Literature summary extracted from

Clarke, T.A.; Kemp, G.L.; Van Wonderen, J.H.; Doyle, R.M.; Cole, J.A.; Tovell, N.; Cheesman, M.R.; Butt, J.N.; Richardson, D.J.; Hemmings, A.M.
Role of a conserved glutamine residue in tuning the catalytic activity of Escherichia coli cytochrome c nitrite reductase (2008), Biochemistry, 47, 3789-3799.

Cloned(Commentary)

EC Number	Cloned (Comment)	Organism
1.7.2.2	gene nrfA, expression of wild-type and mutant enzymes in Escherichia coli strain JCB4083a	Escherichia coli

Crystallization (Commentary)

EC Number	Crystallization (Comment)	Organism
1.7.2.2	purified recombinant wild-type and mutant Q263E, 10 mg/ml protein, under aerobic conditions by the vapor diffusion hanging drop method using 20% v/v PEG 10000 in 100 mM Na-HEPES, pH 7.5, 20% ethylene glycol as cryoprotectant, X-ray diffraction structure determination and analysis at 1.74 A and 2.04 A resolution, respectively, molecular replacement	Escherichia coli

Protein Variants

EC Number	Protein Variants	Comment	Organism
1.7.2.2	Q263E	site-directed mutagenesis, the mutation leads to introduction of a negative charge into the vicinity of the active site heme, and the mutant shows reduced activity compared to the wild-type enzyme. The high spin state of the active site to be preserved, indicating that a water/hydroxide molecule is still coordinated to the heme in the resting state of the enzyme	Escherichia coli

KM Value [mM]

EC Number	KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
1.7.2.2	additional information	-	additional information	kinetics	Escherichia coli
1.7.2.2	0.033	-	nitrite	pH 7.0, 25°C, wild-type enzyme with methyl viologen as reductant	Escherichia coli
1.7.2.2	0.413	-	nitrite	pH 7.0, 25°C, mutant Q263E with methyl viologen as reductant	Escherichia coli

Metals/Ions

EC Number	Metals/Ions	Comment	Organism	Structure
1.7.2.2	Ca2+	the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview. An important function of the unusual Q263-calcium ion pair is to increase substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand	Escherichia coli
1.7.2.2	Fe3+	active site heme Fe(III) iron, the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview	Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number	Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
1.7.2.2	nitrite + ferrocytochrome c + H+	Escherichia coli	-	NH3 + H2O + ferricytochrome c	-	?

Organism

EC Number	Organism	UniProt	Comment	Textmining
1.7.2.2	Escherichia coli	P0ABK9	gene nrfA	-

Purification (Commentary)

EC Number	Purification (Comment)	Organism
1.7.2.2	recombinant wild-type and mutant enzymes from Escherichia coli strain JCB4083a by ammonium sulfate fractionation and anion exchange chromatography	Escherichia coli

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
1.7.2.2	additional information	the NrfA active site consists of a hexacoordinate high-spin heme with a lysine ligand on the proximal side and water/hydroxide or substrate on the distal side. There are four further highly conserved active site residues including a Q263 positioned near the heme iron for which the side chain, unusually, coordinates a conserved, essential calcium ion, overview. Important function of the Q263-calcium ion pair increasing substrate affinity through its role in supporting a network of hydrogen bonded water molecules stabilizing the active site heme distal ligand, active site structures of native and Q263 mutant NrfA enzymes, overview	Escherichia coli	?	-	?
1.7.2.2	nitrite + ferrocytochrome c + H+	-	Escherichia coli	NH3 + H2O + ferricytochrome c	-	?
1.7.2.2	nitrite + reduced methyl viologen	-	Escherichia coli	NH3 + H2O + oxidized methyl viologen	-	?

Synonyms

EC Number	Synonyms	Comment	Organism
1.7.2.2	cytochrome c nitrite reductase	-	Escherichia coli
1.7.2.2	NrfA	-	Escherichia coli

Temperature Optimum [°C]

EC Number	Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
1.7.2.2	25	-	assay at	Escherichia coli

pH Optimum

EC Number	pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
1.7.2.2	7	-	assay at	Escherichia coli

Cofactor

EC Number	Cofactor	Comment	Organism	Structure
1.7.2.2	heme	-	Escherichia coli

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Release 2023.1 (January 2023)