I257T
-
1.4% of wild-type activity
I257V
-
125% of wild-type activity
M150G
-
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
M150G
-
mutant enzyme shows lower catalytic activity than the wild-type enzyme. The type-1 site optical spectrum differs significantly from that of the native enzyme. The midpoint potential of the type-1 site of nitrite reductase M150G is higher than that of the native enzyme
M150H
-
mutant enzyme shows very low catalytic activity
M150T
-
mutation increases the reorganization energy by 0.3 eV (30 kJ/mol)
M150T
-
mutant enzyme has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an about 50-fold slower intramolecular electrontransfer to the type-2 site
M150T
-
mutant enzyme shows lower catalytic activity than the wild-type enzyme
I257A
Alcaligenes faecalis S-6
-
3.7% of wild-type activity
-
I257G
Alcaligenes faecalis S-6
-
2.5% of wild-type activity
-
I257L
Alcaligenes faecalis S-6
-
26% of wild-type activity
-
I257M
Alcaligenes faecalis S-6
-
4% of wild-type activity
-
I257V
Alcaligenes faecalis S-6
-
125% of wild-type activity
-
M150G
Alcaligenes faecalis S-6
-
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
-
M150T
Alcaligenes faecalis S-6
-
mutation increases the reorganization energy by 0.3 eV (30 kJ/mol)
-
C114A
-
lacks the type I copper ion in the N-terminal domain, shows catalytic activity
C260A
-
lacks the type I copper ion in the C-terminal domain, no nitrite-reduction activity
C114A
Hyphomicrobium denitrificans A3151
-
lacks the type I copper ion in the N-terminal domain, shows catalytic activity
-
C260A
Hyphomicrobium denitrificans A3151
-
lacks the type I copper ion in the C-terminal domain, no nitrite-reduction activity
-
H280L
Q9JYE1
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
-
inactive protein, the unusual highly cooperative and strongly hysteretic redox titration of the wild-type is lost in the mutant protein
Y25S
-
unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, O2, and hydroxylamine without a reduuctive activation step
H327A
-
mutant protein has no nitrite reductase activity but maintains the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form; reduction of nitrite is severely compromised
H327A
-
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
H369A
-
mutant protein has no nitrite reductase activity but maintains the ability to reduce O2 to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form; reduction of nitrite is severely compromised
H369A
-
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
H369A
-
mutation impairs the reaction with O2, affecting both the properties and lifespan of the intermediate species
Y10F
-
no change in optical spectrum, nitrite and oxidase activity and heme c to heme d1 electron transfer rates compared to wild-type
Y10F
-
high-field-pulse electron papramagnetic spectroscopy spectra and the derived 14N hyperfine and quadrupole interactions are the same for wild-type and mutant. Residue Y10 does not influence the NO ligand orientation in the reduced state in solution
D129A
-
low activity
D129N
-
low activity
H287A
-
very low activity
I289A
-
activity comparable to wild-type
I289V
-
activity comparable to wild-type
M182T
-
activity comparable to wild-type
M144Q
-
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
additional information
-
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
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
medicine
-
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