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ENZYMOLOGY OF CYTOCHROME P450 REDUCTASE

 

Flavins and Electron Transfer

The first studies on cytochrome P450 reductase (CPR) identified it as a flavoprotein,1 although it was not recognized until the studies of Iyanagi and Mason in 1973 that the enzyme contains both FAD and FMN in equal proportions.2  The role of the flavins in electron flow through the enzyme was elucidated in a series of studies from Coon and associates3 based on the redox midpoint potentials determined by Iyanagi et al.4  These studies established that FAD is the electron acceptor flavin from NADPH and that FMN is the electron donor to acceptor proteins such as the cytochromes:

 CPR electron flow

Under physiologic conditions the enzyme was proposed to cycle between the 1- and 3-electron reduced states, as shown below:

redox cycling: mammalian CPR and SR

The redox cycle of the reductase from Bacillus megaterium P450BM3 and from the housefly appears to differ from that shown above; these reductases cycle between the fully oxidized enzyme and the 2-electron reduced form:5

Redox cycling: BM3 and housefly CPR

A noticeable difference between these two schemes is the redox state of the FMN cofactor during catalysis: With the mammalian enzyme the fully reduced flavin (FMNH2) is the electron donor to P450 (or other electron acceptor protein), whereas with the P450 BM3 reductase the 1-electron reduced semiquinone (FMNH) is the donor.  These differences appear to be related to the flavin midpoint potentials of these enzymes: the table and graph below show the midpoint potentials for mammalian CPR,4 P450BM3 reductase (BM3),6 bacterial sulfite reductase (SR),7 and neuronal nitric oxide synthase (nNOS)8 (the reductase components of P450BM3, SR, and nNOS are homologous with CPR).9  

Redox Midpoint Potentials for Reductase Flavins
Flavin couple

Em7 (mV)

CPR BM3 SR nNOS
FMN/FMNH -110 -213 -152 -49
FMNH/FMNH2 -270 -193 -327 -274
FAD/FADH -290 -292 -382 -232
FADH/FADH2 -365 -372 -322 -280

The electron donor species appears to be the more electronegative of the two FMN couples.  This makes sense from a thermodynamic viewpoint, where the more electronegative species provides a stronger driving force for electron transfer to acceptor proteins.  Thus FMNH2 is the more likely donor for rabbit CPR, as its redox midpoint potential is significantly more negative than that of the semiquinone (-270 mV vs -110 mV).  While this is also true for SR and nNOS, it is not true for the BM3 reductase, where FMNH2 has a slightly higher potential than the semiquinone.  Consistent with this reversal of potentials, it is the lower potential semiquinone (FMNH) in the BM3 reductase that donates electrons to P450 (as noted above).5  Indeed, the FMN hydroquinone (FMNH2) is reported to be catalytically inactive in the BM3 reductase, and it appears from spectroscopic and EPR studies that nicotinamide cofactor binding alters the redox potentials of the BM3 flavins such that the disemiquinoid form of the enzyme is favored, thereby avoiding formation of FMNH2.10  Kinetics studies suggest that this is probably true for the housefly reductase as well.10  It is not clear why the FMN hydroquinone is inactive, but under normal circumstances the tight binding of NADP+ following hydride transfer would prevent its formation.

Flavin midpoint potentials for CPR enzymesThe resting state of these enzymes is also determined by the FMN midpoint potentials, but here it is the more electropositive species that is preferred.  Thus, with CPR, SR, and nNOS the 1-electron reduced FMN semiquinone is thermodynamically preferred over the 2-electron reduced form, and these reductases cycle between the 1- and 3-electron reduced states (as noted above).  Although the 2-electron reduced FMN hydroquinone would appear to be preferred with P450 BM3, this redox form is catalytically inactive, and thus to be avoided; as noted above, nicotinamide cofactor binding may prevent formation of this redox form in favor of the disemiquinoid (FMNH/FADH), allowing the enzyme to cycle between the oxidized and 2-electron reduced states.10  These redox differences between forms of this enzyme from different species may ultimately be resolved as evolutionary adaptations to different intracellular environments and functions; the protein factors that dictate the different redox potentials of these enzymes have yet to be explored. 

 

NADPH Binding

Cofactor Specificity12

  Enzyme Km
NAD(H)
(M)
Km
NADP(H)
(M)
Ratio*
CPR 11600 2.7 4300
GR 2000 21.7 90
DHFR 320 6.5 49
FNR 3770 7.2 520
NR 3000 13 200
GR, glutathione reductase; DHFR, dihydrofolate reductase; FNR, ferredoxin NADP+ reductase; NR, nitrate reductase

 

CPR has an unusually high specificity for NADPH (vs NADH), as shown in the accompanying table.  These two cofactors differ only in the presence of a 2' phosphate instead of a hydroxyl group (OH) on the adenine ribose of NADPH, and thus this moiety must provide the target for cofactor discrimination.  Chemical modification studies have indicated that positively charged amino acids (lysines and arginines) interact with this negatively charged phosphate group,11 and, in support of this hypothesis, Sem and Kasper NADPHhave shown that replacing an arginine at position 597 with an uncharged amino acid significantly increases the the Km for NADPH without affecting the Km for NADH.12  Several other amino acids in this segment of CPR are also likely to be involved in binding the 2' phosphate group, based on the structure and sequence of ferredoxin NADP+ reductase (FNR), a homologous protein.** 13  An alignment of the 2' phosphate binding segment of FNR with the corresponding segments of CPR, NADPH-specific nitrate reductase (NR) and two homologous NADH-specific enzymes, NR from corn and cytochrome b5 reductase (b5R) are shown below:

CPR        594  A F S R E Q A H . . . . K V Y V Q
                |   | | | |             |   |   |
FNR        232  A I S R E Q Q N P E G G K M Y I Q
                    | |           | |   :      
NR (NADPH) 918  T L S R P G A E W E G L R G R L D
                                  | |         
NR (NADH)  203  V I D Q V K R P E E G . W K Y S V
                    |             |     |   |   
b5R        212  T L D R A . . . P E A . W D Y G Q

Relevant single letter amino acid code: S, serine; R, arginine; D, aspartate; K, lysine; Y, tyrosine; W, tryptophan; Q, glutamine.
Sequences are derived from: CPR, rat; FNR, spinach; NR (NADPH), Neurospora crassa; NR (NADH), corn plant; b5R, human.

 

The highlighted amino acids were identified in the FNR structure as interacting with the 2' phosphate of NADP+,13 and the arginine mutated by Sem and Kasper in CPR is indicated in red.  The amino acids highlighted in blue in the NADH-specific enzymes differ consistently from the corresponding positions in NADPH-specific enzymes.  Indeed, substitution of aspartate for the serine at position 920 of NADPH-specific NR (also shown in red) reduced the NADPH-preference ratio from 231 to 1.6, indicating that an acidic residue (aspartate) at this position (as is found in the NADH-specific reductases) opposes the binding of the negatively charged 2' phosphate group of NADPH.15  Current studies in my laboratory are directed at identifying the specific amino acids in CPR that are responsible for its strong preference for NADPH.

The penultimate amino acid in rat CPR, Trp677, plays an important role in controlling NADP(H) binding and hydride transfer to FAD.16  This aromatic residue lies beneath the FAD molecule (re face), shielding the isoalloxazine ring from solvent.  Upon binding of NADPH the Trp side chain is believed to rotate away and allow the nicotinamide ring to slide under the FAD isoalloxazine ring for hydride transfer.  Removal of this amino acid (and some substitutions) have several remarkable effects: dissociation of NADP+ following hydride transfer is greatly impeded (and thus turnover is reduced), and the Km for both NADPH and NADH is greatly decreased.  Thus, nicotinamide cofactors appear to bind much more tightly upon removal of the shielding Trp side-chain; a remarkable result of this increase in affinity is that an alanine mutant at this position has a kcat with NADH that is nearly as high as that with NADPH in the wild-type enzyme!17  Thus CPR does not discriminate against NADH, but rather selects for the 2'-phosphate group of NADPH, while the shielding Trp residue opposes binding by both cofactors.  In the native enzyme only NADPH binds tightly enough to displace the Trp residue to allow hydride transfer; once the flavin is reduced Trp677 promotes the dissociation of NADP+.  


* In this table 'ratio' = Km(NADH)/Km(NADPH) for each enzyme.  Km is an approximate measure of the affinity of the enzyme for the substrate, with the higher Km's indicating lesser affinity (Km is the substrate concentration necessary to achieve 50% of maximal velocity of catalysis).  Thus a high ratio indicates strong preference for NADPH over NADH.

** Although the 3-dimensional structure of CPR has been determined, the specific amino acids that bind the 2' phosphate of NADP(H) were not identified.14

 

Electron Transfer to Cytochrome P450

P450/CPR charge pairingThe interaction of CPR with cytochrome P450 and other electron acceptor proteins is based primarily on electrostatic charge pairing, although there is evidence for an additional  hydrophobic component.18  Chemical cross-linking and modification studies have shown that CPR contains multiple carboxylate groups, presumably contributed by the acidic amino acids aspartate and glutamate.19  These charge groups pair with basic amino acids (lysines, arginines) on the various electron acceptor proteins.  In addition, cytochrome P450 forms a dipole across the molecule, with the positive charge at the proximal face of the protein where the heme makes its closest approach to the surface.20  This is thought to be the surface most suitable for electron transfer from CPR.  While electrostatic forces may serve to connect and orient the pair, hydrophobic forces contributed by nonpolar amino acids (leucine, tryptophan, valine, and others) may be responsible for bringing the two proteins close enough together for electron transfer.18  Other electron acceptor proteins, such as cytochrome b5, heme oxygenase, and squalene monooxygenase probably interact by the same mechanism.21 

Site-directed mutagenesis studies have identified two clusters of acidic amino acids in the FMN domain of CPR (consistent with its role as the electron donor flavin) that, upon mutation to nonacidic amino acids, disrupt the interaction with cytochrome P450 and cytochrome c.22  Mutations differentially affect the interaction with these two proteins, suggesting that P450 and cytochrome c bind in different manners.  Studies with cytochrome b5 indicate that its binding site on CPR is also distinct from that of P450;18 it may be that each redox partner has a slightly different interaction with CPR. 

The 3-dimensional structure of the reductase FMN domain-P450 BM3 complex (shown to the right) supports the above model for interaction of these proteins.23  Electrostatic interactions play a lesser role with this complex, in part because P450 BM3 and reductase are physically linked on the same polypeptide, rather than separate proteins as in most other systems.  In this complex the dimethylbenzene ring of the FMN is oriented perpendicular to the heme plane and the two are separated by 18 and an intervening heme-binding peptide loop.  Electron transfer is proposed to occur through this peptide pathway.  Because the dimethybenzene ring faces the FAD molecule in the CPR structure and is not accessible from the surface,14 this model suggests that the FMN domain must swing about, as on a hinge, in order to pass electrons to P450 and other acceptor proteins. 

 

References
  1. Horecker BL.  Triphosphopyridine nucleotide-cytochrome c reductase in liver.  J Biol Chem 1950, 183:593-605.  Williams CH Jr, Kamin H.  Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver.  J Biol Chem 1962, 237:587-95.  Phillips AH, Langdon RG.  Hepatic triphosphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies.  J Biol Chem 1962, 237:2652-60
  2. Iyanagi T, Mason HS.  Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase.  Biochemistry 1973 Jun 5;12(12):2297-308
  3. Vermilion JL, Coon MJ.  Identification of the high and low potential flavins of liver microsomal NADPH-cytochrome P-450 reductase.  J Biol Chem 1978 Dec 25;253(24):8812-9.  Vermilion JL, Ballou DP, Massey V, Coon MJ.  Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase.  J Biol Chem 1981 Jan 10;256(1):266-77.  Oprian DD, Coon MJ.  Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH.  J Biol Chem 1982 Aug 10;257(15):8935-44
  4. Iyanagi T, Makino N, Mason HS.  Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases.  Biochemistry 1974 Apr 9;13(8):1701-10.  Munro AW, Noble MA, Robledo L, Daff SN, Chapman SK.  Determination of the redox properties of human NADPH-cytochrome P450 reductase.  Biochemistry 2001 Feb 20;40(7):1956-63 [abstract].
  5. Sevrioukova I, Shaffer C, Ballou DP, Peterson JA.  Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3.  Biochemistry 1996 Jun 4;35(22):7058-68 [abstract].  Murataliev MB, Klein M, Fulco A, Feyereisen R.  Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain.  Biochemistry 1997 Jul 8;36(27):8401-12 [abstract].  
  6. Daff SN, Chapman SK, Turner KL, Holt RA, Govindaraj S, Poulos TL, Munro AW.  Redox control of the catalytic cycle of flavocytochrome P-450 BM3.  Biochemistry 1997 Nov 11;36(45):13816-23 [abstract]
  7. Ostrowski J, Barber MJ, Rueger DC, Miller BE, Siegel LM, Kredich NM.  Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.  J Biol Chem 1989 Sep 25;264(27):15796-808 [abstract]
  8. Noble MA, Munro AW, Rivers SL, Robledo L, Daff SN, Yellowlees LJ, Shimizu T, Sagami I, Guillemette JG, Chapman SK. Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase.  Biochemistry 1999 Dec 14;38(50):16413-8 [abstract]
  9. Porter TD, Kasper CB.  NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins.  Biochemistry 1986 Apr 8;25(7):1682-7 [abstract].  Porter TD.  An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals.  Trends Biochem Sci 1991 Apr;16(4):154-8 [abstract]  Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH.  Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.  Nature 1991 Jun 27;351(6329):714-8 [abstract]. 
  10. Murataliev MB, Feyereisen R.  Mechanism of cytochrome P450 reductase from the house fly: evidence for an FMN semiquinone as electron donor.  FEBS Lett 1999 Jun 18;453(1-2):201-4 [abstract].  Murataliev MB, Feyereisen R.  Functional interactions in cytochrome P450BM3. Evidence that NADP(H) binding controls redox potentials of the flavin cofactors.  Biochemistry 2000 Oct 17;39(41):12699-707 [abstract].
  11. Inano H, Tamaoki B.  Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor.  Eur J Biochem 1986 Mar 17;155(3):485-9 [abstract].  Slepneva IA, Weiner LM.  Affinity modification of NADPH-cytochrome P-450 reductase.  Biochem Biophys Res Commun 1988 Sep 15;155(2):1026-32 [abstract].  Muller K, Linder D, Lumper L.  The cosubstrate NADP(H) protects lysine 601 in the porcine NADPH-cytochrome P-450 reductase against pyridoxylation.  FEBS Lett 1990 Jan 29;260(2):289-90 [abstract].
  12. Sem DS, Kasper CB.  Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH.  Biochemistry 1993 Nov 2; 32(43):11548-58 [abstract].  Scrutton NS, Berry A, Perham RN.  Redesign of the coenzyme specificity of a dehydrogenase by protein engineering.  Nature 1990 Jan 4;343(6253):38-43 [abstract].  M. Poe, N. J. Greenfield, J. M. Hirshfield, M. N. Williams & K. Hoogsteen.  Dihydrofolate reductase. Purification and characterization of the enzyme from an amethopterin-resistant mutant of Escherichia coli.  Biochemistry 11: 1023-1030 (1972)  Shin, M. Methods Enzymol 23, 440-447 (1971).  Shiraishi N, Croy C, Kaur J, Campbell WH.  Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase.  Arch Biochem Biophys 1998 Oct 1;358(1):104-15 [abstract]
  13. Karplus PA, Daniels MJ, Herriott JR.  Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family.  Science 1991 Jan 4;251(4989):60-6 [abstract].
  14. Wang M, Roberts DL, Paschke R, Shea TM, Masters BS, Kim JJ.  Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes.  Proc Natl Acad Sci U S A 1997 Aug 5;94(16):8411-6 [abstract].
  15. Shiraishi N, Croy C, Kaur J, Campbell WH.  Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase.  Arch Biochem Biophys 1998 Oct 1;358(1):104-15 [abstract].
  16. Shen AL, Kasper CB.   Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis.  J Biol Chem 2000 Dec 29;275(52):41087-91 [abstract].  Gutierrez A, Doehr O, Paine M, Wolf CR, Scrutton NS, Roberts GC.  Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases.  Biochemistry 2000 Dec 26;39(51):15990-9 [abstract].  Paul A. Hubbard, Anna L. Shen, Rosemary Paschke, Charles B. Kasper, and Jung-Ja P. Kim.  NADPH-cytochrome P450 oxidoreductase: structural basis for hydride and electron transfer.  J. Biol. Chem, [ in press]  
  17. Dohr O, Paine MJ, Friedberg T, Roberts GC, Wolf CR.  Engineering of a functional human NADH-dependent cytochrome P450 system.  Proc Natl Acad Sci U S A 2001 Jan 2;98(1):81-6 [abstract]
  18. Inano H, Tamaoki B.  The presence of essential carboxyl group for binding of cytochrome c in rat hepatic NADPH-cytochrome P-450 reductase by the reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.  J Enzyme Inhib 1985;1(1):47-59 [abstract].  Tamburini PP, Schenkman JB.  Differences in the mechanism of functional interaction between NADPH-cytochrome P-450 reductase and its redox partners.  Mol Pharmacol 1986 Aug;30(2):178-85 [abstract].  Nadler SG, Strobel HW.  Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450.  Arch Biochem Biophys 1991 Nov 1;290(2):277-84 [abstract]. 
  19. Nisimoto Y.  Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase.  J Biol Chem 1986 Oct 25;261(30):14232-9 [abstract].  Bernhardt R, Pommerening K, Ruckpaul K.  Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450 LM2.  Biochem Int 1987 May;14(5):823-32 [abstract].  Nadler SG, Strobel HW.  Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450.  Arch Biochem Biophys 1988 Mar;261(2):418-29 [abstract]. 
  20. Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, Deisenhofer J.  Structure and function of cytochromes P450: a comparative analysis of three crystal structures.  Structure 1995 Jan 15;3(1):41-62 [abstract]
  21. Ono T, Ozasa S, Hasegawa F, Imai Y.  Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.  Biochim Biophys Acta 1977 Mar 25;486(3):401-7 [abstract].  Enoch HG, Strittmatter P.  Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase.  J Biol Chem 1979 Sep 25;254(18):8976-81.  Schacter BA, Nelson EB, Marver HS, Masters BS.  Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system.  J Biol Chem 1972 Jun 10;247(11):3601-7.
  22. Shen AL, Kasper CB.  Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c.  J Biol Chem 1995 Nov 17;270(46):27475-80 [abstract]
  23. Sevrioukova IF, Li H, Zhang H, Peterson JA, Poulos TL.  Structure of a cytochrome P450-redox partner electron-transfer complex.  Proc Natl Acad Sci U S A 1999 Mar 2;96(5):1863-8 [abstract].  Sevrioukova IF, Hazzard JT, Tollin G, Poulos TL.  The FMN to heme electron transfer in cytochrome P450BM-3. Effect Of chemical modification of cysteines engineered at the fmn-heme domain interaction site.  J Biol Chem 1999 Dec 17;274(51):36097-106 [abstract]
 

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Comments to Todd D. Porter, Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, KY 40536-0082.  Phone 859 257-1137; FAX 859 257-7564
Last Modified: November 27, 2001
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