NAD(P)H dehydrogenase (quinone)

 

Quinone reductase exists as a dimer of identical subunits, each comprising of 273 amino acids with two identical catalytic sites at equivalent positions. The enzyme catalyses the FAD dependent reduction of quinones. The dimer binds two FAD cofactors which remain non-covalently bound during catalysis. NAD(P)H and NAD(P)+ cycle in and out of the catalytic site. The catalytic domain has the characteristic twisted central parallel beta sheet surrounded on both sides by connecting helices, as found in other flavoproteins.

 

Reference Protein and Structure

Sequence
P15559 UniProt (1.6.5.2) IPR003680 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1d4a - CRYSTAL STRUCTURE OF HUMAN NAD[P]H-QUINONE OXIDOREDUCTASE AT 1.7 A RESOLUTION (1.7 Å) PDBe PDBsum 1d4a
Catalytic CATH Domains
3.40.50.360 CATHdb (see all for 1d4a)
Cofactors
Fadh2(2-) (1), 1,4-benzoquinone (1)
Click To Show Structure

Enzyme Reaction (EC:1.6.5.2)

NADH(2-)
CHEBI:57945ChEBI
+
1,4-benzoquinones
CHEBI:132124ChEBI
+
hydron
CHEBI:15378ChEBI
→
NAD(1-)
CHEBI:57540ChEBI
+
hydroquinones
CHEBI:24646ChEBI
Alternative enzyme names: p-benzoquinone reductase, DT-diaphorase, NAD(P)H dehydrogenase, NAD(P)H menadione reductase, NAD(P)H-quinone dehydrogenase, NAD(P)H-quinone oxidoreductase, NAD(P)H:(quinone-acceptor)oxidoreductase, NAD(P)H: menadione oxidoreductase, NADH-menadione reductase, Dehydrogenase, reduced nicotinamide adenine dinucleotide (phosphate, quinone), Diaphorase, Flavoprotein NAD(P)H-quinone reductase, Menadione oxidoreductase, Menadione reductase, Naphthoquinone reductase, Phylloquinone reductase, Quinone reductase, Reduced NAD(P)H dehydrogenase, Reduced nicotinamide-adenine dinucleotide (phosphate) dehydrogenase, Viologen accepting pyridine nucleotide oxidoreductase, Vitamin K reductase, Vitamin-K reductase, Azoreductase, NAD(P)H(2) dehydrogenase (quinone), NQO1, QR1,

Enzyme Mechanism

Introduction

The obligatory two-electron reduction is shown here to proceed via two direct hydride transfers.

The mechanism has two distinct steps. Firstly, hydride transfer occurs from NAD(P)H to the enzyme-bound flavin followed by the release of NAD(P)+. Secondly, a hydride transfer takes place from the flavin to the quionone followed by the releases of hydroquinone. The mechanism follows a 'ping-pong' kinetic scheme. The binding of the quinone substrate cannot occur until NAD(P)+ is released because the nictotinamide and quninone share the same binding site.

This enzyme features two independent, equivalent active sites which are located at opposite ends of the dimer interface. Residues from both monomers line the large active sites which extend from the protein surface to the isoalloxazine rings of the FAD cofactors [PMID:11035252].

Catalytic Residues Roles

UniProt PDB* (1d4a)
Gly150 (main-N) Gly149A (main-N) The residue hydrogen bonds to the N1 of the flavin cofactor, stabilising the enolate anion through charge delocalisation. hydrogen bond donor, electrostatic stabiliser
Tyr156 Tyr155A The residue hydrogen bonds to the flavin enolate oxygen. It acts as a proton donor to the anion, with concurrent abstraction of a proton from the close proximity His161. This acquired proton is then removed by His161 in the second reaction step, leading to a hydride transfer from the flavin cofactor to the quinone substrate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
His162 His161A The residue acts as a general acid towards Tyr155 in the first reaction step, and then as a general base towards the same Tyr155 in the second reaction step. This initiates the hydride transfer from the flavin cofactor to the quinone substrate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay
*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

hydride transfer, aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, overall product formed, intermediate formation, proton transfer, proton relay, aromatic bimolecular elimination, native state of cofactor regenerated, native state of enzyme regenerated, native state of cofactor is not regenerated

References

  1. Foster CE et al. (2000), Free Radic Biol Med, 29, 241-245. Structures of mammalian cytosolic quinone reductases. DOI:10.1016/s0891-5849(00)00299-9. PMID:11035252.
  2. Cavelier G et al. (2001), Proteins, 43, 420-432. Mechanism of NAD(P)H:Quinone reductase: Ab initio studies of reduced flavin. DOI:10.1002/prot.1055.abs. PMID:11340659.
  3. Li R et al. (1995), Proc Natl Acad Sci U S A, 92, 8846-8850. The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. DOI:10.1073/pnas.92.19.8846. PMID:7568029.

Step 1. A hydride ion is transferred from NADH to the FAD cofactor, with concomitant double bond rearrangement.

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Catalytic Residues Roles

Residue Roles
Gly149A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr155A hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
His161A hydrogen bond donor

Chemical Components

hydride transfer, ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, overall product formed, intermediate formation

Step 2. The FADH deprotonates Tyr155, which in turn deprotonates His161.

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Catalytic Residues Roles

Residue Roles
His161A hydrogen bond donor
Gly149A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr155A proton relay, hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Tyr155A proton acceptor
His161A proton donor
Tyr155A proton donor

Chemical Components

proton transfer, proton relay, intermediate formation

Step 3. NAD+ is released, followed by binding of the quinine. His161 deprotonates Tyr155, which in turn deprotonates the FADH, resulting in double bond rearrangement and the elimination of a hydride to the quinone.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His161A hydrogen bond acceptor
Gly149A (main-N) hydrogen bond donor, electrostatic stabiliser
Tyr155A proton relay, hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
His161A proton acceptor
Tyr155A proton acceptor, proton donor

Chemical Components

proton transfer, hydride transfer, ingold: aromatic bimolecular elimination, ingold: aromatic bimolecular nucleophilic addition, proton relay, intermediate formation, cofactor used, native state of cofactor regenerated

Step 4. The negatively charged substrate deprotonates His161, which is rapidly re-protonated from the solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His161A hydrogen bond donor, proton relay
Tyr155A hydrogen bond acceptor, hydrogen bond donor
Gly149A (main-N) hydrogen bond donor
His161A proton acceptor, proton donor

Chemical Components

proton transfer, overall reactant used, overall product formed, native state of enzyme regenerated, proton relay, native state of cofactor is not regenerated
Download: Image, Marvin File

Step 1. A hydride ion is transferred from NADH to the FAD cofactor, with concomitant double bond rearrangement.

Step 2. The FADH deprotonates Tyr155, which in turn deprotonates His161.

Step 3. NAD+ is released, followed by binding of the quinine. His161 deprotonates Tyr155, which in turn deprotonates the FADH, resulting in double bond rearrangement and the elimination of a hydride to the quinone.

Step 4. The negatively charged substrate deprotonates His161, which is rapidly re-protonated from the solvent.

Products.

Contributors

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