Electrostatic Interaction between Cytochrome P450 and NADPH-P450 Reductase: Comparison of Mixed and Fused Systems Consisting of Rat Cytochrome P450 1A1 and Yeast NADPH-P450 Reductase

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  • Electro oNADP MFused roP450 1 ct

    Shunya K K*Division o toSakyo-ku, EnFaculty of 657

    Received Fe

    The elechrome Pwas analycontaininThe Vmaxthe recomtochrome(the mixestrengthever, onP450 1A1system), tcreasing ialso diffetems. Basthat cytohave mority of theexplainedhand, theing mode due to the limited topology of cytochromeP450 andAcademic Pres

    Cytochrtems areEukaryotof two m(P450) anP450 monare transfP450. In

    tioedge-nhaly,ruy sthenucneu

    thheveseng. Oons

    ductase can transfer electrons to rat P450 1A1 as

    1 To whom6265. E-mai


    Biochemical and Biophysical Research Communications 257, 273278 (1999)

    Article ID bbrc.1999.0455, available online at http://www.idealibrary.com onNADPH-P450 reductase domains. 1999s

    ome P450-dependent monooxygenase sys-widely distributed in the biological kingdom.ic microsomal P450 monooxygenases consistembrane-bound enzymes, cytochrome P450d NADPH-P450 reductase (reductase). Theooxygenation requires two electrons whicherred from NADPH through the reductase tothe last two decades, many reports on the

    efficiently as rat NADPH-P450 reductase (12), the re-combinant yeast microsomal membrane could be con-sidered as a kind of a good reconstituted membranecontaining a single species of P450 and NADPH-P450reductase. In addition, we have succeeded in the ex-pression of genetically engineered P450 1A1-reductasefused enzyme in S. cerevisiae cells and revealed therapid intra-molecular electron transfer from the reduc-tase part to P450 1A1 part of the fused enzyme withhigh monooxygenase activity under low ionic strength(12). Thus, the fused enzyme appears a suitable modelto reveal the direct interaction between P450 and re-ductase. In this report, we will discuss the interactionbetween P450 and reductase by comparing the effect ofcorrespondence should be addressed. Fax: 181-75-753-static Interaction between CytochrH-P450 Reductase: Comparison ofSystems Consisting of Rat CytochA1 and Yeast NADPH-P450 Redu

    ondo,* Toshiyuki Sakaki,* Hideo Ohkawa, andf Applied Life Sciences, Graduate School of Agriculture, KyoKyoto 606-8502, Japan; and Department of Biological andAgriculture, Kobe University, Rokkodai-cho, Nada-ku, Kobe

    bruary 20, 1999

    ctrostatic interaction between rat cyto-450 1A1 and yeast NADPH-P450 reductasezed by using recombinant yeast microsomesg both native enzymes or their fused enzyme.of the 7-ethoxycoumarin O-deethylation inbinant microsomes containing both rat cy-P4501A1 and yeast NADPH-P450 reductased system) was maximal when the ionic

    of the reaction mixture was 0.1-0.15. How-the fused enzyme between rat cytochromeand yeast NADPH-P450 reductase (the fusedhe activity was uniformly reduced with in-onic strength. The pH profiles of Vmax wererent between the mixed and the fused sys-ed on these results, we propose a hypothesischrome P450 and NADPH-P450 reductasee than one binding mode. The maximal activ-mixed system at ionic strength of 0.1-0.15 isby change of the binding mode. On the otherfused enzyme appears to have only one bind-

    interacpublishof characids otively cRecenttiary ststrongltial onVoznesthe redby thegestingtween t

    To inreductaproduciductasehave nconditioionic stremixed sys

    l: inouye@kais.kyoto-u.ac.jp.ion used: P450, cytochrome P450.

    273me P450 andixed andmease

    uniyo Inouye*,1

    University, Kitashirakawa, Oiwake-cho,vironmental Science,-0013, Japan

    n between P450 and the reductase have been. Most of the reports suggest the importancepairing involving negatively charged amino

    the surface of the reductase (1-3) and posi-rged amino acids on the surface of P450 (4-8).x-ray crystallographic study revealed the ter-cture of rat NADPH-P450 reductase (9), anduggested the significance of its surface poten-e interaction with P450. On the other hand,sky and Schenkman (10, 11) indicated thattion rate of P450 by the reductase increasedtralization of electrostatic interactions, sug-e significance of electrostatic repulsion be-two enzymes.

    stigate the interaction between P450 and the, we used the recombinant yeast microsomes

    rat P450 1A1 and yeast NADPH-P450 re-n the basis of the facts that S. cerevisiae cells

    detectable endogenous P450 in the cultureof this study, and yeast NADPH-P450 re-ngth on the monoxygenase activity of thetem and the fused system.

    0006-291X/99 $30.00Copyright 1999 by Academic PressAll rights of reproduction in any form reserved.


    Materials. The expression plasmids pAMR2 for rat P450 1A1 andyeast NADPtween rat Pand pARR3constructedused as a hofrom AldricSigma ChemYeast (Tokycially availa

    PreparatioThe recombAH22/pARRimal mediumout amino aMicrosomaldescribed pr

    Western brecombinantlinear graditransferredThe filter wathen followemethod (Am

    Determinareductase.reduced CO-mM21 cm21 (microsomalreductase ac

    7-ethoxycocoumarin insayed by mestimate therescence chatation wavelusing a HitaThe reactionpH 7.4, 0-40and 0-60 mNADPH andcells containmicrosomes)reductase fuated by thecontinuouslymonitoring,reaction miThe concenNADPH wemM21 cm21

    cm21 at 339imum velocimethod app

    NADPH obetween 7-hthe initial raing 100 mMtions of 0 anbance at 33(Kyoto, Japa50 and 0 mMdependent NMR microso

    hef Neriumwh




    f rali










    . Fctaely

    Vol. 257, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSH-P450 reductase, pAFCR1 for the fused enzyme be-450 1A1 and yeast NADPH-cytochrome P450 reductase,for only yeast NADPH-cytochrome P450 reductase wereas reported previously (12, 13). S. cerevisiae AH22 wasst. The following chemicals were used: 7-ethoxycoumarinh (Milwaukee, WI, U.S.A.); 7-hydroxycoumarin fromical Co. (St. Louis, MO, U.S.A.); NADPH from Oriental

    o, Japan). All other chemicals were of the best commer-ble grade.

    n of microsomal fractions of the recombinant yeast cells.inant S. cerevisiae AH22/pAMR2, AH22/pAFCR1 and3 cells were cultivated in a concentrated synthetic min-

    containing 8% glucose, 5.4% yeast nitrogen base with-cids and 160 mg/l histidine as described previously (14).fractions of the recombinant yeast cells were prepared aseviously (15).

    lot analysis. Microsomal fractions prepared from thecells were subjected to electrophoresis on a 4 to 20%

    ent polyacrylamide-sodium dodecyl sulfate gel and thenelectrophoretically from the gel to nitrocellulose filter.s probed with anti-yeast NADPH-P450 reductase Ig andd by Enhanced Chemiluminescence immunodetectionersham, Buckinghamshire, England).

    tion of the concentration of the P450 and NADPH-P450The concentration of P450 1A1 was determined from thedifference spectrum using an extinction coefficient of 9116). The concentration of NADPH-P450 reductase in thefraction was estimated on the basis of cytochrome ctivity as described previously (12).

    umarin O-deethylation. O-Deethylation of 7-ethoxy-the recombinant yeast microsomal fractions was as-

    onitoring the formation of 7-hydroxycoumarin (17). Toinitial rate of 7-hydroxycoumarin formation, the fluo-

    nge was continuously monitored at 37C with an exci-ength at 366 nm and an emission wavelength at 452 nmchi fluorescence spectrophotometer 850 (Tokyo, Japan).mixture contains 10-200 mM sodium phosphate buffer,

    0 mM sodium chloride or 0-400 mM potassium chlorideM of 7-ethoxycoumarin, 1.0% methanol, 400 mM of

    the microsomal fraction prepared from AH22/pAMR2ing P450 1A1 and yeast NADPH-P450 reductase (MR, or AH22/pAFCR1 cells containing the P450 1A1-sed enzyme (FCR microsomes). The reaction was initi-addition of NADPH, and the fluorescence change was

    monitored for more than 5 min. At the end of thea known amount of 7-hydroxycoumarin was added to thexture in order to calibrate the fluorescence change.tration of 7-ethoxycoumarin, 7-hydroxycoumarin, andre determined by using extinction coefficients of 13.56at 320 nm, 14.5 mM21 cm21 at 324 nm, and 6.2 mM21

    nm, respectively. The Michaelis constant, Km, and max-ty, Vmax were determined using a nonlinear least-squareslied to the Michaelis-Menten equation (18).

    xidation. In order to calculate the coupling efficiencyydroxycoumarin formation and NADPH consumption,te of NADPH oxidation in the reaction mixture contain-NADPH was measured at 7-ethoxycoumarin concentra-d 50 mM, respectively, by continuous recording of absor-9 nm with a Shimadzu UV-2200 spectrophotometern) at 37C. The difference of NADPH oxidation between

    of 7-ethoxycoumarin was considered as the P4501A1-ADPH oxidation because the difference was observed in

    mes and FCR microsomes, but not in the control micro-

    somes. Tchange ochange d7-thoxycooxidation


    Exprand P4contentmicroso0.27 anbasis oting anenzymewith nNADPHmatedcytochrNADPH

    EffecactivityenzymeMR mireductareductamicrososuredtrol m7-hydroFCR mthe fus

    The eture wfrom 0somesin the r

    FIG. 1from recophoresedIg. LanepAFCR11A1-redurespectiv

    274initial rate was calculated using a molar absorptionADPH oxidation, D 5 6,200 M21 cm21. The absorptionved from the formation of 7-hydroxycoumarin fromarin was subtracted to determine the exact NADPHile it gave a small influence.

    ion of P450 1A1, NADPH-P450 reductase,1A1-reductase fused enzyme in yeast. The

    f P450 1A1 and the fused enzyme in MRes and FCR microsomes were estimated to be0.11 nmol/mg protein, respectively, on the

    educed CO difference spectra. Western blot-ysis suggested that the most of the fusedn FCR microsomes remains intact (Fig. 1)

    proteolytic degradation. The content of450 reductase in MR microsome was esti-be 0.24 nmol/mg protein on the basis of thee c reductase activity of the purified yeast450 reductase sample (12, 19).

    of salts on 7-ethoxycoumarin O-deethylationf P450 1A1 and P450 1A1-reductase fused7-Ethoxycoumarin O-deethylation activity ofsomes containing both P450 1A1 and the

    , FCR microsomes containing P450 1A1-fused enzyme and the control AH22/pARR3

    es containing no P450 1A1 was each mea-the reconstituted system. Since the con-osomes showed no detectable activity,coumarin formation in MR microsomes andosomes was found to depend on P450 1A1 andenzyme, respectively.ct of the ionic strength in the reaction mix-examined with the substrate concentration60 mM. The catalytic activity of MR micro-

    s strongly affected by the salt concentrationction mixture. While the Km was not affected,

    Western blot analysis of microsomal fractions preparedinant yeast cells. The microsomal fraction was electro-d then probed with anti-yeast NADPH-P450 reductaseAH22/pARR3; lane 2, AH22/pAMR2; lane 3, AH22/

    E and YR indicate the migrating points of P450se fused enzyme and yeast NADPH-P450 reductase,.

  • the Vmax insodium pcreased wphosphatemum activWhen Nabuffer (pHing the codecreasedfrom 50 toKCl whilemM (Fig.salts, it isan essent

    7-Ethoxsomes wations as Mmicrosom(Fig. 2) anever, theconcentraactivationfrom 0 mMas shown

    ThroughO-deethylno significthe additiovalue of 7

    gave no significant effect on the coupling efficiency be-tween 7-hydroxycoumarin formation and NADPH oxida-tion, except for FCR microsomes in the presence of 400


    e rminss)e

    t of45prasRin




    .val1 aMR20f 0,

    FIG. 2.tration on tO-deethylatenzyme. Thmicrosomesand 200 mM

    Vol. 257, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONScreased with increasing the concentration ofhosphate from 10 mM to 40 mM, and de-ith increasing the concentration of sodiumfrom 80 mM to 200 mM (Fig. 2). The maxi-ity was obtained in the range of 40 ; 80 mM.

    Cl was added to 20 mM sodium phosphate7.4) (Fig. 3), the Vmax increased with increas-ncentration of NaCl from 0 to 50 mM, andwith increasing the concentration of NaCl400 mM. Similar results were obtained withthe maximum activity was obtained at 1004). From these results with three kinds ofstrongly suggested that the ionic strength is

    ial factor to determine Vmax.ycoumarin O-deethylation of FCR micro-s measured under the same reaction condi-R microsomes. The catalytic activity of FCR

    es was also affected by the sodium phosphated KCl (Fig. 4) in the reaction mixture, how-maximum Vmax was obtained at lower salttion than MR microsomes and no significantby salts was observed. The addition of NaClto 400 mM uniformly decreased the activity

    in Fig. 3.out the measurement of 7-ethoxycoumarin

    ation of MR microsomes and FCR microsomes,ant change of the Km value was observed withn of the salts (Figs. 2-4). Salts affected the Vmax-ethoxycoumarin O-deethylation, however, it

    mM ofstronglyfrom ththat froenzymeence wacrosomeinfluenc

    Effecity of PThe pHtivity wand FCenzymepH proing thereductacrosomsuggestbinding



    FIG. 3Vmax (b)P450 1AAH22/pAsured intration o

    The effect of sodium phosphate buffer (pH 7.4) concen-he Km (a) and the Vmax (b) values of 7-ethoxycoumarinion catalyzed by P450 1A1 and P450 1A1-reductase fusede activities of AH22/pAMR2 (E) and AH22/pAFCR1 (n)were measured in 10, 20, 30, 40, 60, 80, 100, 120, 150,sodium phosphate buffer (pH 7.4).

    275Cl or KCl as shown in Fig. 5. These resultsggest that salts influence the electron transfereductase to P4501A1 in MR microsomes andthe reductase domain to P450 domain of fused

    FCR microsomes. However, the clear differ-observed between the mixed system (MR mi-and the fused system (FCR microsomes) on theof the salts.

    pH on 7-ethoxycoumarin O-deethylation activ-0 1A1 and P450 1A1-reductase fused enzyme.ofile of 7-ethoxycoumarin O-deethylation ac-clearly different between MR microsomes

    microsomes as shown in Fig. 6. The fusedFCR microsomes showed a bell-shape-like

    with a maximum Vmax at pH 7.0-7.5, suggest-ple binding mode between P4501A1 and theparts. However, the mixed system (MR mi-showed the rather complicated pH profile,

    g the presence of pH-dependent multipleodes between P4501A1 and the reductase.


    eports have suggested the importance ofiring on the interactions between electron

    The effect of NaCl concentration on the Km (a) and theues of 7-ethoxycoumarin O-deethylation catalyzed bynd P450 1A1-reductase fused enzyme. The activities of2 (E) and AH22/pAFCR1 (F) microsomes were mea-mM sodium phosphate buffer (pH 7.4) at NaCl concen-50, 100, 200, and 400 mM.

  • donors anusing solucomplex bNADH-cytochromeinteractiotase, site-ical modisuggestedpositive chof NADPstructurenegative ctant roletochrome

    On theal. (27) regenase acThese stimseveral Preconstituand NAD(10, 11, 26repulsionon the suAccordingtions by swith neupears inco

    mentioned above. The repulsion model appears reason-able up to 0.15 of the ionic strength, however, it couldnot clearly explain the reduction of the activity by salts

    erhis1-erinphad402-4ratt mmad2

    FIG. 4. T(b) values o1A1 and P4pAMR2 (E)mM sodiumand 400 mM

    . CNA



    Vol. 257, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONSd acceptors. Chemical cross-linking studiesble carbodiimide revealed the formation of ay charge-pairing between cytochrome b5 andtochrome b5 reductase (20), and between cy-c and NADPH-P450 reductase (21). On then between P450 and NADPH-P450 reduc-directed mutagenesis (3, 5, 7, 22) and chem-fication studies (1, 2, 4, 6, 8, 23-25) havethe importance of charge-pairings betweenarges of...


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