NADPH-hemoprotein reductase

 

Rat NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyses the transfer of electrons from NADPH to a range of cytochrome P450 proteins. Other electron acceptors include heme oxygenase, cytochrome b5 and fatty-acid elongase. It is a 78kDa protein and is attached to the cytoplasmic side of the endoplasmic reticulum and the outer membrane of the nuclear envelope by a hydrophobic N-terminal membrane anchor.

The FAD and FMN containing domains are linked by a flexible peptide hinge and it is thought that CYPOR evolved after a fusion of genes encoding for a FMN-containing flavodoxin and a FAD- binding ferredoxin-NADP reductase.

 

Reference Protein and Structure

Sequence
P00388 UniProt (1.6.2.4) IPR023208 (Sequence Homologues) (PDB Homologues)
Biological species
Rattus norvegicus (Norway rat) Uniprot
PDB
1amo - THREE-DIMENSIONAL STRUCTURE OF NADPH-CYTOCHROME P450 REDUCTASE: PROTOTYPE FOR FMN-AND FAD-CONTAINING ENZYMES (2.6 Å) PDBe PDBsum 1amo
Catalytic CATH Domains
2.40.30.10 CATHdb 3.40.50.80 CATHdb (see all for 1amo)
Cofactors
Fadh2(2-) (1), Fmnh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.6.2.4)

NADPH(4-)
CHEBI:57783ChEBI
+
iron(3+)
CHEBI:29034ChEBI
→
NADP(3-)
CHEBI:58349ChEBI
+
iron(2+)
CHEBI:29033ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: CPR, FAD-cytochrome c reductase, NADP--cytochrome c reductase, NADP--cytochrome reductase, NADPH-dependent cytochrome c reductase, NADPH:P-450 reductase, NADPH:ferrihemoprotein oxidoreductase, NADPH--cytochrome P-450 oxidoreductase, NADPH--cytochrome c oxidoreductase, NADPH--cytochrome c reductase, NADPH--cytochrome p-450 reductase, NADPH--ferricytochrome c oxidoreductase, NADPH--ferrihemoprotein reductase, TPNH(2) cytochrome c reductase, TPNH-cytochrome c reductase, Aldehyde reductase (NADPH-dependent), Cytochrome P-450 reductase, Cytochrome c reductase (reduced nicotinamide adenine dinucleotide phosphate, NADPH, NADPH-dependent), Dihydroxynicotinamide adenine dinucleotide phosphate-cytochrome c reductase, Ferrihemoprotein P-450 reductase, Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, Reductase, cytochrome c (reduced nicotinamide adenine dinucleotide phosphate), Cytochrome P450 reductase, NADPH--cytochrome P450 reductase, NADPH--cytochrome P450 oxidoreductase, NADPH:P450 reductase, POR,

Enzyme Mechanism

Introduction

The overall reaction is the oxidation of NADPH to NADP and the reduction of cytochrome P450, or other protein substrate. Both two-electron and four-electron reduced enzyme species have been detected in vitro. It is not yet known if CYPOR is reduced to the two-, three- or four-electron form during catalytic turnover in vivo. However, it has been proposed that the electron transfer leading to the four-electron reduced species is unlikely to have any biological significance.
A hydride ion is transferrred from NADPH to FAD. FAD then transfers electrons to FMN which in turn passes them on to the heme of cytochrome P450. Electrons are transferred from FADH2 one at a time resulting in the formation of FMNH* and FADH* radical intermediates.

Catalytic Residues Roles

UniProt PDB* (1amo)
Ser457 Ser457(394)A Ser457 stabilises the semiquinone form of FAD. Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, proton relay, radical stabiliser
Cys630 Cys630(567)A Cys630 stabilises the transition state carbocation of the nicotinamide ring during the hydride transfer step. Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD. hydrogen bond donor, proton acceptor, proton donor, proton relay, van der waals interaction, electrostatic stabiliser, steric role
Trp677 Trp677(614)A Plays a role in the release of oxidised co-enzyme from the FAD-binding domain (Trp is positioned over the re-face of the FAD isoalloxazine ring). steric role
Asp675 Asp675(612)A Asp675, Ser457 and Cys630 form a hydrogen bond network that may promote proton release from the semiqunione form of FAD. A carboxylate oxygen on Asp675 acts as the hydrogen bond acceptor for hydrogen bonding to Ser457 and Cys630 in the abasence of NADPH. When NADPH is bound Asp675 forms hydrogen bonds to its amide group instead. modifies pKa, hydrogen bond acceptor
*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

aromatic unimolecular elimination by the conjugate base, hydride transfer, aromatic bimolecular nucleophilic addition, proton transfer, overall reactant used, cofactor used, overall product formed, intermediate formation, electron transfer, radical formation, radical termination, native state of cofactor regenerated, intermediate terminated, proton relay, redox reaction, native state of enzyme regenerated

References

  1. Hubbard PA et al. (2001), J Biol Chem, 276, 29163-29170. NADPH-Cytochrome P450 Oxidoreductase: STRUCTURAL BASIS FOR HYDRIDE AND ELECTRON TRANSFER. DOI:10.1074/jbc.m101731200. PMID:11371558.
  2. Rwere F et al. (2016), J Biol Chem, 291, 14639-14661. Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone. DOI:10.1074/jbc.m116.724625. PMID:27189945.
  3. Hedison TM et al. (2015), FEBS J, 282, 4357-4375. Real-time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochromec. DOI:10.1111/febs.13501. PMID:26307151.
  4. Meints CE et al. (2013), FEBS J, 280, 1460-1474. Aromatic substitution of the FAD-shielding tryptophan reveals its differential role in regulating electron flux in methionine synthase reductase and cytochrome P450 reductase. DOI:10.1111/febs.12141. PMID:23332101.
  5. Iyanagi T et al. (2012), Arch Biochem Biophys, 528, 72-89. NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family. DOI:10.1016/j.abb.2012.09.002. PMID:22982532.
  6. Xia C et al. (2011), J Biol Chem, 286, 16246-16260. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding. DOI:10.1074/jbc.m111.230532. PMID:21345800.
  7. Aigrain L et al. (2011), Biochem J, 435, 197-206. Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH–cytochrome P450 reductase. DOI:10.1042/bj20101984. PMID:21265736.
  8. Hamdane D et al. (2009), J Biol Chem, 284, 11374-11384. Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450. DOI:10.1074/jbc.m807868200. PMID:19171935.
  9. Brenner S et al. (2008), FEBS J, 275, 4540-4557. Inter-flavin electron transfer in cytochrome P450 reductase - effects of solvent and pH identify hidden complexity in mechanism. DOI:10.1111/j.1742-4658.2008.06597.x. PMID:18681889.
  10. Gutierrez A et al. (2003), Biochem Soc Trans, 31, 497-501. Electron transfer in human cytochrome P450 reductase. PMID:12773143.
  11. Wang M et al. (1997), Proc Natl Acad Sci U S A, 94, 8411-8416. Three-dimensional structure of NADPH-cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes. DOI:10.1073/pnas.94.16.8411. PMID:9237990.

Step 1. NADP eliminates a hydride ion, which adds to FAD, with concomitant deprotonation of water at the FAD N1. Cys630 is partially responsible for the proper orientation of the nicotinamide ring by making a van der Waals contact with the C4 atom and promoting hydride transfer by stabilising the transient carbocation formed upon release of the hydride ion. The nicotinamide ring of NADP+ is displaced by Trp677.

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Catalytic Residues Roles

Residue Roles
Ser457(394)A hydrogen bond acceptor
Asp675(612)A hydrogen bond acceptor
Cys630(567)A van der waals interaction, steric role, electrostatic stabiliser
Trp677(614)A steric role

Chemical Components

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

Step 2. Water deprotonates the N1, initiating a single electron transfer from FAD to FMN, with concomitant deprotonation of water at the FMN N5.

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Catalytic Residues Roles

Residue Roles
Trp677(614)A steric role
Ser457(394)A hydrogen bond acceptor, hydrogen bond donor
Asp675(612)A hydrogen bond acceptor
Cys630(567)A hydrogen bond donor

Chemical Components

proton transfer, electron transfer, radical formation, cofactor used, intermediate formation

Step 3. Water deprotonates Cys630, which deprotonates Ser457 which deprotonates FAD facilitating the second single electron transfer from FAD to FMN. Ser457 stabilises the semiquinone form of FAD. It has also been proposed that Ser457, in conjunction with Asp675 and Cys630, forms a hydrogen bond network that may promote proton release from the semiquinone [PMID:11371558]. The identity of the species accepting this proton is unclear, and we assume water fulfils this role.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp675(612)A modifies pKa
Ser457(394)A hydrogen bond acceptor, hydrogen bond donor, proton relay, radical stabiliser
Asp675(612)A hydrogen bond acceptor
Cys630(567)A hydrogen bond donor, proton relay
Cys630(567)A proton acceptor, proton donor
Ser457(394)A proton donor, proton acceptor

Chemical Components

proton transfer, electron transfer, radical termination, native state of cofactor regenerated, intermediate terminated, intermediate formation, overall product formed, proton relay

Step 4. FMN transfers a single electron to the hemoprotein.

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Catalytic Residues Roles

Residue Roles
Ser457(394)A hydrogen bond donor
Asp675(612)A hydrogen bond acceptor
Cys630(567)A hydrogen bond donor

Chemical Components

redox reaction, radical formation, overall reactant used, intermediate formation, electron transfer

Step 5. Water deprotonates the FMN, facilitating the transfer of the second single electron to the hemoprotein.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser457(394)A hydrogen bond donor
Asp675(612)A hydrogen bond acceptor
Cys630(567)A hydrogen bond donor

Chemical Components

proton transfer, electron transfer, radical termination, intermediate terminated, overall product formed, native state of cofactor regenerated, native state of enzyme regenerated, redox reaction
Download: Image, Marvin File

Step 1. NADP eliminates a hydride ion, which adds to FAD, with concomitant deprotonation of water at the FAD N1. Cys630 is partially responsible for the proper orientation of the nicotinamide ring by making a van der Waals contact with the C4 atom and promoting hydride transfer by stabilising the transient carbocation formed upon release of the hydride ion. The nicotinamide ring of NADP+ is displaced by Trp677.

Step 2. Water deprotonates the N1, initiating a single electron transfer from FAD to FMN, with concomitant deprotonation of water at the FMN N5.

Step 3. Water deprotonates Cys630, which deprotonates Ser457 which deprotonates FAD facilitating the second single electron transfer from FAD to FMN. Ser457 stabilises the semiquinone form of FAD. It has also been proposed that Ser457, in conjunction with Asp675 and Cys630, forms a hydrogen bond network that may promote proton release from the semiquinone [PMID:11371558]. The identity of the species accepting this proton is unclear, and we assume water fulfils this role.

Step 4. FMN transfers a single electron to the hemoprotein.

Step 5. Water deprotonates the FMN, facilitating the transfer of the second single electron to the hemoprotein.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid