Nitrite reductase (copper type)

 

This family of enzymes is found in both denitrifying bacteria and fungi which reside in soil and aquatic ecosystems.

Denitrification, the reduction of dissolved nitrate and nitrite to gaseous NO, N20 and N2 is an important process in the recycling of nitrogen in the biosphere, and is a key step in the nitrogen cycle resulting in the loss of terrestrial nitrogen to the atmosphere.

It is chiefly carried out by denitrifying bacteria, which contain nitrate and nitrite reductases. The copper-containing family of enzymes is found in both denitrifying bacteria and fungi which reside in soil and aquatic ecosystems and uses copper ions as cofactors rather than the more common haem cd1

The enzymes in question catalyse the reduction of nitrite (NO2-) to NO + H2O. They contain two copper centres, a Type I centre which receives electrons from pseudoazurin (a copper containing protein), and a type II centre which is the site of nitrite reduction.

 

Reference Protein and Structure

Sequence
P25006 UniProt (1.7.2.1) IPR001287 (Sequence Homologues) (PDB Homologues)
Biological species
Achromobacter cycloclastes (Bacteria) Uniprot
PDB
1nia - THE STRUCTURE OF CU-NITRITE REDUCTASE FROM ACHROMOBACTER CYCLOCLASTES AT FIVE PH VALUES, WITH NITRITE BOUND AND WITH TYPE II CU DEPLETED (2.5 Å) PDBe PDBsum 1nia
Catalytic CATH Domains
2.60.40.420 CATHdb (see all for 1nia)
Cofactors
Copper(2+) (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.7.2.1)

nitrous acid
CHEBI:25567ChEBI
+
copper(2+)
CHEBI:29036ChEBI
+
hydron
CHEBI:15378ChEBI
→
water
CHEBI:15377ChEBI
+
nitric oxide
CHEBI:16480ChEBI
+
copper(1+)
CHEBI:49552ChEBI
Alternative enzyme names: cd-cytochrome nitrite reductase, (Nitrite reductase (cytochrome)), Cytochrome c-551:O(2), NO(2)(+) oxidoreductase, Cytochrome cd, Cytochrome cd1, Hydroxylamine (acceptor) reductase, Methyl viologen-nitrite reductase, Nitrite reductase (cytochrome; NO-forming), Pseudomonas cytochrome oxidase, Nitrite reductase, Nitrite reductase (cytochrome),

Enzyme Mechanism

Introduction

The resting oxidised state of the enzyme contains a water molecule coordinated to a Cu2+ ion at the type II copper centre. Nitrite then binds to the type II copper ion and and displaces the water molecule. This makes electron transfer from pseudoazurin to the type II copper centre via the type I copper centre energetically favourable. Nitrite reduction occurs at the type II centre. The type II copper transfers the electron it received to the nitrite, which together with protonation of the nitrite leads to reduction of nitrite to NO + H2O.

Two protons are needed for the reduction of NO2- to NO + H2O, which are likely to come from bulk solvent as the Asp98A catalytic residue is at the end of a proton tunnel connected to the bulk solvent.

Catalytic Residues Roles

UniProt PDB* (1nia)
Asp136 Asp98A Involved in supplying proton(s) for the reduction of NO2- to NO + H2O. activator, hydrogen bond acceptor, proton acceptor, proton donor
His173 His135A Binds the Cu(II) ion in the type II site and is part of the electron transfer pathway to the Cu(II) type II site from the type I site. single electron relay, single electron acceptor, single electron donor, metal ligand
Cys174 Cys136A Binds the Cu(II) ion in the type I site and is part of the electron transfer pathway to the Cu(II) type II site from the type I site. single electron relay, single electron acceptor, single electron donor, metal ligand
His293 His255B Has been variously proposed to have roles in providing a positive charge to assist the pushing of an electron onto the nitrite during the reduction, protonating the substrate during the reduction, and modifying the properties of Asp 98 via the intervening water molecule. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu317 (main-C), Thr318 Glu279B (main-C), Thr280B Activates His255. electrostatic stabiliser
His183, Met188, His133 His145A, Met150A, His95A Binds the Cu(II) ion in the type I site. metal ligand
His344, His138 His306B, His100A Binds the Cu(II) ion in the type II site. metal ligand
*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

overall reactant used, intermediate formation, proton transfer, electron transfer

References

  1. Fukuda Y et al. (2016), Proc Natl Acad Sci U S A, 113, 2928-2933. Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography. DOI:10.1073/pnas.1517770113. PMID:26929369.
  2. Li Y et al. (2015), Biochemistry, 54, 1233-1242. Enzymatic Mechanism of Copper-Containing Nitrite Reductase. DOI:10.1021/bi5007767. PMID:25594136.
  3. Impagliazzo A et al. (2005), Chembiochem, 6, 1648-1653. Pseudoazurin-Nitrite Reductase Interactions. DOI:10.1002/cbic.200500082. PMID:16138306.
  4. Astier Y et al. (2005), Chemphyschem, 6, 1114-1120. Sensing Nitrite through a Pseudoazurin-Nitrite Reductase Electron Transfer Relay. DOI:10.1002/cphc.200400384. PMID:15900523.
  5. Kataoka K et al. (2004), J Biol Chem, 279, 53374-53378. Structure-based Engineering of Alcaligenes xylosoxidans Copper-containing Nitrite Reductase Enhances Intermolecular Electron Transfer Reaction with Pseudoazurin. DOI:10.1074/jbc.m410198200. PMID:15475344.
  6. Pinho D et al. (2004), Eur J Biochem, 271, 2361-2369. Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135. Evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the type 2 copper center. DOI:10.1111/j.1432-1033.2004.04155.x. PMID:15182351.
  7. Boulanger MJ et al. (2000), J Biol Chem, 275, 23957-23964. Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase. DOI:10.1074/jbc.m001859200. PMID:10811642.
  8. Suzuki S et al. (2000), Acc Chem Res, 33, 728-735. Metal Coordination and Mechanism of Multicopper Nitrite Reductase. DOI:10.1021/ar9900257. PMID:11041837.
  9. Murphy ME et al. (1997), J Biol Chem, 272, 28455-28460. Structure of Nitrite Bound to Copper-containing Nitrite Reductase from Alcaligenes faecalis: MECHANISTIC IMPLICATIONS. DOI:10.1074/jbc.272.45.28455. PMID:9353305.
  10. Adman ET et al. (1995), J Biol Chem, 270, 27458-27474. The Structure of Copper-nitrite Reductase from Achromobacter cycloclastes at Five pH Values, with NO(2)[IMAGE] Bound and with Type II Copper Depleted. DOI:10.1074/jbc.270.46.27458. PMID:7499203.
  11. Kukimoto M et al. (1994), Biochemistry, 33, 5246-5252. X-ray Structure and Site-Directed Mutagenesis of a Nitrite Reductase from Alcaligenes Faecalis S-6: Roles of Two Copper Atoms in Nitrite Reduction. DOI:10.1021/bi00183a030. PMID:8172899.

Step 1. Asp98 is protonated from bulk solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor, activator
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond donor
His306B metal ligand
His100A metal ligand
His145A metal ligand
Met150A metal ligand
His95A metal ligand
Asp98A proton acceptor

Chemical Components

overall reactant used, intermediate formation, proton transfer

Step 2. The nitrite substrate accepts a proton from Asp98.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond donor
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His100A metal ligand
His306B metal ligand
Asp98A proton donor

Chemical Components

proton transfer

Step 3. A single electron is transferred from Pseudoazurin, via the type I copper ion, Cys136, and His135 to the type II copper ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond acceptor
His100A metal ligand
His306B metal ligand
His145A metal ligand
His95A metal ligand
Met150A metal ligand
Cys136A single electron donor
His135A single electron relay
Cys136A single electron relay
His135A single electron donor
Cys136A single electron acceptor
His135A single electron acceptor

Chemical Components

electron transfer

Step 4. His255 rotates upon reduction of the type II copper ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His306B metal ligand
His100A metal ligand
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His135A metal ligand
Cys136A metal ligand

Chemical Components

Step 5. The conserved water deprotonates the rotated His255.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His306B metal ligand
His100A metal ligand
Cys136A metal ligand
His135A metal ligand
His255B proton donor

Chemical Components

proton transfer

Step 6. The nitrous acid substrate collapses to copper bound N=O and water by deprotonating the activated water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His100A metal ligand
His306B metal ligand
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His135A metal ligand
Cys136A metal ligand
Thr280B electrostatic stabiliser
Glu279B (main-C) electrostatic stabiliser

Chemical Components

proton transfer

Step 7. The type II copper ion donates an electron to the NO+ intermediate, forming the final product (NO radical).

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His100A metal ligand
His306B metal ligand
His135A metal ligand
Cys136A metal ligand
Thr280B electrostatic stabiliser
Glu279B (main-C) electrostatic stabiliser

Chemical Components

electron transfer

Step 8. Water displaces the NO product and His255 rotates back to its starting position with reprotonation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His306B metal ligand
His100A metal ligand
Met150A metal ligand
His95A metal ligand
His145A metal ligand
His135A metal ligand
Cys136A metal ligand
His255B proton acceptor

Chemical Components

proton transfer
Download: Image, Marvin File

Step 1. Asp98 is protonated from bulk solvent.

Step 2. The nitrite substrate accepts a proton from Asp98.

Step 3. A single electron is transferred from Pseudoazurin, via the type I copper ion, Cys136, and His135 to the type II copper ion.

Step 4. His255 rotates upon reduction of the type II copper ion.

Step 5. The conserved water deprotonates the rotated His255.

Step 6. The nitrous acid substrate collapses to copper bound N=O and water by deprotonating the activated water molecule.

Step 7. The type II copper ion donates an electron to the NO+ intermediate, forming the final product (NO radical).

Step 8. Water displaces the NO product and His255 rotates back to its starting position with reprotonation.

Products.

Introduction

The resting oxidised state of the enzyme contains a water molecule coordinated to a Cu2+ ion at the type II copper centre. Nitrite then binds to the type II copper ion and and displaces the water molecule. The type II copper now receives an electron from the type I copper and transfers it to the nitrite, which together with protonation of the nitrite leads to reduction of nitrite to NO + H2O.

Two protons are needed for the reduction of NO2- to NO + H2O, but the details of proton transfer have still not been firmly established. Proposed sources for the protons include His 255 (although it has also been proposed that this residue is not positioned appropriately for this role); the water molecule that bridges His 255 and Asp 98; the water molecule initially bound to copper (which is suggested to be deprotonated by Asp 98 prior to its displacement by NO2-, with Asp 98 later supplying the proton to the nitrite); and the nitrite entering as HNO2 with one proton already present.

Catalytic Residues Roles

UniProt PDB* (1nia)
Asp136 Asp98A Activates His255 to act as the general acid/base through a water molecule. activator, hydrogen bond acceptor
His173 His135A Binds the Cu(II) ion in the type II site and is part of the electron transfer pathway from the type I Cu(II) site to the type II Cu(II) site. single electron relay, single electron acceptor, single electron donor, metal ligand
Cys174 Cys136A Binds the Cu(II) ion in the type I site and is part of the electron transfer pathway from the type I Cu(II) site to the type II Cu(II) site. single electron relay, single electron acceptor, single electron donor, metal ligand
His293 His255B General acid/base hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Glu317 (main-C), Thr318 Glu279B (main-C), Thr280B Help modify the pKa of His255. modifies pKa
His183, Met188, His133 His145A, Met150A, His95A Bind the Cu(II) ion in the type I site. metal ligand
His344, His138 His306B, His100A Bind the Cu(II) ion in the type II site. metal ligand
*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

electron transfer, coordination, overall reactant used, coordination to a metal ion, intermediate formation, electron relay, radical formation, redox reaction, elimination (not covered by the Ingold mechanisms), proton transfer, heterolysis, overall product formed, dehydration, decoordination from a metal ion, native state of enzyme regenerated

References

  1. Suzuki S et al. (2000), Acc Chem Res, 33, 728-735. Metal Coordination and Mechanism of Multicopper Nitrite Reductase. DOI:10.1021/ar9900257. PMID:11041837.
  2. Antonyuk SV et al. (2005), Proc Natl Acad Sci U S A, 102, 12041-12046. Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism. DOI:10.1073/pnas.0504207102. PMID:16093314.
  3. Tocheva EI et al. (2004), Science, 304, 867-870. Side-On Copper-Nitrosyl Coordination by Nitrite Reductase. DOI:10.1126/science.1095109. PMID:15131305.
  4. Boulanger MJ et al. (2000), J Biol Chem, 275, 23957-23964. Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase. DOI:10.1074/jbc.m001859200. PMID:10811642.
  5. Inoue T et al. (1999), J Biol Chem, 274, 17845-17852. Crystal Structure Determinations of Oxidized and Reduced Pseudoazurins from Achromobacter cycloclastes: CONCERTED MOVEMENT OF COPPER SITE IN REDOX FORMS WITH THE REARRANGEMENT OF HYDROGEN BOND AT A REMOTE HISTIDINE. DOI:10.1074/jbc.274.25.17845. PMID:10364229.
  6. Adman ET et al. (1995), J Biol Chem, 270, 27458-27474. The Structure of Copper-nitrite Reductase from Achromobacter cycloclastes at Five pH Values, with NO(2)[IMAGE] Bound and with Type II Copper Depleted. DOI:10.1074/jbc.270.46.27458. PMID:7499203.
  7. Kukimoto M et al. (1994), Biochemistry, 33, 5246-5252. X-ray Structure and Site-Directed Mutagenesis of a Nitrite Reductase from Alcaligenes Faecalis S-6: Roles of Two Copper Atoms in Nitrite Reduction. DOI:10.1021/bi00183a030. PMID:8172899.

Step 1. Pseudoazurin donates an electron, through the type I copper ion, Cys136 and His135 to the type II copper ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor, activator
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond donor
Cys136A single electron acceptor
His100A metal ligand
His306B metal ligand
His145A metal ligand
His95A metal ligand
Met150A metal ligand
His135A single electron acceptor
Cys136A single electron donor, single electron relay
His135A single electron relay, single electron donor

Chemical Components

electron transfer, coordination, overall reactant used, coordination to a metal ion, intermediate formation, electron relay

Step 2. Cu502 donates the electron to the bound nitrous acid, which collapses to form water, deprotonating His255B, and a Cu-bound NO radical.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond donor
Glu279B (main-C) modifies pKa
Thr280B modifies pKa
His255B proton donor

Chemical Components

radical formation, redox reaction, elimination (not covered by the Ingold mechanisms), proton transfer, heterolysis, overall product formed, dehydration, decoordination from a metal ion

Step 3. Water displaces the NO radical, and is deprotonated by His255B, to regenerate the enzyme's resting state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp98A hydrogen bond acceptor
His135A metal ligand
Cys136A metal ligand
His255B hydrogen bond acceptor
Glu279B (main-C) modifies pKa
Thr280B modifies pKa
His255B proton acceptor

Chemical Components

proton transfer, overall reactant used, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Pseudoazurin donates an electron, through the type I copper ion, Cys136 and His135 to the type II copper ion.

Step 2. Cu502 donates the electron to the bound nitrous acid, which collapses to form water, deprotonating His255B, and a Cu-bound NO radical.

Step 3. Water displaces the NO radical, and is deprotonated by His255B, to regenerate the enzyme's resting state.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Alex Gutteridge, Craig Porter, Katherine Ferris