The Interaction of NADPH-P450 Reductase with P450: An Electrochemical Study of the Role of the Flavin Mononucleotide-Binding Domain

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 333, No. 1, September 1, pp. 308–315, 1996Article No. 0395

The Interaction of NADPH-P450 Reductase with P450:An Electrochemical Study of the Role of the FlavinMononucleotide-Binding Domain1

Ronald W. Estabrook,*,2 Manjunath S. Shet,* Charles W. Fisher,*Christopher M. Jenkins,† and Michael R. Waterman†*Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,Dallas, Texas 75235-9038; and †Department of Biochemistry, Vanderbilt UniversitySchool of Medicine, Nashville, Tennessee 37232-0146

Received April 25, 1996, and in revised form June 19, 1996

pyridine nucleotide (NADPH) to the heme of the P450The electrochemically reduced mediator cobalt se- via a flavoprotein called NADPH-P450 reductase (OR

pulchrate requires the presence of a flavoprotein for or reductase). Numerous studies have been carried outthe rapid transfer of electrons to cytochrome P450. to evaluate those factors that influence the interactionThis electrochemical method has been used here to of the reductase with the P450 since protein–proteinshow the interaction of NADPH-P450 reductase (either interaction is essential for electron transfer (1). It hasthe detergent-solubilized form, d-OR, or the proteo- been proposed that negatively charged amino acids onlytic-cleaved truncated form, t-OR), as well as Esche- the surface of the reductase and positively chargedrichia coli flavodoxin (FLD), with P450c17 by measur- amino acids on the surface of the P450 play a role ining the rate of 17a-hydroxylation of progesterone. the docking of these proteins (2–7). Further, a 1:1 com-When NADPH is used as electron donor with a recon- plex of the proteins is essential for electron transfer tostituted system composed of d-OR and P450c17, the occur (8, 9).addition of t-OR, flavodoxin, or cytochrome c inhibited The membrane-bound microsomal flavoprotein,the rate of formation of 17a-hydroxyprogesterone.

NADPH-P450 reductase (d-OR),3 contains both flavinThese results suggest the presence of a common pro-mononucleotide (FMN) and flavin adenine nucleotidetein binding site on the surface of d-OR, t-OR, and fla-FAD as essential cofactors for electron transport (10–vodoxin which plays a role in the interaction of the12). The sequence of electron transfer has been estab-flavoproteins with the P450. It is speculated that a do-lished to be from NADPH to FAD and then to FMNmain composed of acidic amino acids, located near thevia a series of internal one-electron transfer reactionsflavin mononucleotide-binding region of the flavopro-involving semiquinone intermediates (10, 13). In addi-teins, may serve as this site. No inhibition by t-OR,tion to the domains on the reductase identified withflavodoxin, or cytochrome c is observed when compa-the binding of the two flavins (FAD and FMN), a do-rable experiments are carried out using the artificialmain has been characterized as the site for interactionrecombinant fusion protein rF450[mBov17A/mRa-

tOR]L1 containing the heme-domain of P450c17 linked of NADPH (14, 15). Of considerable interest is a hy-to the flavin-domains of NADPH-P450 reductase. q 1996 drophobic membrane-binding domain at the amino-ter-Academic Press, Inc. minus of the reductase (16, 17) since this domain ap-

pears to be essential for electron transfer from the re-ductase to P450 (18). This hydrophobic domain hasbeen characterized and is readily removed from the

The functioning of microsomal cytochrome P450s(P450) requires the transfer of electrons from reduced

3 Abbreviations used: d-OR, detergent-solubilized NADPH-P450reductase; FMN, flavin mononucleotide; t-OR, truncated NADPH-

1 This work was supported in part by grants from the National P450; DLPC, dilauroyl L-a-phosphatidyl choline; NTA, nitrilotri-acetic acid; IPTG, isopropyl-b-D-thiogalactopyranoside; FLD, flavo-Institutes of Health (GM16488, GM37942, and ES07628).

2 To whom correspondence should be addressed. doxin.

308 0003-9861/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

AID ARCH 9597 / 6b1F$$$481 07-31-96 12:18:26 arca AP: Archives

309INTERACTION OF NADPH-P450 REDUCTASE WITH P450

dioxane-free, (isopropyl-b-D-thiogalactopyranoside) was obtainedenzyme by proteolytic digestion to form a water-solu-from Research Organics Inc. (Cleveland, OH). Bacto-Tryptone andble, truncated form of the reductase (t-OR) (19).Bacto-Yeast Extract were purchased from Difco Laboratories (De-

Recently, Shen and Kasper (7) have provided evi- troit, MI). All other chemicals were of the highest quality available.dence supporting the earlier proposal of Strobel and Expression vectors and plasmids. The plasmids pIBI25 (Interna-co-workers (20, 21) that there are unique clusters of tional Biotechnologies, Inc., New Haven, CT) and pCRII (Invitrogen,

San Diego, CA) were used for cloning and sequence analysis of PCRacidic amino acids on the surface of the reductase nearproducts. The expression vector pCWori/ : :Bov17A-rORfus has beenthe FMN-binding domain that might serve as bindingdescribed (25). The cDNA for rat liver NADPH-P450 reductasesites for interaction with cytochrome c or P450. Fur- (pOR263) was obtained from Charles Kasper (26), University of Wis-

ther, Jenkins and Waterman have shown (22) that the consin (Madison, WI). Escherichia coli DH5a was obtained from BRLFMN-containing flavoprotein Escherichia coli flavo- (Bethesda, MD).

Recombinant DNA manipulations. The plasmid pCWori/ : :doxin, when combined with flavodoxin reductase, isBov17AHis was prepared from pCWori/ : :Bov17A-rORfus by excis-capable of transferring electrons from NADPH toing the P450 domain (NdeI to SalI) and inserting the sequences intoP450c17 for the reconstitution of progesterone 17a-hy-a pCWori6His expression vector which retains the codons for the

droxylase activity. serine–threonine linker. The desired plasmid was identified andRecently we have described the use of an electro- used for DNA sequence analysis.

DNA modifying and restriction enzymes were obtained from Pro-chemical method whereby P450-catalyzed reactionsmega (Madison, WI), BRL, or NEB (Beverly, MA) unless stated other-can occur in the absence of NADPH (23, 24). Our re-wise.sults demonstrate the essential role of the flavoprotein

Cell growth, disruption, and purification of the enzymes. Esche-moiety for the transfer of electrons from an electromo- richia coli DH5a were transformed, plated, selected for growth, andtively active dye mediator to the P450. In these experi- grown at 287C as previously described (25, 27). Cells were generallyments we were able to reconstitute the omega hydroxyl- harvested after 72 h of growth. The recombinant protein (termed

either P450c17(His)6 or P45017A(His)6) was expressed at a level ofation of lauric acid using purified P4504A1 and purified200 nmol of P450/liter of growth medium as determined spectropho-NADPH-P450 reductase or the recombinant fusion pro-tometrically. Cells were disrupted by sonic treatment and mem-tein rF450(mRat4A1/mRatOR)L1. The use of the elec- branes containing the P450c17 or the fusion protein were prepared

trochemical method as the source of electrons for P450 by differential centrifugation, suspended in 50 mM Tris-HCl, (pH7.5), containing 20% glycerol and 0.5 mM EDTA (Buffer A) and frozenfunction now permits us to examine the role of variousat 0807C. Membranes were thawed and diluted to approximately 2flavoproteins as electron transfer carriers to P450 with-mg of protein/ml in Buffer A and solubilized by addition of the deter-out concern for the role of NADPH and the NADPH-gent Emulgen 911 as described previously (27). The fusion protein

binding site of the reductase protein. rF450[mRat4A1/mRatOR]L1 was purified by chromatography usingThis paper describes experiments showing that a a 2 *,5*-ADP Sepharose affinity column as previously described (25).

P450c17(His)6 was purified using a Ni2/-NTA agarose column follow-proteolytic-cleaved form of NADPH-P450 reductaseing the procedure described (28) for recombinant P450s and for the(t-OR) as well as the flavodoxin purified from E. colipurification of recombinant human b5 (29). Recombinant NADPH-(22) can serve for the transfer of electrons from theP450 reductase (d-OR) was expressed in E. coli and isolated and

electrochemically reduced dye mediator, cobalt se- purified from membranes essentially as described by Shen et al. (26).pulchrate, to P450c17 thereby catalyzing the 17a- The proteolytically digested, water-soluble form of the reductase

(t-OR) was purified from the supernatent of disrupted E. coli byhydroxylation of progesterone. Further, these studieschromatography using a 2 *,5*-ADP Sepharose affinity column (30).show that t-OR, flavodoxin, and cytochrome c inhibitRecombinant flavodoxin was overexpressed in E. coli and purifiedthe NADPH-dependent hydroxylation of progester- (C. Jenkins, unpublished).

one when studying the activity of a reconstituted sys- Spectrophotometric measurements. The amount of recombinanttem containing d-OR and P450c17. These results sug- P450 expressed in intact E. coli cells and in samples prepared during

purification was determined by difference spectrophotometry usinggest a common binding site for d-OR with the FMN-an Aminco DW2a wavelength scanning recording spectrophotometercontaining domain of t-OR and flavodoxin. Failure ofconnected to an IBM computer. A millimolar extinction coefficient offlavodoxin or cytochrome c to inhibit the 17a-hydrox-91 was applied for the absorbance difference at 450 minus 490 nm

ylation of progesterone, as catalyzed by the fusion for the carbon monoxide complex of the reduced hemeprotein (31).protein rF450(mBov17A/mRatOR)L1, indicates a Measurements of enzymatic activities and HPLC analysis of prod-structural conformation for the fusion protein which ucts formed. Samples of the purified P450-containing proteins

(stock solutions of 20 to 50 nmol P450/ml) were preincubated aslimits accessibility to the site of domain interactionindicated. In some instances aliquots of DLPC (50 mg/ml) were addedrequired for electron transfer.to give the final concentrations indicated. The concentrated enzymeswere mixed, preincubated at 377C for 10 min, and then diluted tothe concentrations designated with a buffer mixture containing 50MATERIALS AND METHODSmM Tris-Cl (pH 7.5) and 10 mM MgCl2. [1,2,6,7-3H]Progesterone(30,000 cpm/ml) was then added to the concentrations indicated fol-Chemicals. [1,2,6,7-3H]Progesterone (64.8 Ci/mmol) was pur-

chased from Amersham International. L-a-Phosphatidyl choline, di- lowed by the addition of 1.0 mM NADPH (final concentration) andan NADPH regenerating system (0.8 mM sodium isocitrate, 0.1 units/lauroyl (DLPC), FMN, FAD, cytochrome c, and NADPH were pur-

chased from Sigma Chemical Co. (St. Louis). Emulgen 911 was a gift ml of isocitrate dehydrogenase). The diluted mixture of reactantswas incubated with gentle stirring at 377C and 0.5 ml-samples werefrom Kao Chemicals (Tokyo, Japan). Ni2/-NTA (nitrilotriacetic acid)

agarose was purchased from QIAGEN Inc. (Chatsworth, CA). IPTG, removed at the times indicated and added to 5 ml of methylene


310 ESTABROOK ET AL.

reductase (d-OR) are required per molecule of purifiedP450c17 to obtain a half-maximal rate of 17a-hydroxyl-ation of progesterone when using NADPH as electrondonor. Using reaction conditions of low ionic strengthand a temperature of 377C, in the absence of addedphospholipid, an equilibrium constant (Keq) for the re-action of reductase with P450 of 0.76 mM can be calcu-lated. This equilibrium constant is about 10-foldgreater than that reported by Miwa et al. (8) from stud-ies with rat liver P450 2B4 catalyzing the N-demethyl-ation of benzphetamine. When this experiment was re-peated using conditions where the reductase and P450are preincubated with phospholipid (100 mg DLPC/ml),one observes only a slight enhancement in the maximalrate of steroid hydroxylation (cf. 33, 34) with a smalldecrease in the calculated equilibrium constant. Con-versely, when the reaction is carried out in a buffermixture fortified with 150 mM KCl, a small decrease in

FIG. 1. Effect of varying flavoprotein concentrations on the recon- the maximal rate of steroid hydroxylation is observed.stitution of progesterone 17a-hydroxylase activity of P450c17 in the However, the statistical significance of these differ-presence of DLPC or 150 mM KCl. An aliquot of purified P450c17 ences has not been studied and is considered to be mar-(30 nmol/ml) was mixed with the designated concentration of purified

ginal.reductase (65 nmol/ml) and incubated for 10 min at 377C prior todilution with 50 mM Tris-Cl buffer, pH 7.5, containing 10 mM MgCl2 In agreement with earlier reports (17, 18), no mea-to give a final concentration of 0.5 mM P450. When DLPC was present, surable activity is observed when the truncated form2 ml of a 50 mg/ml stock solution was added for each milliliter of of the reductase (t-OR) is used for reconstitution withfinal reaction solution. [3H]Progesterone was added to provide a final

purified P450c17. Likewise, no activity is observedconcentration of 50 mM. The reaction was initiated by addition ofwhen purified E. coli flavodoxin is used (in the absenceNADPH (1 mM final concentration) and an NADPH regenerating

system. Samples were removed at 2.5, 5, 10, 15, and 20 min and the of flavodoxin reductase).initial rate of progesterone 17a-hydroxylation was determined by

Electrochemically driven 17a-hydroxylation of pro-HPLC analysis of the sample extracts. Purified flavodoxin was usedgesterone. We have recently described (23) the use ofin the absence of flavodoxin reductase.electrochemistry to drive the omega hydroxylation oflauric acid by P4504A1. In these studies the electro-chemically active mediator, cobalt sepulchrate, is re-chloride. The samples were mixed vigerously for at least 1 min and

the methylene chloride layer was removed and gently evaporated to duced at a platinum electrode and the reduced media-dryness under a stream of nitrogen. The residue was dissolved in tor, in turn, reduces a component of the flavoprotein100 ml of methanol and analyzed by reversed-phase HPLC using a

reductase. In this way electrons are transferred to aWaters 380 computer-controlled apparatus with a C18-mBondapackP450 without the need for NADPH. These studies havereverse-phase column coupled to a Radiometer Flow-1 radioactive

detector essentially as described (25). shown that the best results (maximal activity and long-The electrochemical method. The use of an electrochemical poten- term stability) are obtained when a recombinant fusion

tial to drive a P450 reaction has been described in detail (24). Briefly, protein is used as catalyst, although reconstitution ex-the reaction mixture, of 10 ml volume, is placed in a temperature- periments using purified NADPH-P450 reductase withcontrolled (377C) cell containing a platinum gauze electrode together

P4504A1 did result in a system capable of catalyzingwith a silver/silver chloride reference electrode and a platinum auxil-lary electrode. A polarizing voltage of0650 mV is applied by a Bioan- the omega-hydroxylation of a fatty acid.alytical Systems, Inc. (West Lafayette, IN) CV-27 Voltammograph. Figure 2 shows the ability of the fusion proteinThe reaction vessel also contains a Yellow Springs Instrument (Yel- rF450(mBov17A/mRatOR)L1, to catalyze the 17a-hy-low Springs, OH) YSI 5331 Oxygen probe and a gassing tube con-

droxylation of progesterone. The data presented in thisnected to a tank of compressed oxygen so that a constant level offigure illustrate that the rate of steroid hydroxylationoxygen is maintained in the reaction mixture. Samples (0.5 ml) are

removed from the stirred reaction mixture at the times indicated. obtained by the electrochemical method is about theOther. Protein concentrations were estimated by the Bradford same as that determined when NADPH is used as elec-

method (32) with bovine serum albumin as the standard. tron donor. Further, high levels of steroid can be me-tabolized by this method for extended periods of time,

RESULTS i.e., at least 2 h.When a series of reconstitution experiments similarReconstitution of progesterone 17a-hydroxylation

to those illustrated in Figure 1 was carried out usingwith varying concentrations of d-OR. Figure 1 showsthat approximately two or three molecules of purified the electrochemical method, it was observed (Fig. 3)



servation that purified flavodoxin as well as t-OR iscapable of catalyzing electron transfer to the P450,when using the electrochemically driven system, sug-gests that these flavoproteins may be interacting withthe P450 at or near the same site on the surface of theP450 protein normally occupied by d-OR. Therefore,a series of experiments was carried out to determinewhether flavodoxin or t-OR might serve as inhibitorsof such an interaction. As shown in Figure 4, both fla-vodoxin and t-OR inhibit the NADPH-dependent activ-ity obtained when the system is reconstituted withP450c17 and d-OR. For these studies, the designatedamounts of flavodoxin or t-OR were preincubated withaliquots of the reductase and P450 prior to dilution ofthe mixtures for the assay of activity.

Inhibition of the NADPH-dependent activity of theFIG. 2. The 17a-hydroxylation of progesterone by the recombinant reconstituted system by cytochrome c. It has beenfusion protein rF450 (mBov17A/mRatOR)L1 using NADPH (open

known for many years that cytochrome c inhibits thecircles) or an electrochemically driven system (closed circles). Sam-functioning of P450s in drug metabolism (35, 36). Itples of the fusion protein (final concentration 0.2 mM) were diluted

in 50 mM Tris-Cl buffer, pH 7.5, containing 10 mM MgCl2 and 50 was not always apparent whether this inhibition wasmM [3H]progesterone, and the reaction was initiated by addition of the result of an electron competition associated withNADPH (1 mM final concentration) and an NADPH regenerating the reduction of cytochrome c by NADPH via the reduc-system. Samples were removed at the times indicated and analyzed

tase or whether the cytochrome c might serve to com-by HPLC. Comparable experiments were carried out in the electro-pete with P450 for a binding site on the reductase.chemical reaction vessel with the buffer mixture supplemented by

addition of 1.0 mM cobalt sepulchrate and 150 mM KCl as described Recent results by Shen and Kasper (7) suggest that the(23, 24). The initial rates of the reactions are indicated in brackets binding site of a P450 and cytochrome c may be in closeas nmol product formed/min/nmol P450. proximity on the surface of the flavoprotein reductase.

As shown in Fig. 5, the addition of cytochrome c, albeitat high concentrations, does inhibit the 17a-hydroxy-

that the initial rate of 17a-hydroxylation of progester-one was dependent on the ratio of reductase toP450c17. The maximal rate of progesterone hydroxyla-tion by the electrochemical method, using a reconstitu-tion system composed of d-OR and P450c17, is about20% of that seen when NADPH is used as electrondonor (cf. Fig. 1) and about 35% of that seen when thefusion protein is used as catalyst (cf. Fig. 2). Surpris-ingly, it was observed that the 17a-hydroxylation ofprogesterone also occurs by the electrochemical methodwhen E. coli flavodoxin or the proteolytic form (t-OR)of the reductase are used with P450c17. Although therates observed (turnover numbers of the P450) are con-siderably slower than seen when d-OR is used, theseexperiments show that the electromotively active co-balt mediator is capable of reducing these flavoproteinsand that the reduced flavoproteins, in turn, are able totransfer electrons to the P450. This result confirms theearlier report of Jenkins and Waterman (22) that E.coli flavodoxin, together with flavodoxin reductase andNADPH, is able to support the hydroxylation of proges- FIG. 3. Progesterone 17a-hydroxylase activity using an electro-terone by P450c17. Replacement of the flavoproteins chemically driven system with varying concentrations of flavopro-with 50 mM FMN or FAD, alone or in combination, did teins. Aliquots of purified P450c17 were preincubated at 377C for 10

min with the designated amounts of the flavoproteins and then di-not support the electrochemically driven hydroxylationluted with a buffer mixture containing 50 mM Tris-Cl buffer, pH 7.5,of progesterone.10 mM MgCl2, 50 mM [3H]progesterone, 150 mM KCl, and 1.0 mM

Inhibition of the NADPH-dependent activity of the cobalt sepulchrate as described (23, 24). Samples were removed at2.5, 5, and 10 min and analyzed by HPLC.reconstituted system by flavodoxin and t-OR. The ob-



tase dictates the need for a rapid association and disso-ciation of the reductase protein as it travels from oneP450 to the next (42).

How is the complex formed when a molecule of reduc-tase interacts with a molecule of P450? A number offacts have been established:

(a) The membrane-bound form of the reductase(d-OR) is required for reconstitution of the NADPH-supported enzymatic activity for a P450 (Fig. 1 and Ref.(16, 17). However, little is known about the manner inwhich the hydrophobic amino acid sequence at theN-terminus of d-OR influences the proper orientationof the reductase as it interacts to give a functional com-plex with a P450. Is it merely the hydrophobic associa-tion of two molecules, or is it more complex and depen-dent on the two-dimensional properties of a lipidsurface which may influence binding of proteins to a

FIG. 4. Inhibition of the NADPH-dependent reconstitution of pro- micelle? Of interest is the recent report (43) that thegesterone 17a-hydroxylation by flavodoxin and t-OR. Experiments hydrophobic domain of the P450 is not required forsimilar to those described in the legend to Fig. 1 were carried out reconstitution of activity.using 0.5 mM P450c17, 1.0 mM reductase, 20 mM progesterone, and the

In contrast, the presence of the hydrophobic mem-indicated amounts of purified flavodoxin or proteolytically digestedbrane-binding sequence of d-OR is not required for thereductase (t-OR). The reaction was initiated by addition of NADPH

(1 mM final concentration) and an NADPH regenerating system. function of artificial fusion proteins containing theSamples were removed at 2.5, 5, 10, 15, and 20 min and the initial heme-domain of a P450 linked to the N-terminal regionrate of progesterone 17a-hydroxylation determined by HPLC analy- of NADPH-P450 reductase (44, 45). Indeed, the pres-sis of the sample extracts.

lase activity of the reconstituted reaction system, whenNADPH is used as electron donor. It was noted thatthe cytochrome c was immediately reduced on additionof NADPH and the pink color of the reduced hemopro-tein persisted throughout the reaction.

Of interest is the observation that 100 mM cyto-chrome c did not inhibit the NADPH-dependent func-tion of the fusion protein rF450(mBov17A/mRatOR)L1,as it catalyzed the 17a-hydroxylation of progesterone(data not shown). In a similar way, high concentrationsof flavodoxin (4 mM) also did not inhibit the NADPH-dependent activity of the fusion protein (data notshown).

DISCUSSION

Our understanding of the mechanism(s) of interac-tion of d-OR with various P450s remains unclear inspite of an extensive literature describing numerousexperiments designed to examine this reaction (e.g.,37–40). Protein–protein interaction is essential for FIG. 5. Inhibition of the NADPH-dependent reconstitution of pro-electron transfer to occur from the reduced FMN of the gesterone 17a-hydroxylation by ferrous cytochrome c. Experimentsflavoprotein to the substrate-bound ferric form of the similar to those described in the legend to Fig. 4 were carried out

using 0.5 mM P450c17, 1.0 mM reductase, 20 mM progesterone, andhemeprotein. This reaction is considered to be the rate-the indicated amounts of cytochrome c. The reaction was initiatedlimiting step in the enzymatic functioning of manyby addition of NADPH (1 mM final concentration) and an NADPHP450s (41). In situations like those occurring on the regenerating system. Note that all the cytochrome c was rapidly

surface of liver microsomes the stoichiometry of 10 to reduced in the first few seconds of the reaction. Samples were re-moved at the times indicated and analyzed by HPLC.20 molecules of different P450s per molecule of reduc-



reductase is only now being studied (49). The X-raystructures of four P450s have now been determined(50). These studies show that there are many commonfeatures when the structures of these P450s are com-pared in the region of the heme-core of the molecules.However, there are significant differences in the sur-face structure when comparing each of the P450s. Onemight expect differences in the properties of the surfaceof each P450, since differences in the affinity of a P450for the reductase, when reconstituting enzymatic activ-ities with different P450s, have been noted (38).

(c) Cytochrome c has long been recognized as an in-hibitor of P450 reactions (35, 36). Although cytochromec is an effective acceptor of electrons from the reductaseit is also a very positively charged molecule. One canFIG. 6. A schematic representation of the interaction of P450 andenvision a molecule of cytochrome c interacting withNADPH-P450 reductase (d-OR) during electron transfer. The paths

of electron flow from NADPH or the electrochemical mediator, cobalt the clusters of negatively charged amino acids associ-sepulchrate (Co), are shown. The role of flavodoxin (FLD) as an elec- ated with the reductase and in this way serve to impedetron carrier and the proposed mechanism of inhibition of electron the interaction of the reductase with the P450 (51).flow from the reductase to P450 by FLD and cytochrome c (c) are

This latter interpretation appears to be the case in theillustrated.studies described here.

We propose the schematic model shown in Fig. 6 toence of the hydrophobic domain of the reductase inhib-represent the interaction of the reductase with P450its expression of a functional P450 domain of the fusionduring electron transfer. This representation shows aproteins in transformed E. coli (unpublished experi-role for negatively charged regions in the vicinity ofments).the FMN-binding domain of NADPH-P450 reductaseAs shown here, the hydrophobic membrane-bindingas important for the interaction with the P450. At leastsequence of d-OR is not essential for electron transfertwo possible alignments of the amino acids in this re-when an electromotively active dye is reduced electro-gion of the reductase protein can be made to a similarchemically and used as the source of electrons for re-region of the flavodoxin molecule (Fig. 7). It has beenduction of the flavoprotein (see Fig. 3). Coon and col-pointed out that two clusters of acidic amino acids,leagues have shown that t-OR is ineffective in reducing(207Asp–Asp–Asp209) and (213Glu–Glu–Asp215) (51–P450s with NADPH (11). Electrochemically, however,53), are present in mammalian and plant NADPH-t-OR is as efficient as flavodoxin in transfering elec-

trons to P450c17. This indicates that the FMN-con-taining domain of t-OR is able to bind productively withP450c17 and as long as electrons can be transferred tothis domain, t-OR can function as a P450 reductase.Perhaps the reason that t-OR cannot reduce P450 viaNADPH is because electrons transfered from the FADto the FMN domains are somehow altered whereaswith the electrochemical system only the FMN domainparticipates in electron transfer.

(b) It has been shown (e.g., 1, 6, 7) that modifyingthe ionic strength of the reaction medium by addition ofvarious salts can influence the rate of electron transfer

FIG. 7. Two possible amino acid alignments in the region of thefrom NADPH to a P450. Although the interpretationFMN-binding site that define the negatively charged domains of theof these results is controversial, it is now generallyrat NADPH-P450 reductase (OR) and E. coli flavodoxin (FLD).accepted that ionic interactions do occur during the (Alignment 1) The alignment based on two clusters of acidic amino

collision of an electron-transfer protein (adrenodoxin acids (underlined and identified by delta) as proposed by Shen andor a P450 reductase) with a P450 (21, 46, 47). It has Kasper (7). (Alignment 2) A similar alignment showing the highly

conserved residues in this region of the molecule for all flavodoxinsbeen proposed (7) that clusters of negatively charged(#); vertical lines indicate identical residues in the alignment whileamino acids on the surface of the reductase react withcolons indicate similar residues. The 21-amino acid residue loop in-positively charged amino acids on the surface of the sertion (WPTAGYHFEASKGLADDDHFV) found in long-chain fla-

P450 (48). The details of this interaction remain specu- vodoxins is symbolized as 21. Alignment is partially based on thatreported by Grandori and Carey (53) and eyesight.lative since the structural analysis of NADPH-P450



8. Miwa, G. T., West, S. B., Huang, M. T., and Lu, A. Y. (1979) J.P450 reductases. Shen and Kasper (7), using site-di-Biol. Chem. 254, 5695–5700.rected mutagenesis, have provided evidence that this

9. French, J. S., Guengerich, F. P., and Coon, M. J. (1980) J. Biol.region of the reductase molecule may be the site ofChem. 255, 4112–4119.interaction with cytochrome c as well as P450. Of inter-

10. Iyanagi, T., Makino, N., and Mason, H. S. (1974) Biochemistryest is the observation that flavodoxin from E. coli has 13, 1701–1710.two similar amino acid clusters in the region of the

11. Vermilion, J. L., and Coon, M. J. (1978) J. Biol. Chem. 253,FMN-binding site (54) (Fig. 7, alignment 1). The alter- 2694–2704.nate alignment of amino acids of the reductase and 12. Yasukochi, Y., Okita, R. T., and Masters, B. S. (1980) Arch. Bio-flavodoxin (Fig. 7, alignment 2) proposes a role for one chem. Biophys. 202, 491–498.cluster of acidic amino acids but considers a number of 13. Vermilion, J. L., Ballou, D. P., Massey, V., and Coon, M. J. (1981)other amino acids in this region that are highly con- J. Biol. Chem. 256, 266–277.served in all flavodoxins (54). This latter proposal (C. 14. Porter, T. D. (1991) Trends Biochem. Sci. 16, 154–158.Jenkins and M. Waterman, unpublished) recognizes a 15. Karplus, P. A., Daniels, M. J., and Herriott, J. R. (1991) Science

251, 60–66.role for a 21-amino acid loop insertion found in manyflavodoxins. Additional experiments will be required to 16. Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemis-

try 14, 607–613.determine which model is most appropriate.17. Black, S. D., and Coon, M. J. (1982) J. Biol. Chem. 257, 5929–The fact that flavodoxin can serve to transfer elec-

5938.trons to P450, either via the NADPH-reduced flavo-18. Lu, A. Y., Junk, K. W., and Coon, M. J. (1969) J. Biol. Chem.doxin reductase (22) or when reduced by an electromo-

244, 3714–3721.tively active dye (shown here), lends further support to19. Kamin, H., Masters, B. S., Gibson, Q. H., and Williams, C. H.,the concept that specific recognition sites on the protein

Jr. (1965) Fed. Proc. 24, 1164–1171.molecules play a role in the ionic interaction of the20. Nadler, S. G., and Strobel, H. W. (1991) Arch. Biochem. Biophys.proteins. The observation that t-OR and flavodoxin 290, 277–284.

(FLD) inhibit the interaction of d-OR with the P450,21. Shen, S., and Strobel, H. W. (1993) Arch. Biochem. Biophys. 304,

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The Interaction of NADPH-P450 Reductase with P450: An Electrochemical Study of the Role of the Flavin Mononucleotide-Binding Domain