Nitrate reductase

 

Nitrate reductase is a molybdenum-bis(molybdopterin guanine dinucleotide) dependent enzyme. It catalyses the reduction of nitrate to nitrite and water. The protein associates with NapB, a soluble haem-containing protein and NapC, a membrane-bound cytochrome c. The periplasmic nitrate reductases are not involved in the assimilation of nitrogen, and are not directly involved in the formation of electrochemical gradients (i.e. respiration) either. Rather, the purpose of this enzyme is either dissimilatory (i.e. to dispose of excess reductive equivalents) or indirectly respiratory by virtue of the consumption of electrons derived from NADH via the proton translocating NADH dehydrogenase.

 

Reference Protein and Structure

Sequence
Q53176 UniProt (1.9.6.1) IPR010051 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodobacter sphaeroides 2.4.1 (Bacteria) Uniprot
PDB
1ogy - Crystal structure of the heterodimeric nitrate reductase from Rhodobacter sphaeroides (3.2 Å) PDBe PDBsum 1ogy
Catalytic CATH Domains
3.40.228.10 CATHdb 3.30.200.210 CATHdb (see all for 1ogy)
Cofactors
Tetra-mu3-sulfido-tetrairon (1), Molybdopterin guanine dinucleotide (2), Molybdenum(6+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:1.7.99.4)

nitrate
CHEBI:17632ChEBI
+
iron(2+)
CHEBI:29033ChEBI
+
hydron
CHEBI:15378ChEBI
→
water
CHEBI:15377ChEBI
+
nitrite
CHEBI:16301ChEBI
+
iron(3+)
CHEBI:29034ChEBI
Alternative enzyme names: Nitrate reductase (acceptor), Respiratory nitrate reductase, Nitrite:(acceptor) oxidoreductase,

Enzyme Mechanism

Introduction

The nitrate oxyanion attacks the molybdenum coordination in a displacement reaction, eliminating the Cys152 sulfur which results in the formation of a disulfide bridge within the coordination sphere. The nitrate ligand undergoes heterolytic dissociation from molybdenum to form the nitrite product. The oxygen ligand is left behind at the metal centre. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue. A solvent molecule acts as a proton donor to the dianionic molybdenum centre. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue. A solvent molecule acts as a proton donor, forming a water ligand within the metal coordination sphere. The Cys152 sulfur atom substitutes the water molecule coordinated to the molybdenum centre.

Catalytic Residues Roles

UniProt PDB* (1ogy)
Cys181 Cys152E Forms part of the molybdenum coordination sphere. covalently attached, nucleofuge, metal ligand, nucleophile, activator, electrostatic stabiliser
Lys85 Lys56E Acts as a single electron relay. single electron relay, hydrogen bond donor, activator, single electron donor, single electron acceptor
Gly412 (main-N), Gln413 Gly383E (main-N), Gln384E Help stabilise the reactive intermediates formed during the course of the reaction. hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Met375, Met182 Met346E, Met153E Help stabilise the reactive intermediates and also ensure that all the substrates are correctly positioned for the reaction to occur. The conformational change adopted by the coordination sphere of the molybdenum ion on addition of the nitrate molecule is stabilised by the steric bulk and electronic charge delocalisation resulting from the position of Met308 and Met141 [PMID:10368307]. steric role, electrostatic stabiliser, polar/non-polar interaction
*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

bimolecular nucleophilic substitution, coordination to a metal ion, intermediate formation, decoordination from a metal ion, overall reactant used, cofactor used, redox reaction, intramolecular elimination, overall product formed, electron transfer, native state of cofactor regenerated, electron relay, proton transfer, intramolecular nucleophilic substitution, native state of enzyme regenerated, intermediate terminated

References

  1. Romão MJ (2009), Dalton Trans, 4053-4068. Molybdenum and tungsten enzymes: a crystallographic and mechanistic overview. DOI:10.1039/b821108f. PMID:19452052.
  2. Coelho C et al. (2015), Protein Sci, 24, 1901-1911. Structural and mechanistic insights on nitrate reductases. DOI:10.1002/pro.2801. PMID:26362109.
  3. Sparacino-Watkins C et al. (2014), Chem Soc Rev, 43, 676-706. Nitrate and periplasmic nitrate reductases. DOI:10.1039/c3cs60249d. PMID:24141308.
  4. Biaso F et al. (2012), Inorg Chem, 51, 3409-3419. DFT investigation of the molybdenum cofactor in periplasmic nitrate reductases: structure of the Mo(V) EPR-active species. DOI:10.1021/ic201533p. PMID:22397692.
  5. Cerqueira NM et al. (2009), J Comput Chem, 30, 2466-2484. The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase. DOI:10.1002/jcc.21280. PMID:19360810.
  6. Dias JM et al. (1999), Structure, 7, 65-79. Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods. DOI:10.1016/s0969-2126(99)80010-0. PMID:10368307.

Step 1. The nitrate oxyanion attacks the molybdenum coordination in a displacement reaction, eliminating the Cys152 sulfur which results in the formation of a disulfide bridge within the coordination sphere. Physiological conditions allow an equilibrium between Mo(V) and Mo(VI) to exist [PMID:19360810].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E hydrogen bond donor
Cys152E activator, covalently attached
Gly383E (main-N) electrostatic stabiliser, hydrogen bond acceptor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role
Gln384E electrostatic stabiliser, hydrogen bond acceptor
Cys152E nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, coordination to a metal ion, intermediate formation, decoordination from a metal ion, overall reactant used, cofactor used

Step 2. The nitrate ligand undergoes heterolytic dissociation from molybdenum to form the nitrite product. The oxygen ligand is left behind at the metal centre. Theoretical studies have shown an inner sphere mechanism to be more favourable than a potential second sphere alternative [PMID:19360810].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E hydrogen bond donor
Cys152E activator, electrostatic stabiliser, covalently attached
Gly383E (main-N) hydrogen bond acceptor, electrostatic stabiliser
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role
Gln384E hydrogen bond acceptor, electrostatic stabiliser

Chemical Components

redox reaction, ingold: intramolecular elimination, intermediate formation, overall product formed

Step 3. The electron is relayed to the iron-sulfur cluster from a mobile electron carrier present in the quinol oxidation respiratory electron transport chain. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E activator, hydrogen bond donor
Cys152E activator, metal ligand, covalently attached
Gly383E (main-N) hydrogen bond donor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E single electron relay, single electron donor, single electron acceptor

Chemical Components

electron transfer, cofactor used, native state of cofactor regenerated, intermediate formation, electron relay, overall reactant used

Step 4. A solvent molecule acts as a proton donor to the dianionic molybdenum centre. The proton donor is presumed to be a solvent molecule because of the identification of solvent channels close to the metal coordination site. However, the possibility of a residue acting as a general acid has not been ruled out [PMID:10368307].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E hydrogen bond donor
Cys152E activator, electrostatic stabiliser, metal ligand, covalently attached
Gly383E (main-N) hydrogen bond donor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role

Chemical Components

proton transfer, intermediate formation, overall reactant used

Step 5. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue. The electron is relayed to the iron-sulfur cluster from a mobile electron carrier present in the quinol oxidation respiratory electron transport chain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E activator, hydrogen bond donor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E single electron relay, single electron donor, single electron acceptor

Chemical Components

electron transfer, cofactor used, native state of cofactor regenerated, electron relay, intermediate formation

Step 6. A solvent molecule acts as a proton donor, forming a water ligand within the metal coordination sphere. The proton donor is presumed to be a solvent molecule because of the identification of solvent channels close to the metal coordination site. However, the possibility of a residue acting as a general acid has not been ruled out [PMID:10368307].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E hydrogen bond donor
Cys152E activator, electrostatic stabiliser, covalently attached
Gly383E (main-N) hydrogen bond donor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 7. The Cys152 sulfur atom substitutes the water molecule coordinated to the molybdenum centre. The lone pair of electrons on the bridging sulfur substitutes the coordinated water. Both sulfur atoms of the disulfide bond are coordinated to the metal. In the absence of nitrate, the metal coordination sphere returns to its ground state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Met153E electrostatic stabiliser, polar/non-polar interaction, steric role
Lys56E hydrogen bond donor
Cys152E activator, covalently attached, electrostatic stabiliser, metal ligand
Gly383E (main-N) hydrogen bond acceptor
Met346E electrostatic stabiliser, polar/non-polar interaction, steric role
Cys152E nucleophile

Chemical Components

ingold: intramolecular nucleophilic substitution, native state of cofactor regenerated, overall product formed, native state of enzyme regenerated, intermediate terminated
Download: Image, Marvin File

Step 1. The nitrate oxyanion attacks the molybdenum coordination in a displacement reaction, eliminating the Cys152 sulfur which results in the formation of a disulfide bridge within the coordination sphere. Physiological conditions allow an equilibrium between Mo(V) and Mo(VI) to exist [PMID:19360810].

Step 2. The nitrate ligand undergoes heterolytic dissociation from molybdenum to form the nitrite product. The oxygen ligand is left behind at the metal centre. Theoretical studies have shown an inner sphere mechanism to be more favourable than a potential second sphere alternative [PMID:19360810].

Step 3. The electron is relayed to the iron-sulfur cluster from a mobile electron carrier present in the quinol oxidation respiratory electron transport chain. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue.

Step 4. A solvent molecule acts as a proton donor to the dianionic molybdenum centre. The proton donor is presumed to be a solvent molecule because of the identification of solvent channels close to the metal coordination site. However, the possibility of a residue acting as a general acid has not been ruled out [PMID:10368307].

Step 5. The close proximity iron sulfur cluster provides an electron to the molybdenum centre via a bridging lysine residue. The electron is relayed to the iron-sulfur cluster from a mobile electron carrier present in the quinol oxidation respiratory electron transport chain.

Step 6. A solvent molecule acts as a proton donor, forming a water ligand within the metal coordination sphere. The proton donor is presumed to be a solvent molecule because of the identification of solvent channels close to the metal coordination site. However, the possibility of a residue acting as a general acid has not been ruled out [PMID:10368307].

Step 7. The Cys152 sulfur atom substitutes the water molecule coordinated to the molybdenum centre. The lone pair of electrons on the bridging sulfur substitutes the coordinated water. Both sulfur atoms of the disulfide bond are coordinated to the metal. In the absence of nitrate, the metal coordination sphere returns to its ground state.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday