NADP nitrous oxide-forming nitric oxide reductase

 

Cytochrome P450 nitric oxide reductase (P450nor) is involved in a dissimilatory reduction of nitrite. Acts as a nitric oxide reductase. Is able to reduce nitrate and nitrite to a gaseous form of N2O when oxygen supply is limited or discontinued. May function as a detoxification mechanism.

This protein is found in denitrifying organisms such as Fusarium oxysporum. It is a member of the cytochrome P450 superfamily, which are heme-thiolate enzymes. There are two isoforms of P450nor in F. oxysporum, one of which uses NADH as cofactor exclusively, while the other uses NADH or NADPH.

P450nor catalyses the NAD(P)H-dependent reduction of two molecules of the free radical, nitric oxide (NO), to nitrous oxide (N2O). The reaction is unusual because it involves direct electron transfer, in the form of a hydride, from NAD(P)H to a redox protein (heme) that contains only a one-electron redox centre.

P450nor appears to have an important role in protecting the fungus from NO inhibition of mitochondria and other cellular damage that may result from the reaction of NO with other molecules such as oxygen or superoxide to form biologically hazardous compounds.

 

Reference Protein and Structure

Sequence
P23295 UniProt (1.7.1.14) IPR002397 (Sequence Homologues) (PDB Homologues)
Biological species
Fusarium oxysporum (Fungus) Uniprot
PDB
1xqd - Crystal structure of P450NOR complexed with 3-pyridinealdehyde adenine dinucleotide (1.8 Å) PDBe PDBsum 1xqd
Catalytic CATH Domains
1.10.630.10 CATHdb (see all for 1xqd)
Cofactors
Heme b (1)
Click To Show Structure

Enzyme Reaction (EC:1.7.1.14)

dinitrogen oxide
CHEBI:17045ChEBI
+
NAD(+)
CHEBI:15846ChEBI
+
water
CHEBI:15377ChEBI
→
oxidonitrate(1-)
CHEBI:29121ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH
CHEBI:16908ChEBI
Alternative enzyme names: Fungal nitric oxide reductase, Cytochrome P450nor, NOR,

Enzyme Mechanism

Introduction

A molecule of NO binds to the resting state enzyme, where the heme iron is in its ferric (Fe3+) state, to form an [Fe3+ NO] complex.

This is reduced to form the intermediate thought to be [Fe3+ NO 2H+]. Reduction occurs by hydride transfer from NAD(P)H, and the addition of a proton, derived from the bulk solvent and probably transferred via a hydrogen bonding network involving [Solvent Water -- Asp393 -- Water -- Ser286 -- Water] to the heme.

NAD(P)+ is rapidly released from the active site, and the intermediate reacts with another molecule of NO to regenerate the enzyme and form N2O and water.

Catalytic Residues Roles

UniProt PDB* (1xqd)
Thr243 Thr243A Interacts with the carboxyl of nicotinic acid ring to fix it stereochemically. This interaction of Thr243 together with the propionate of haem moving upward restricts the conformation of the nicotinic acid ring so that the pro-R side of C4-hydrogens faces the haem, which is consistent with the pro-R hydrogen-specific hydride transfer. steric role
Cys352 Cys352A Is the axial ligand to the heme prosthetic group. activator, metal ligand
Asp393, Ser286 Asp393A, Ser286A Involved in forming a delivery pathway of a proton from the bulk solvent to the heme iron to yield the reaction intermediate. proton shuttle (general acid/base)
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Riplinger C et al. (2014), Chemistry, 20, 1602-1614. New Insights into the Nature of Observable Reaction Intermediates in Cytochrome P450 NO Reductase by Using a Combination of Spectroscopy and Quantum Mechanics/Molecular Mechanics Calculations. DOI:10.1002/chem.201302443. PMID:24453075.
  2. McQuarters AB et al. (2014), Curr Opin Chem Biol, 19, 82-89. Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor). DOI:10.1016/j.cbpa.2014.01.017. PMID:24658055.
  3. Shoun H et al. (2012), Philos Trans R Soc Lond B Biol Sci, 367, 1186-1194. Fungal denitrification and nitric oxide reductase cytochrome P450nor. DOI:10.1098/rstb.2011.0335. PMID:22451104.
  4. Riplinger C et al. (2011), Chemphyschem, 12, 3192-3203. The Reaction Mechanism of Cytochrome P450 NO Reductase: A Detailed Quantum Mechanics/Molecular Mechanics Study. DOI:10.1002/cphc.201100523. PMID:22095732.
  5. Lehnert N et al. (2006), J Comput Chem, 27, 1338-1351. Electronic structure of iron(II)-porphyrin nitroxyl complexes: Molecular mechanism of fungal nitric oxide reductase (P450nor). DOI:10.1002/jcc.20400. PMID:16788909.
  6. Oshima R et al. (2004), J Mol Biol, 342, 207-217. Structural Evidence for Direct Hydride Transfer from NADH to Cytochrome P450nor. DOI:10.1016/j.jmb.2004.07.009. PMID:15313618.
  7. Shimizu H et al. (2000), J Biol Chem, 275, 4816-4826. Proton Delivery in NO Reduction by Fungal Nitric-oxide Reductase. CRYOGENIC CRYSTALLOGRAPHY, SPECTROSCOPY, AND KINETICS OF FERRIC-NO COMPLEXES OF WILD-TYPE AND MUTANT ENZYMES. DOI:10.1074/jbc.275.7.4816. PMID:10671516.
  8. Shiro Y et al. (1995), J Biol Chem, 270, 1617-1623. Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism. PMID:7829493.

Step 1.

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Catalytic Residues Roles

Residue Roles
Cys352A metal ligand, activator
Asp393A proton shuttle (general acid/base)
Ser286A proton shuttle (general acid/base)
Thr243A steric role

Chemical Components

Step 1.

Contributors

Gemma L. Holliday