Information on EC 1.7.3.6 - hydroxylamine oxidase (cytochrome)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.7.3.6
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RECOMMENDED NAME
GeneOntology No.
hydroxylamine oxidase (cytochrome)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydroxylamine + 2 ferricytochrome c = nitroxyl + 2 ferrocytochrome c + 2 H+
show the reaction diagram
(1a)
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hydroxylamine + O2 = nitrite + H2O + H+
show the reaction diagram
overall reaction
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nitroxyl + 2 ferrocytochrome c + O2 + H+ = nitrite + 2 ferricytochrome c + H2O
show the reaction diagram
spontaneous
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dehydrogenation
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oxidation
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redox reaction
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reduction
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SYSTEMATIC NAME
IUBMB Comments
hydroxylamine:oxygen oxidoreductase
The enzyme from the heterotrophic nitrifying bacterium Paracoccus denitrificans contains three to five non-heme, non-iron-sulfur iron atoms and interacts with cytochrome c556 and pseudoazurin [2,3]. Under anaerobic conditions in vitro only nitrous oxide is formed [3]. Presumably nitroxyl is released and combines with a second nitroxyl to give nitrous oxide and water. When oxygen is present, nitrite is formed.
CAS REGISTRY NUMBER
COMMENTARY hide
9075-43-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
enzyme HAO catalyzes the conversion of hydroxylamine to nitrite in nitrifying bacteria, that is key reaction in the nitrogen cycle. The enzyme HAO, protein NE1300, may play a structural role in the ternary complex with cytochrome c554, the physiological electron acceptor of HAO. Two of HAO's product electrons are subsequently transferred back to ammonia monooxygenase as substrate electrons, and the other two electrons contribute to the electrochemical gradient through a terminal oxidase in the cytoplasmic membrane
additional information
one heme in each HAO monomer is a highly unusual heme P460 that is the site of catalysis, Enzyme structure analysis and molecular docking, modelling
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
hydroxylamine + 2 ferricytochrome c
nitroxyl + 2 ferrocytochrome c + 2 H+
show the reaction diagram
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?
hydroxylamine + ferricytochrome c550
nitroxyl + ferrocytochrome c550 + H+
show the reaction diagram
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under aerobic assay conditions nitrite is the major reaction product but N2O is also detected at low levels. Under anaerobic conditions no nitrite is produced but N2O is detected, though at non-stoichiometric levels. The production of nitrite by Paracoccus denitrificans hydroxylamine oxidase is an oxygen-dependent reaction
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hydroxylamine + ferricytochrome c551
nitroxyl + ferrocytochrome c551 + H+
show the reaction diagram
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?
hydroxylamine + horse heart ferricytochrome c
nitroxyl + horse heart ferrocytochrome c + H+
show the reaction diagram
hydroxylamine + O2
nitrite + H2O + H+
show the reaction diagram
hydroxylamine + pseudoazurin
nitrite + reduced pseudoazurin
show the reaction diagram
nitroxyl + 2 ferrocytochrome c + O2 + H+
nitrite + 2 ferricytochrome c + H2O
show the reaction diagram
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?
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
hydroxylamine + 2 ferricytochrome c
nitroxyl + 2 ferrocytochrome c + 2 H+
show the reaction diagram
Q82V11
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?
nitroxyl + 2 ferrocytochrome c + O2 + H+
nitrite + 2 ferricytochrome c + H2O
show the reaction diagram
Q82V11
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c554
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cytochrome P-460
one heme in each HAO monomer is a highly unusual heme P460 that is the site of catalysis. Heme P460 contains two covalent cross-links between the porphyrin and a Tyr residue, structure analysis, overview
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additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe
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non-heme iron enzyme, the enzyme could possess a non-heme ferric iron centre that is lost when the 20000 Da polypeptide is subjected to SDS-PAGE, but that can be reconstituted by addition of Fe3+ ions to the refolded enzyme
Fe2+
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the enzyme is activated 6fold and 21fold by 0.01 mM and 0.1 mM ferrous ions, respectively
Iron
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contains 3-5 nonhaem, non-iron-sulfur iron atoms as prosthetic groups, predominantly coordinated by carboxylate ligands
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cyanide
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the cyanide-sensitive form of the enzyme exists only during turnover
H2O2
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irreversible, inactivation most rapid at pH 9-10, overview: substrates which protect against inactivation
NaCl
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15 mM, 50% inhibition of activity with horse heart cytochrome, activity with pseudoazurin or cytochrome c550 is inhibited only above 100 mM
additional information
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hardly inhibited by 0.1 mM KCN
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
ferricytochrome c551
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pH 7.8, 25°C
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0.07
horse heart ferricytochrome c
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in 10 mM Tris-HC1 (pH 8.0), at 30°C
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0.35
hydroxylamine
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in 10 mM Tris-HC1 (pH 8.0), at 30°C
0.033
pseudoazurin
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pH 7.8, 25°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.53
hydroxylamine
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pH 8, temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
EDTA
Arthrobacter globiformis
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at pH 9.0 and 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.6
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in 10 mM Tris-HC1 (pH 8.0), at 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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activity with horse heart cytochrome c
10
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activity with pseudoazurin
11
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activity with cytochrome c550 continues to increase to pH 11
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3 - 11.5
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pH 7.3: about 35% of maximal activity, pH 11.5: about 50% of maximal activity activity with pseudoazurin
7.6 - 10
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pH 7.6: about 50% of maximal activity, pH 10.0: about 35% of maximal activity with horse heart cytochrome c
8 - 11
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activity with cytochrome c550 continues to increase to pH 11, activity at pH 8 is about 50% of the activity at pH 11
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19000
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x * 19000, SDS-PAGE
20000
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x * 20000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotrimer
3 * 67000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified native enzyme, mixing of 0.002 ml of protein in 20 mM Tris-HCl, pH 8.1, with 0.002 ml of crystallization solution containing 0.1 M potassium nitrate, 0.1 M MES-Na buffer, pH 7.5, and 46% v/v PEG 400, X-ray diffraction structure determination and analysis at 2.1 A resolution, modelling
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
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90% loss of activity after 1 min at 70°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme by ammonium sulfate fractionation, gel filtration, ultracentrifugation, again gel filtration, followed by anion exchange chromatography
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
in energy-depleted cells, hao 3 gene expression is increased in the presence of 30 mM NH4+