Found as a multiheme cytochrome in many bacteria. The enzyme from Escherichia coli contains five hemes c and requires Ca2+. It also reduces nitric oxide and hydroxylamine to ammonia, and sulfite to sulfide.
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SYSTEMATIC NAME
IUBMB Comments
ammonia:ferricytochrome-c oxidoreductase
Found as a multiheme cytochrome in many bacteria. The enzyme from Escherichia coli contains five hemes c and requires Ca2+. It also reduces nitric oxide and hydroxylamine to ammonia, and sulfite to sulfide.
the periplasmic cytochrome c nitrite reductase, Nrf, system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. NO is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate
important role for the enzyme in nitric oxide management in oxygen-limited environments. Nitric oxide is a key element in host defense against invasive pathogens
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
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
NrfA from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium, it might also participate in NO radical detoxification, detoxifying exogenously generated NO radical encountered during invasion of a human host, overview
the enzyme shows a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO radical reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO radical reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine
important role for the enzyme in nitric oxide management in oxygen-limited environments. Nitric oxide is a key element in host defense against invasive pathogens
NrfA from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium, it might also participate in NO radical detoxification, detoxifying exogenously generated NO radical encountered during invasion of a human host, overview
the periplasmic cytochrome c nitrite reductase, Nrf, system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. NO is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate
10 heme centres range in reduction potential from -30 to 320 mV. The heme oxidation state has a profound effect on the interactions with substrate molecules, nitrite and hydroxylamine
crystal structure of penta-heme NrfA. Four of the NrfA hemes have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand
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
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
inhibition in the absence of Ca2+ is noted at potentials where heme 4 and/or 5 is reduced, this may implicate Ca2+ association with the propionate of heme 4 as being essential for maintaining steady-state activity from the fully reduced enzyme-substrate complex
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant H264N, to 2.15 A resolution. Homodimeric protein, the constellation of His/His and Lys-coordinated c-hemes is indistinguishable from those of the native enzyme
purified NrfA, hanging drop vapor diffusion method, five conditions that result in several different crystal packing motifs, e.g. 0.001 ml of protein solution containing 10 mg/ml of NrfA mixed with 0.001 ml of mother liquor containing 100 mM HEPES, pH 7.5, 20% PEG 10000 and incubated in hanging drop trays with a 1 ml well solution at 4°C, 5-14 days, other conditions, overview. X-ray diffraction structure determination and analysis at 1.7-2.5 A resolution
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
vapour diffusion method. Crystals belong to space group P2(1)2(1)2(1) with apparent cell parameters of a = 81.47 A, b = 90.84 and c = 294.87 A and contain four molecules of NrfA per asymmetric unit
mutant is unable to catalyze nitrite reduction but able to reduce hydroxylamine. The mutant simultaneously binds nitrite and electrons at the catalytic heme
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
Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry
Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo