Download pdf - 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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Biochemical and Biophysical Research Communications 257, 273–278 (1999)

Article ID bbrc.1999.0455, available online at http://www.idealibrary.com on

lectrostatic Interaction between Cytochrome P450 andADPH-P450 Reductase: Comparison of Mixed andused Systems Consisting of Rat Cytochrome450 1A1 and Yeast NADPH-P450 Reductase

hunya Kondo,* Toshiyuki Sakaki,* Hideo Ohkawa,† and Kuniyo Inouye*,1

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho,akyo-ku, Kyoto 606-8502, Japan; and †Department of Biological and Environmental Science,aculty of Agriculture, Kobe University, Rokkodai-cho, Nada-ku, Kobe 657-0013, Japan

eceived February 20, 1999

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The electrostatic interaction between rat cyto-hrome P450 1A1 and yeast NADPH-P450 reductaseas analyzed by using recombinant yeast microsomes

ontaining both native enzymes or their fused enzyme.he Vmax of the 7-ethoxycoumarin O-deethylation in

he recombinant microsomes containing both rat cy-ochrome P4501A1 and yeast NADPH-P450 reductasethe mixed system) was maximal when the ionictrength of the reaction mixture was 0.1-0.15. How-ver, on the fused enzyme between rat cytochrome450 1A1 and yeast NADPH-P450 reductase (the fusedystem), the activity was uniformly reduced with in-reasing ionic strength. The pH profiles of Vmax werelso different between the mixed and the fused sys-ems. Based on these results, we propose a hypothesishat cytochrome P450 and NADPH-P450 reductaseave more than one binding mode. The maximal activ-

ty of the mixed system at ionic strength of 0.1-0.15 isxplained by change of the binding mode. On the otherand, the fused enzyme appears to have only one bind-

ng mode due to the limited topology of cytochrome450 and NADPH-P450 reductase domains. © 1999

cademic Press

Cytochrome P450-dependent monooxygenase sys-ems are widely distributed in the biological kingdom.ukaryotic microsomal P450 monooxygenases consistf two membrane-bound enzymes, cytochrome P450P450) and NADPH-P450 reductase (reductase). The450 monooxygenation requires two electrons whichre transferred from NADPH through the reductase to450. In the last two decades, many reports on the

1 To whom correspondence should be addressed. Fax: 181-75-753-265. E-mail: [email protected] used: P450, cytochrome P450.

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nteraction between P450 and the reductase have beenublished. Most of the reports suggest the importancef charge-pairing involving negatively charged aminocids on the surface of the reductase (1-3) and posi-ively charged amino acids on the surface of P450 (4-8).ecently, x-ray crystallographic study revealed the ter-

iary structure of rat NADPH-P450 reductase (9), andtrongly suggested the significance of its surface poten-ial on the interaction with P450. On the other hand,oznesensky and Schenkman (10, 11) indicated that

he reduction rate of P450 by the reductase increasedy the neutralization of electrostatic interactions, sug-esting the significance of electrostatic repulsion be-ween the two enzymes.

To investigate the interaction between P450 and theeductase, we used the recombinant yeast microsomesroducing rat P450 1A1 and yeast NADPH-P450 re-uctase. On the basis of the facts that S. cerevisiae cellsave no detectable endogenous P450 in the cultureonditions of this study, and yeast NADPH-P450 re-uctase can transfer electrons to rat P450 1A1 asfficiently as rat NADPH-P450 reductase (12), the re-ombinant yeast microsomal membrane could be con-idered as a kind of a good reconstituted membraneontaining a single species of P450 and NADPH-P450eductase. In addition, we have succeeded in the ex-ression of genetically engineered P450 1A1-reductaseused enzyme in S. cerevisiae cells and revealed theapid intra-molecular electron transfer from the reduc-ase part to P450 1A1 part of the fused enzyme withigh monooxygenase activity under low ionic strength12). Thus, the fused enzyme appears a suitable modelo reveal the direct interaction between P450 and re-uctase. In this report, we will discuss the interactionetween P450 and reductase by comparing the effect ofonic strength on the monoxygenase activity of “the

ixed system” and “the fused system”.

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

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Vol. 257, No. 2, 1999 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATERIALS AND METHODS

Materials. The expression plasmids pAMR2 for rat P450 1A1 andeast NADPH-P450 reductase, pAFCR1 for the fused enzyme be-ween rat P450 1A1 and yeast NADPH-cytochrome P450 reductase,nd pARR3 for only yeast NADPH-cytochrome P450 reductase wereonstructed as reported previously (12, 13). S. cerevisiae AH22 wassed as a host. The following chemicals were used: 7-ethoxycoumarinrom Aldrich (Milwaukee, WI, U.S.A.); 7-hydroxycoumarin fromigma Chemical Co. (St. Louis, MO, U.S.A.); NADPH from Orientaleast (Tokyo, Japan). All other chemicals were of the best commer-ially available grade.

Preparation of microsomal fractions of the recombinant yeast cells.he recombinant S. cerevisiae AH22/pAMR2, AH22/pAFCR1 andH22/pARR3 cells were cultivated in a concentrated synthetic min-

mal medium containing 8% glucose, 5.4% yeast nitrogen base with-ut amino acids and 160 mg/l histidine as described previously (14).icrosomal fractions of the recombinant yeast cells were prepared as

escribed previously (15).

Western blot analysis. Microsomal fractions prepared from theecombinant cells were subjected to electrophoresis on a 4 to 20%inear gradient polyacrylamide-sodium dodecyl sulfate gel and thenransferred electrophoretically from the gel to nitrocellulose filter.he filter was probed with anti-yeast NADPH-P450 reductase Ig andhen followed by Enhanced Chemiluminescence immunodetectionethod (Amersham, Buckinghamshire, England).

Determination of the concentration of the P450 and NADPH-P450eductase. The concentration of P450 1A1 was determined from theeduced CO-difference spectrum using an extinction coefficient of 91M21 cm21 (16). The concentration of NADPH-P450 reductase in theicrosomal fraction was estimated on the basis of cytochrome c

eductase activity as described previously (12).

7-ethoxycoumarin O-deethylation. O-Deethylation of 7-ethoxy-oumarin in the recombinant yeast microsomal fractions was as-ayed by monitoring the formation of 7-hydroxycoumarin (17). Tostimate the initial rate of 7-hydroxycoumarin formation, the fluo-escence change was continuously monitored at 37°C with an exci-ation wavelength at 366 nm and an emission wavelength at 452 nmsing a Hitachi fluorescence spectrophotometer 850 (Tokyo, Japan).he reaction mixture contains 10-200 mM sodium phosphate buffer,H 7.4, 0-400 mM sodium chloride or 0-400 mM potassium chloridend 0-60 mM of 7-ethoxycoumarin, 1.0% methanol, 400 mM ofADPH and the microsomal fraction prepared from AH22/pAMR2

ells containing P450 1A1 and yeast NADPH-P450 reductase (MRicrosomes), or AH22/pAFCR1 cells containing the P450 1A1-

eductase fused enzyme (FCR microsomes). The reaction was initi-ted by the addition of NADPH, and the fluorescence change wasontinuously monitored for more than 5 min. At the end of theonitoring, a known amount of 7-hydroxycoumarin was added to the

eaction mixture in order to calibrate the fluorescence change.he concentration of 7-ethoxycoumarin, 7-hydroxycoumarin, andADPH were determined by using extinction coefficients of 13.56M21 cm21 at 320 nm, 14.5 mM21 cm21 at 324 nm, and 6.2 mM21

m21 at 339 nm, respectively. The Michaelis constant, Km, and max-mum velocity, Vmax were determined using a nonlinear least-squares

ethod applied to the Michaelis-Menten equation (18).

NADPH oxidation. In order to calculate the coupling efficiencyetween 7-hydroxycoumarin formation and NADPH consumption,he initial rate of NADPH oxidation in the reaction mixture contain-ng 100 mM NADPH was measured at 7-ethoxycoumarin concentra-ions of 0 and 50 mM, respectively, by continuous recording of absor-ance at 339 nm with a Shimadzu UV-2200 spectrophotometerKyoto, Japan) at 37°C. The difference of NADPH oxidation between0 and 0 mM of 7-ethoxycoumarin was considered as the P4501A1-ependent NADPH oxidation because the difference was observed inR microsomes and FCR microsomes, but not in the control micro-

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omes. The initial rate was calculated using a molar absorptionhange of NADPH oxidation, D« 5 6,200 M21 cm21. The absorptionhange derived from the formation of 7-hydroxycoumarin from-thoxycoumarin was subtracted to determine the exact NADPHxidation while it gave a small influence.

ESULTS

Expression of P450 1A1, NADPH-P450 reductase,nd P450 1A1-reductase fused enzyme in yeast. Theontent of P450 1A1 and the fused enzyme in MRicrosomes and FCR microsomes were estimated to be

.27 and 0.11 nmol/mg protein, respectively, on theasis of reduced CO difference spectra. Western blot-ing analysis suggested that the most of the fusednzyme in FCR microsomes remains intact (Fig. 1)ith no proteolytic degradation. The content ofADPH-P450 reductase in MR microsome was esti-ated to be 0.24 nmol/mg protein on the basis of the

ytochrome c reductase activity of the purified yeastADPH-P450 reductase sample (12, 19).

Effects of salts on 7-ethoxycoumarin O-deethylationctivity of P450 1A1 and P450 1A1-reductase fusednzyme. 7-Ethoxycoumarin O-deethylation activity ofR microsomes containing both P450 1A1 and the

eductase, FCR microsomes containing P450 1A1-eductase fused enzyme and the control AH22/pARR3icrosomes containing no P450 1A1 was each mea-

ured in the reconstituted system. Since the con-rol microsomes showed no detectable activity,-hydroxycoumarin formation in MR microsomes andCR microsomes was found to depend on P450 1A1 andhe fused enzyme, respectively.

The effect of the ionic strength in the reaction mix-ure was examined with the substrate concentrationrom 0 to 60 mM. The catalytic activity of MR micro-omes was strongly affected by the salt concentrationn the reaction mixture. While the Km was not affected,

FIG. 1. Western blot analysis of microsomal fractions preparedrom recombinant yeast cells. The microsomal fraction was electro-horesed and then probed with anti-yeast NADPH-P450 reductaseg. Lane 1, AH22/pARR3; lane 2, AH22/pAMR2; lane 3, AH22/AFCR1. FE and YR indicate the migrating points of P450A1-reductase fused enzyme and yeast NADPH-P450 reductase,espectively.

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he Vmax increased with increasing the concentration ofodium phosphate from 10 mM to 40 mM, and de-reased with increasing the concentration of sodiumhosphate from 80 mM to 200 mM (Fig. 2). The maxi-um activity was obtained in the range of 40 ; 80 mM.hen NaCl was added to 20 mM sodium phosphate

uffer (pH 7.4) (Fig. 3), the Vmax increased with increas-ng the concentration of NaCl from 0 to 50 mM, andecreased with increasing the concentration of NaClrom 50 to 400 mM. Similar results were obtained withCl while the maximum activity was obtained at 100M (Fig. 4). From these results with three kinds of

alts, it is strongly suggested that the ionic strength isn essential factor to determine Vmax.7-Ethoxycoumarin O-deethylation of FCR micro-

omes was measured under the same reaction condi-ions as MR microsomes. The catalytic activity of FCRicrosomes was also affected by the sodium phosphate

Fig. 2) and KCl (Fig. 4) in the reaction mixture, how-ver, the maximum Vmax was obtained at lower saltoncentration than MR microsomes and no significantctivation by salts was observed. The addition of NaClrom 0 mM to 400 mM uniformly decreased the activitys shown in Fig. 3.Throughout the measurement of 7-ethoxycoumarin-deethylation of MR microsomes and FCR microsomes,o significant change of the Km value was observed withhe addition of the salts (Figs. 2-4). Salts affected the Vmax

alue of 7-ethoxycoumarin O-deethylation, however, it

FIG. 2. The effect of sodium phosphate buffer (pH 7.4) concen-ration on the Km (a) and the Vmax (b) values of 7-ethoxycoumarin-deethylation catalyzed by P450 1A1 and P450 1A1-reductase fusednzyme. The activities of AH22/pAMR2 (E) and AH22/pAFCR1 (■)icrosomes were measured in 10, 20, 30, 40, 60, 80, 100, 120, 150,

nd 200 mM sodium phosphate buffer (pH 7.4).

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ave no significant effect on the coupling efficiency be-ween 7-hydroxycoumarin formation and NADPH oxida-ion, except for FCR microsomes in the presence of 400M of NaCl or KCl as shown in Fig. 5. These results

trongly suggest that salts influence the electron transferrom the reductase to P4501A1 in MR microsomes andhat from the reductase domain to P450 domain of fusednzyme in FCR microsomes. However, the clear differ-nce was observed between the mixed system (MR mi-rosomes) and the fused system (FCR microsomes) on thenfluence of the salts.

Effect of pH on 7-ethoxycoumarin O-deethylation activ-ty of P450 1A1 and P450 1A1-reductase fused enzyme.he pH profile of 7-ethoxycoumarin O-deethylation ac-ivity was clearly different between MR microsomesnd FCR microsomes as shown in Fig. 6. The fusednzyme in FCR microsomes showed a bell-shape-likeH profile with a maximum Vmax at pH 7.0-7.5, suggest-ng the simple binding mode between P4501A1 and theeductase parts. However, the mixed system (MR mi-rosomes) showed the rather complicated pH profile,uggesting the presence of pH-dependent multipleinding modes between P4501A1 and the reductase.

ISCUSSION

Many reports have suggested the importance ofharge-pairing on the interactions between electron

FIG. 3. The effect of NaCl concentration on the Km (a) and themax (b) values of 7-ethoxycoumarin O-deethylation catalyzed by450 1A1 and P450 1A1-reductase fused enzyme. The activities ofH22/pAMR2 (E) and AH22/pAFCR1 (F) microsomes were mea-ured in 20 mM sodium phosphate buffer (pH 7.4) at NaCl concen-ration of 0, 50, 100, 200, and 400 mM.

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onors and acceptors. Chemical cross-linking studiessing soluble carbodiimide revealed the formation of aomplex by charge-pairing between cytochrome b5 andADH-cytochrome b5 reductase (20), and between cy-

ochrome c and NADPH-P450 reductase (21). On thenteraction between P450 and NADPH-P450 reduc-ase, site-directed mutagenesis (3, 5, 7, 22) and chem-cal modification studies (1, 2, 4, 6, 8, 23-25) haveuggested the importance of charge-pairings betweenositive charges of P450 molecule and negative chargesf NADPH-P450 reductase molecule. The tertiarytructure of rat NADPH-P450 reductase revealed itsegative charged surface which would play an impor-ant role in the formation of charge-pairings with cy-ochrome c, b5 and P450 (9).

On the other hand, Schenkman et al. (26) and Yun etl. (27) revealed the enhancement of P450 monooxy-enase activity by salts up to 0.15 of the ionic strength.hese stimulatory effects of salts were observed witheveral P450 species in both microsomal fraction andeconstituted systems consisting of the purified P450nd NADPH-P450 reductase (26). Schenkman et al.10, 11, 26) explained these phenomena by giving “theepulsion model” suggesting the existence of repulsionn the surfaces of P450 and NADPH-P450 reductase.ccording to this model, the stimulation of P450 reac-

ions by salts is caused by a reduction of the repulsionith neutralization of charges. This hypothesis ap-ears inconsistent with the “the charge-pairing model”

FIG. 4. The effect of KCl concentration on the Km (a) and the Vmax

b) values of 7-ethoxycoumarin O-deethylation catalyzed by P450A1 and P450 1A1-reductase fused enzyme. The activities of AH22/AMR2 (E) and AH22/pAFCR1 (F) microsomes were measured in 20M sodium phosphate buffer at KCl concentration of 0, 50, 100, 200,

nd 400 mM.

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entioned above. The repulsion model appears reason-ble up to 0.15 of the ionic strength, however, it couldot clearly explain the reduction of the activity by saltst higher ionic strength.In this report, the great influence of salts on

4501A1-dependent 7-ethoxycoumarin O-deethylationas observed. In the mixed system, as the salt concen-

ration increased, the activity increased up to 60 mModium phosphate or 100 mM KCl or 50 mM NaCl, andhen gradually decreased up to 200 mM sodium phos-hate, 400 mM NaCl and 400 mM KCl, respectivelyFigs. 2-4). Although our heterologous system con-ained rat P450 1A1 and yeast NADPH-P450 reductasen yeast microsomes, these results are quite similar tochenkman’s studies using rabbit microsomal frac-ions and reconstituted systems consisting of P450 1A2r P450 2B4, and rabbit NADPH-P450 reductase (26).

FIG. 5. Coupling efficiency between 7-hydroxycoumarion forma-ion and NADPH oxidation. The coupling efficiency in the reactionixture containing AH22/pAMR2 (E) and AH22/pAFCR1 (F) micro-

omes in 10-200 mM sodium phosphate buffer (pH 7.4) (a), in 20 mModium phosphate buffer (pH 7.4) at a NaCl concentration of 0-400M (b), and at a KCl concentration of 0-400 mM (c) was calculated.

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hus, it is suggested that the interaction mode be-ween P450 and the reductase is basically commoneyond species difference.Our previous data strongly suggest that the fused sys-

em mimics 1:1 functional complex of P450 and NADPH-450 reductase with efficient electron transfer at low

onic strength (12). Based on the putative topologicaleature of the fused enzyme, the interaction between450 and the reductase domains seems more simplified

han the mixed system. No significant enhancement ofhe activity by the salt was observed in the fused systemFigs. 2-4). To explain these results in both mixed andused systems consistently, we propose a hypothesis that450 and NADPH-P450 reductase have more than oneinding mode including both suitable and unsuitableodes for electron transfer in the mixed system. Namely,

ome charge-pairings on the surface of P450 and reduc-ase may prevent the complex formation for an efficientlectron transfer. At extremely low ionic strength,harge-pairings unsuitable for electron transfer appearo be dominantly formed. Neutralization of the chargesy salts leads to the formation of the suitable complex forlectron transfer. The reduction of the activity by salts athe ionic strength higher than 0.15 could be explained bynhibition of formation of the suitable complex for elec-ron transfer. Thus, these two contrary effects of saltsould generate the optimum ionic strength for the P450

eaction. It is noteworthy that the ionic strength for theaximum activity is nearly the same as that of physio-

ogical condition (27). In the case of the fused enzyme, on

FIG. 6. The effect of pH on the Km (a) and the Vmax (b) values of-ethoxycoumarin O-deethylation catalyzed by P450 1A1 and P450A1-reductase fused enzyme. The activities of AH22/pAMR2 (E) andH22/pAFCR1 (F) microsomes were measured in 20 mM sodiumhosphate buffer at pH of 6.5, 7.0, 7.4, and 8.0.

277

he other hand, the topological freedom of P450 and theeductase domains appeared to be limited as comparedith the mixed system. No significant stimulation of thectivity by salts suggests that only one binding modeetween P450 and the reductase parts should be permit-ed for the fused enzyme. The difference of the pH profileetween the mixed and the fused systems (Fig. 6) mayuggest the difference of the binding mode between thewo systems. Recently, Yun et al. (27) suggested thatalts caused the conformational change of P450 1A2 andncreased the affinity of P450 1A2 for the reductase.hose observations seem consistent with our resultsince conformational change is directly related with theinding mode between P450 and the reductase. Thus, theonformational change of P450 by salts might be essen-ial for the formation of charge-pairings and the bindingodes between P450 and the reductase.In this report, we propose a new concept on the

nteraction between P450 and NADPH-P450 reductaseuggesting the presence of at least two binding modesased on the comparison between the mixed systemnd the fused system. Thus, the genetically engineeredused enzyme gives us useful information on the inter-ction between P450 and NADPH-P450 reductase.

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