Literature summary for 1.7.2.1 extracted from

Fukuda, Y.; Tse, K.M.; Lintuluoto, M.; Fukunishi, Y.; Mizohata, E.; Matsumura, H.; Takami, H.; Nojiri, M.; Inoue, T.
Structural insights into the function of a thermostable copper-containing nitrite reductase (2014), J. Biochem., 155, 123-135 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene nirK, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Geobacillus thermodenitrificans

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified wild-type enzyme with chloride- and formate-bound, as well as purified enzyme mutant C135A with nitrite-bound, hanging drop vapour diffusion method, mixng of 0.0015 ml of 100 mg/ml protein solution with 0.001 5 ml of well solution containing 0.1 M acetate buffer, pH 4.5, 5.0% w/v PEG 4000, 75 mM CuSO4, and 200 mM sodium formate, and equilibration against 0.45 ml well solution, soaking of C135A mutant crystals in nitrite solution, at 20°C, X-ray diffraction structure determination and analysis at 1.15 A and 1.90 A resolution, respectively	Geobacillus thermodenitrificans

Crystallization (Comment)

Organism

purified wild-type enzyme with chloride- and formate-bound, as well as purified enzyme mutant C135A with nitrite-bound, hanging drop vapour diffusion method, mixng of 0.0015 ml of 100 mg/ml protein solution with 0.001 5 ml of well solution containing 0.1 M acetate buffer, pH 4.5, 5.0% w/v PEG 4000, 75 mM CuSO4, and 200 mM sodium formate, and equilibration against 0.45 ml well solution, soaking of C135A mutant crystals in nitrite solution, at 20°C, X-ray diffraction structure determination and analysis at 1.15 A and 1.90 A resolution, respectively

Geobacillus thermodenitrificans

Protein Variants

Protein Variants	Comment	Organism
C135A	site-directed mutagenesis, the crystal structure of mutant C135A with nitrite displays a unique eta1-O coordination mode of nitrite at the catalytic copper site (T2Cu) unlike the wild-type enzyme	Geobacillus thermodenitrificans

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
periplasm	-	Geobacillus thermodenitrificans	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Cu2+	homotrimeric CuNIR contains one type 1 (T1Cu) and one type 2 Cu (T2Cu) site per monomer. The T1Cu atom is coordinated by four amino acid residues (two histidine residues, cysteine and methionine) and functions as a receptor site for the electron supplied by an electron-donor protein such as c-type heme-containing cytochrome or blue-copper proteins. The T2Cu site is a catalytic centre composed of three histidine residues and an axial ligand water molecule. Copper centre structure, overview	Geobacillus thermodenitrificans

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
nitrite + ferrocytochrome c + 2 H+	Geobacillus thermodenitrificans	-	nitric oxide + H2O + ferricytochrome c	-	?
nitrite + ferrocytochrome c + 2 H+	Geobacillus thermodenitrificans NG80-2	-	nitric oxide + H2O + ferricytochrome c	-	?

Organism

Organism	UniProt	Comment	Textmining
Geobacillus thermodenitrificans	A4IL26	-	-
Geobacillus thermodenitrificans NG80-2	A4IL26	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, hydrophobic interaction chromatography, dialysis, and anion exchange chromatography, followed by gel filtration	Geobacillus thermodenitrificans

Reaction

Reaction	Comment	Organism	Reaction ID
nitric oxide + H2O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H+	reaction mechanism, overview. Mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. Analysis of the hydrogen-bond networks around His244 and the flow path of protons consumed by nitrite reduction. The electron transfer reaction is coupled with the proton transfer reaction	Geobacillus thermodenitrificans	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
nitrite + ferrocytochrome c + 2 H+	-	Geobacillus thermodenitrificans	nitric oxide + H2O + ferricytochrome c	-	?
nitrite + ferrocytochrome c + 2 H+	-	Geobacillus thermodenitrificans NG80-2	nitric oxide + H2O + ferricytochrome c	-	?

Subunits

Subunits	Comment	Organism
homotrimer	3 * 35478, sequence calculation	Geobacillus thermodenitrificans
More	CuNIR typically has a homotrimeric structure containing one type 1 (T1Cu) and one type 2 Cu (T2Cu) site per monomer	Geobacillus thermodenitrificans

Synonyms

Synonyms	Comment	Organism
copper-containing nitrite reductase	-	Geobacillus thermodenitrificans
CuNIR	-	Geobacillus thermodenitrificans
GtNiR	-	Geobacillus thermodenitrificans
NirK	-	Geobacillus thermodenitrificans

Cofactor

Cofactor	Comment	Organism	Structure
cytochrome c	-	Geobacillus thermodenitrificans

General Information

General Information	Comment	Organism
metabolism	the enzyme catalyzes the key reaction in denitrification as the nitrogen compound is changed from an ionic state to a gaseous molecule	Geobacillus thermodenitrificans
physiological function	copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO2 ) to nitric oxide (NO) during denitrification	Geobacillus thermodenitrificans