THE JOURNAL OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 22, Issue of June 3, pp. 15724-15731, 1994 Printed in U.S.A.

Quantitative Analyses of Electrostatic Interactions between NADPH-Cytochrome P450 Reductase and Cytochrome P450 Enzymes*

(Received for publication, October 18, 1993, and in revised form, February 7, 1994)

Andrei I. Voznesensky and John B. SchenkmanS From the Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030

A decline in the ionic interactions in the medium with increasing ionic strength (decrease in the ionic activity coefficients) was accompanied by an increase in the fast phase rate constants of CYP2B4 and CYPlA2 reduction. The stimulations were observed both in reconstituted P450 systems and in microsomes. An increase in the ionic strength from 10 to 100 n m sodium phosphate re- sulted in a 7-fold decrease in the K,,, of CYPlA2 for NADPH-cytochrome P450 reductase, while the V,, was unchanged. When ionic interactions were neutralized without changing the ionic strength by addition of charged oligopeptides (polylysine and polyglutamic acid), stimulations of CYPlA2 and CYP2B4 reduction were observed. Increase in the ionic strength also en- hanced the rate of cytochrome P450 reduction in control and phenobarbital-induced rat liver microsomes and in reconstituted systems containing purified rat liver CYP2C6, CYP2C12, CYP2C13, and CYP2E1, and rat re- ductase. A method was devised for the quantification of the number of charges involved in protein-protein inter- actions based on the estimation of the ionic activity co- efficients. Different numbers of charged residues are in- volved in the repulsion between different P450 forms and the reductase. The product of the number of charges involved in the interaction between rabbit reductase and CYP2B4 is 10.84 compared with the value of 6.64 for the reductase-CYPlA2 interaction.

~ ___ _ _ _ _ _ ~

Microsomal cytochrome P450 (P450)’ isozymes are integral membrane proteins responsible for the metabolism of the vari- ety of endogenous and exogenous compounds. The monooxy- genation cycle carried out by P450 requires sequential input of two electrons. These electrons are supplied by another integral membrane protein, NADPH-cytochrome P450 reductase (re- ductase). Reduction of P450 is a relatively slow process requir- ing the formation of a hemoprotein-flavoprotein complex. Forces holding the two proteins in the complex have not been fully defined. It was suggested that electrostatic interactions play a major role in the complex formation and that P450 and reductase are held together by attraction of complementary

* This work was supported in part by United States Public Health Service Grant GM26114 from the National Institute of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. i To whom corresDondence should be addressed. Tel.: 203-679-3694:

Fax-203-679-2473. * The abbreviations used are: P450, cytochrome P450; CYPlA2,

CYP2B4, CYP2C6, CYP2C12, CYP2C13, CYP2E1, cytochrome P450 forms are referred to according to the latest nomenclature (Nelson et al., 1993); DLPC, dilauroyl phosphatidylcholine; reductase, NADPH-cyto- chrome P450 reductase.

charged residues (Bernhardt et al . , 1988; Nadler and Strobel, 1988; Shimizu et al., 1991). Recently we showed that this is not the case for CYP2B4; electrostatic forces inhibit the interaction of CYP2B4 and reductase (Voznesensky and Schenkman, 1992a, 1992b).

P450 is a superfamily of proteins consisting of a number of families (Nelson et al., 1993). P450 enzymes have considerable differences in their amino acid sequence and an effect observed with one of them would not necessarily apply to other micro- somal P450 forms. Neutralization of electrostatic interactions facilitated the formation of the reductase-CYP2B4 complex (Voznesensky and Schenkman, 1992a). Since this effect could be a unique property of this particular form or the P450 2B family, we studied the influence of charge neutralization on the interaction of reductase and several individual forms of P450. We report that in all cases electrostatic forces were inhibitory of the reductase-P450 interactions but the extents of inhibition were different for the individual P450 forms. A method was developed which allowed us to quantitate the number of charges involved in the electrostatic interaction.

MATERIALS AND METHODS Purification ofProteins-In this study 3-kg male New Zealand rab-

bits were used. p-Naphthoflavone-induced liver microsomes were pre- pared after daily treatment of rabbits with 17 mg of p-naphthoflavone per kg for 2 days. The microsomal P450 content was 3.3 nmoVmg pro- tein. Phenobarbital-induced rabbit liver microsomes were prepared af- ter daily treatments with 80 mg of sodium phenobarbital per kg intra- peritoneally for 4 days. The P450 content was 3.4 nmol/mg protein and CYP2B4 was purified as described (Haugen and Coon, 1976). CYPlA2 was isolated from untreated rabbit liver microsomes by a modified method (Alterman and Dowgii, 1990). Briefly the procedure was as follows: 500 ml of microsomes (7.5 mg/ml) in buffer A (50 mM Tris-HC1, pH 7.6, 0.1 mM EDTA, 20% glycerol) were solubilized with a combina- tion of detergents (sodium cholate, 0.625% final, and Triton N-101, 1.25% final). The solution was then applied to sequentially connected DEAE-Sepharose (2.6 x 33-cm) and CM-Sepharose (2.6 x 32-cm) col- umns equilibrated with buffer A containing 0.1% Triton N-101. The columns were washed with buffer A containing 0.1% Triton N-101 and then disconnected. The CM-Sepharose was washed with 50 mM sodium phosphate, pH 7.4,20% glycerol, 0.3% Triton N-101,O.l mM EDTA(200 ml), then with 100 mM sodium phosphate, pH 7.4, 20% glycerol, 0.3% Triton N-101, 0.1 mM EDTA (100 ml). CYPlA2 was eluted from the CM-Sepharose column with a linear gradient of NaCl(550 ml total, 0 to 150 mM) in 100 mM sodium phosphate, pH 7.4,20% glycerol, 0.3% Triton N-101, 0.1 mM EDTA. Fractions containing CYPlA2 (middle of the gradient) were pooled, dialyzed against 30 mM sodium phosphate, pH 7.4,20% glycerol, 0.3% Triton N-101,O.l mM EDTA, and reapplied to the CM-Sepharose column and eluted with the same NaCl gradient. CYPlA2 containing fractions were pooled, dialyzed against 30 mM so- dium phosphate, pH 7.4, applied to a hydroxyapatite column (1.6 x 20 cm), and eluted with a gradient of 30 to 200 mM sodium phosphate, pH 7.4, 20% glycerol, 0.3% Triton N-101, 0.1 mM EDTA. Detergent was removed from the pure CYPlA2 on a hydroxyapatite column (1.6 x 12 cm). Rabbit NADPH-cytochrome P450 reductase was purified by the method of Dignam and Strobel (1975). Rat hepatic microsomal CYP2C6, CYP2C12 (Schenkman et al., 1986), CYP2C13 (Cheng and Schenkman,

15724

Quantifying Interacting P450 and Reductase Charges 15725 1982), and CYP2E1 (Favreauet al., 1987) were purified as described. All enzymes were electrophoretically homogeneous on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970).

Analytical Measurements-Conductivity of solutions was measured at 20 "C with a YSI model 32 conductance meter (Yellow Springs In- strument Co., Yellow Springs, OH). Absorbance spectra were recorded on a Shimadzu UV3000 Spectrophotometer (Shimadzu Co., Japan). P450 levels were quantitated using an extinction coefficient of E450-49n = 91 mM -l cm" for the reduced CO-complexed minus reduced form in difference spectra (Omura and Sato, 1964). NADPH-PI50 reductase was quantitated using the extinction coefficient, E,,, nm = 21.4 mM-l cm-' (Vermilion and Coon, 1978). Protein was measured by bicinchoninic acid protein assay (Pierce Chemical Co.). NADPH-cytochrome -c reduc- tase activity was measured at 20 "C as described (Voznesensky and Schenkman, 1992a). Electron transfer to P450 was monitored at 20 "C with a Dionex stopped-flow spectrophotometer exactly as described ear- lier (Voznesensky and Schenkman, 1992a). The DLPC reconstituted system used in the experiments with pure P450 forms was prepared as described earlier (Jansson et al., 1985). Briefly, P450 and reductase were mixed with DLPC vesicles (DLPC/protein = 8011, P450/reductase = loll) and preincubated for 1 h at 20 "C before initiation of the reac- tion. Poly-L-lysine (average M, 3900) and poly-L-glutamic acid (average M , 6200) were obtained from Sigma. All reagents were of the highest purity available and were not purified further.

Determination of log1,(y)4onductivity of the medium used for the study of protein-protein interactions was directly measured in a series of solutions which differed only by the salt Concentration. Analysis of the data using the procedure described below then provided the infor- mation about log,,(y ) under exact experimental conditions.

Concept-The Debye-Huckel theory predicts that activity coefficient of an ion of a unit charge relates to the mean activity coefficient of the medium (y,) as

log1ncY) = (log,ncY~ ))/(I 2- 12,) (Eq. la)

where z_ and z, are the charges of the ions of the medium. The mean activity coefficient y, can be estimated by measurement of the conduc- tivity of the medium.

For a solution of a univalent salt at infinite dilution conductivity due to positive and negative ions (k, and k-) is

k, + A,c+, k - = k c _ (Eq. 1b)

Where c,, c-, and A+, A_ are the concentrations and molar ion conduc- tivities of cations and anions, respectively. Under conditions other than infinite dilution we have to use activities instead of concentrations (a, = c+y+, a_ = c-y-). Activity coefficients y, and y- reflect the deviations from ideal behavior due the interaction of ions with the ionic atmos- phere in the solution.

k, = A+c+y+, k _ = A-c-y- (Eq. IC)

multiplying the equations we have

k+k- = A + L c , c - y ~ ~ (Eq. Id)

taking into account that k, = k_ = Yik, where k is the total conductivity, that for a univalent salt c, = c_ = c, where c is the salt concentration and that by definition y,2 = y ~ -

k2/c2 = 4h+A_y,' or klc =By, (Eq. le)

where B = (4A+AJx is a constant equal to k/c at infinite dilution. The value of B can be calculated by extrapolation of k/c to zero ionic strength. This relationship allows us to estimate y, and log,,( yJ

Y ? = - and log,,(y_) = log,, - B ( (ki) 1 (Eq. 2a)

log,,(y) can then be calculated using Equation la. The above equations for the analysis of the conductivity data were derived for a univalent salt. To apply them to other substances, such as sodium phosphate, we have to correct for incomplete dissociation using the pK, and treat the compound as a univalent salt dissociating into positive ions and nega- tive ions of a fractional charge corresponding to the average charge of the anion.

Procedure-The procedures used for the estimation of log,,(y) is as follows. 1) A series of solutions having the composition of the assay medium, but of varying salt concentration, was prepared and their conductivity (k ) was measured. 2) We calculated klc, where c is the concentration of cations, and determined klc at zero ionic strength (I)

from the plot of log,,(klc) as a function of Is. This gave the value of B in Equation 2. 3) We calculated log,,(yJ using Equation 2a and then log,,( y using Equation la.

The procedure was verified using a series of sodium chloride and sodium phosphate solutions. There was a less then 3% difference be- tween the published values for y, of NaCl in water (Moore, 1965) and the values obtained by the procedure described above (data not shown). The plot of log,,( y,) uersus I% for sodium phosphate had a slope 1.6 times higher than the one for sodium chloride which agrees with the differ- ence in anion charges (1.6 is an average anion charge of sodium phos- phate under our experimental conditions).

RESULTS

Influence of Salt on Ionic Znteractions-One of the ways to study the role of electrostatic forces in protein-protein interac- tions is to analyze the effect of charge neutralization on the complex formation. Protein-protein ionic interactions can be gradually and gently neutralized by increasing the concentra- tion of salt ions in the medium. The physicochemical basis of the effect is a decrease in the ion's activity coefficient with increasing ionic content of the medium. If there were no elec- trostatic interactions the behavior of the ions would be ideal and the Gibbs energy of an ion of type i would be described by

G, = GP + kT ln(c,) (Eq. 2b)

where ci is the concentration of the ion, k is the Boltzmann constant, and T is the absolute temperature. In reality Gibbs energy of an ion is

G, = G; + kT ln(c,y,) = GP + kTln(c,) + kT lnb,) (Eq. 2c)

An additional term KT ln(y,) has to be introduced because part of the ion's energy is consumed by the interactions with the ionic atmosphere. yi is the ion's activity coefficient. For practi- cal purposes the situation can be simplified to partial neutral- ization of ions by the ionic atmosphere, with yi as the coefficient reflecting the degree of such neutralization. The effect is not ion-specific and equally affects all ions in the medium. Activity coefficients of charges on the protein surface are also propor- tionally decreased, because from an electrostatic point of view there is no difference between salt ions in solution and charged amino acid residues. The rate constant of a reaction between charged molecules is proportional to the ion's activity coeffi- cient (Laidler, 1978). In order to relate effects of charges on protein-protein interactions one needs a way to measure a change in the activity coefficients of charged residues on the protein surface. The Debye-Huckel limiting law predicts log,,(y) to be proportional to the square root of ionic strength. Calculation of the activity coefficient of an ion as a function of ionic strength is subject to errors because the Debye-Huckel limiting law is satisfactorily obeyed only at very low salt con- centration (ionic strength below 10 m). In most biochemical studies a much higher ionic strength has to be used. To solve this problem one needs to choose a measurable rather than a calculated parameter reflecting the change in the activity co- efficients. We found that the variation of molar conductivity can serve as a measurable parameter for the estimation of the change in the activity coefficients of the ions under experimen- ta l conditions. The conductivity of the medium can be easily measured in a series of solutions with increasing salt concen- tration. The change in the logarithm of the activity coefficient (log,,(y)) can then be calculated as described under "Materials and Methods." Since activity coefficients of all ions in solution change proportionally, the value obtained reflects not only the variation in the activity Coefficients of salt ions but also the change in the activity coefficients of charges on the protein surface.

Effect of Salt on Reductase-P450 Interactions-Reduction of P450 by the reductase is biphasic, with a rapid initial phase

15726 Quantifying Interacting P450 and Reductase Charges

2.5

2.0

1.5

1 . 0

0.5

0.00 0.05 0.10 0.15 J

FIG. 1. Dependence of the fast phase rate constant of microso-

somes of the phenobarbital-treated rabbit were suspended to a final mal P450 reduction on the ionic activity coefficient. Liver micro-

P450 concentration of 1 p ~ . Ionic activity coefficient was changed by varying the concentration of sodium phosphate buffer (from 1 to 200 mM, pH 7.4) (0) or by varying the concentration of sodium chloride (from 0 to 300 mM) in 10 mM sodium phosphate (V). log&) was calculated

Methods.” and P450 reduction was monitored as described under “Materials and

followed by a subsequent slower phase. The amount of P450 reduced in the fast phase correlates with the high spin content of some forms of the hemoprotein (Backes and Eyer, 1989; Backes et al., 1985; Schwarze et al., 1985). The rate constant of the spin shift, however, is much faster than the reduction rate constant and the mechanism responsible for the lack of monophasic kinetics remains to be elucidated. In the range of ionic strength used we did not observe any ionic strength-de- pendent spectrophotometrically detectable variation in the spin state equilibrium of the P450 (data not shown); this would indicate that reductase-P450 interactions rather than the changes in the spin state of the P450 are responsible for the changes in the hemoprotein reduction.

If the reductase-P450 complex is formed by attraction of oppositely charged amino acid residues on the two proteins, neutralization of surface charges will lead to a decreased rate of reduction. On the other hand, if there is a charge repulsion between the two proteins, a decline in the ionic activity coeffi- cients will facilitate the reaction. In agreement with our pre- vious reports on the inhibitory influence of electrostatic forces on reductase-CYP2B4 interactions (Voznesensky and Schenk- man, 1992a, 1992b), a 0.12 reduction of log,,(y) (an increase in sodium phosphate concentration from 1 to 200 mM) was accom- panied by an 11-fold increase in the apparent rate constant of the fast phase of P450 reduction in rabbit liver phenobarbital- induced microsomes (Fig. 1). The same effect was observed when sodium chloride was used instead of sodium phosphate as an ion source (Fig. 1). The fraction of the fast phase of the reduction and the apparent rate constant of the slow phase were affected to a lesser extent (Table I).

The same approach was used to investigate the role of

TABLE I Influence of the change in the ionic activity coefficients on the

reduction of CYF’2B4 Phenobarbital-treated rabbit liver microsomes were suspended to 1

1.1~ P450 in the indicated medium. P450 reduction was monitored as described under “Materials and Methods.”

Composition of the medium -logdy) kfeat kdow fast phase

Percent as

s” s-1 lb Sodium phosphate (1 to 200 m ~ ) , 0.006 0.2 4.26 50

pH 7.4 0.016 0.45 4.56 55 0.051 0.4 6.46 60 0.070 1.01 8.39 67 0.101 1.87 9.43 70 0.129 2.28 7.68 72

Sodium chloride (0 to 300 mM), 0.047 0.46 6.98 58 in 10 mM sodium phosphate, 0.048 1.07 9.50 65 pH 7.4 0.065 1.10 9.81 68

0.079 1.33 10.43 71 0.106 1.77 8.13 74

charged residues in the interaction of the reductase with a member of a different P450 family, CYPlA2. We monitored the reduction of rabbit CYPlA2 in the reconstituted system at dif- ferent ionic strengths (Table 11). When the concentration of the sodium phosphate buffer was increased, the reduction of CYPlA2 was facilitated. In all cases reduction of P450 re- mained biphasic and P450 was fully reducible by the reductase. An increase in -log,,(y) by 0.065 (increase in the phosphate buffer concentration from 10 to 100 mM) was accompanied by a 16-fold increase in the apparent rate constant of the fast phase of CYPlA2 reduction. The fraction of the P450 reduced in the fast phase of the reaction and the rate constant of the slow phase were much less affected.

The observed effect of charge neutralization on CYPlA2 re- duction was not unique to the reconstituted system. Reduction of P450 in the p-naphthoflavone-induced rabbit liver micro- somes was also facilitated when ion activity coefficients were deceased. /3-Naphthoflavone induces CYPlA2 in the liver en- doplasmic reticulum and this isozyme is a predominant cyto- chrome P450 isoform in the liver microsomes of p-naphthofla- vone-treated rabbits (Wagner et al., 1987). An increase in -log,,(y ) by 0.054 (increase in ionic strength from 10 to 100 mM sodium phosphate) was accompanied by a 4-fold increase in the rate constant of the fast phase (Table 111). The fraction of the P450 reduced in the fast phase of the reaction and the rate constant of the slow phase were only slightly affected.

Qualitatively, the effect of increasing salt concentration on CYPlA2 reduction in microsomes was similar to that observed in the reconstituted system. In both cases when charges on the proteins were neutralized by increasing ionic strength from 10 to 100 mM sodium phosphate, a substantial increase in the rate constant of the fast phase was observed. When ionic strength was raised even higher, a decline in the rate constant of the fast phase was seen (data not shown). Interpretation of the decline is very difficult because at such a high ionic strength, salt- induced changes in the protein structure become a real possi- bility. Rate constants of the slow phase of the reduction were less sensitive to ionic strength. Quantitative differences in the extent of the stimulation between P450 in microsomes and in the reconstituted system are possibly due to the presence of other microsomal proteins that interact with CYPlA2 and the reductase.

Influence of Polyanionic and Polycationic Probes-To confirm that the observed effect is due to electrostatic interactions be- tween the proteins, we used short homooligopeptides of charged amino acids, polylysine and polyglutamic acid. Polyly- sine bears multiple positive charges and binds to anionic cen-

Quantifying Interacting P450 and Reductase Charges 15727

TABLE 11 Influence of the change in the ionic activity coeficients on the

reduction of CYPlA2 in the reconstituted system Reduction of CYPlA2 in DLPC reconstituted system (CYPlA2/

reductase = lO/l, DLPC/protein = 80/1 mol/mol) at varying concentra- tion of sodium phosphate buffer (from 10 to 100 mM), pH 7.4, 20% glycerol was monitored as described under “Materials and Methods.”

-log,,(Y) kr,, ks,ow fast phase Percent as

s-* 10 s-I 102

0.005 0.37 1.19 21 0.032 2.50 1.29 47 0.055 5.39 1.07 60 0.070 5.86 1.06 56

TABLE I11 Influence of the change in the ionic activity coeficients on the reduction of P450 in P-naphthoflavone-induced rabbit liver

microsomes P-Naphthoflavone-induced rabbit liver microsomes were suspended

to 1 p P450 in varying concentration (from 10 to 100 mM) of the sodium phosphate buffer, pH 7.4. P450 reduction was monitored as described under “Materials and Methods.”

-log,,(Y) kt,,, kd,, Percent as fast phase

s-I 10 s-I 102

0.016 1.17 3.53 66 0.029 3.30 3.44 73 0.051 4.46 3.42 84 0.070 4.99 4.44 76

ters on the protein surface (Cheddar and Tollin, 19901, while negatively charged polyglutamic acid binds to cationic centers. Such binding of polylysine to cytochrome-c peroxidase was shown to interfere with the charge-pairing of cytochrome c with cytochrome-c peroxidase (Mochan, 1970). Micromolar concen- trations of polylysine had a negligible influence on the ionic strength of the medium but resulted in an almost %fold in- crease in the initial rate of the CYP2B4 reduction. I t also in- creased the initial rate of CYPlA2 reduction by 60% (Table IV). When polyglutamic acid was used to block protein surface charges, a 2-fold increase in the initial rate of CYPlA2 reduc- tion was observed. The initial rate of CYP2B4 reduction, how- ever, was not affected by polyglutamic acid (Table IV). Stimu- lation of CYPlA2 and CYP2B4 reduction by polylysine and stimulation of CYPlA2 reduction by polyglutamic acid indi- cates that electrostatic forces inhibit interaction of both P450 isozymes with the reductase. “he lack of effect of polyglutamic acid on CYP2B4 reduction suggests that the local distribution of charged residues on the protein surface involved in the in- teraction with the reductase is different for the two forms of P450.

Earlier we reported that neutralization of charges by in- crease in the ionic strength of the medium decreased the ap- parent K, of CYP2B4 for the reductase almost 26-fold without affecting the V,,, suggesting tighter binding of the P450 to the flavoprotein at higher ionic strength (Voznesensky and Schenk- man, 1992a). To determine the influence of ionic strength on the K,,, of CYPlA2 for the reductase, initial rates of CYPlA2 reduction were measured at various reductase concentrations in 10 and 100 mM sodium phosphate buffer. At all concentra- tions of the reductase, rates in 100 mM phosphate were higher than the corresponding rates in 10 mM buffer (Fig. 2). The iterative fit of the data to the Michaelis-Menten equation gave an apparent K,,, value of 0.88 p~ for CYPlA2 reduction in 10 mM phosphate, while the K, in 100 mM buffer was 7-fold lower (0.12 p ~ ) . At the same time the V,, did not change (0.032 p~ s-’ both in 10 and 100 mM buffer). These data indicate that the phenomenon that we observed with CYP2B4 also applies to

TABLE N Influence of polylysine and polyglutamate on the initial rate of

CYPIA2 and CYP2B4 reduction CYPlA2 or CYP2B4 was reconstituted with the reductase in DLPC

(P450/reductase = lO/l, DLPC/protein = 80/1 mol/mol). Initial rate of

glycerol or in the above buffer containing either 1 PM polylysine or 2 PM reduction was monitored in 5 m~ sodium phosphate buffer, pH 7.4,20%

polyglutamate as described under “Materials and Methods.” Initial rate in 5 mM sodium phosphate served as a control and was 3.2 x p d s for CYPlA2 and 2.9 x p d s for CYP2B4.

P450 form “0

1 pM Poly-Lys 2 p~ Poly-Glu

CYPlA2 % of control

160 200 CYP2B4 280 105

NADPH-cytochrome P450 reductase (pM)

FIG. 2. Relationship between the initial rates of CYPlA2 re- duction and the concentration of the reductase. CYPlA2 and reductase were incorporated into the DLPC vesicles at the molar ratio, protein/DLPC, of 1/80 and vesicles were suspended in 10 mM (0) or 100 mM (a) sodium phosphate buffer, pH 7.4,20% glycerol. All preparations contained 0.5 p P450 and varying amounts of reductase (from 0.01 to 1.5 pd. P450 reduction was monitored as described under “Materials and Methods.” Solid lines were drawn using K, and V,, obtained by an iterative fit of the data to the Michaelis-Menten equation.

CYPlA2, i.e. P450 has higher affinity for the reductase in the medium with higher ionic strength.

Influence of Ionic Strength on the Reduction of Rat Forms of P450”Since shielding of charges by increasing ionic strength facilitated reduction of rabbit CYPlA2 and CYP2B4, we inves- tigated the influence of ionic strength on the reduction of sev- eral rat P450 enzymes. Control rat liver microsomes contain a number of P450 forms (Schenkman et al., 1987). Increase in the concentration of sodium phosphate buffer from 5 to 100 mM resulted in a facilitated reduction of the microsomal P450s (Fig. 3). Treatment of rats with phenobarbital primarily induces CYP2Bl and CYP2B2 in the liver endoplasmic reticulum (Guengerich et al., 1982). Rats were treated with phenobarbital and liver microsomes containing increased levels of CYPZBl and CYP2B2 were prepared. We compared P450 reduction in phenobarbital-treated rat liver microsomes at low (5 m~ so- dium phosphate) and high (100 mM sodium phosphate) ionic strength. The effect was similar to the one observed with CYP2B4, CYPlA2, and control rat liver microsomes, i.e. an increase in ionic strength stimulated P450 reduction (data not shown).

15728 Quantifying Interacting P450 and Reductase Charges Vo ( Z of control)

A450-490

0.03

0.02

0.01

0.00

mM

,,/ 5 mM

0 10 20 30 40

Time (s) FIG. 3. Intluence of ionic strength on the reduction of P450 in

the liver microsomes of control rats. Rat liver microsomes of control rats were suspended in 5 or 100 mM sodium phosphate, pH 7.4, to a final P450 concentration of 0.5 p. P450 reduction was monitored as de- scribed under “Materials and Methods.”

We also examined the influence of ionic strength on the re- duction of several purified rat liver P450 enzymes, CYP2C6, CYP2C12, CYP2C13, and CYP2El reconstituted with the re- ductase in the DLPC vesicles (molar ratios protein/DLPC = 1/80, P450lreductase = 1011). Reduction of P45O was monitored in 5 and 100 mM sodium phosphate buffer and initial rates of the reaction were compared. In all cases reduction proceeded faster at higher ionic strength (Fig. 4), but the extent of the stimulation varied with the form of P450. Highest stimulation was observed with CYP2C13 and CYP2El (6-fold increases in the initial rates). A 3-fold increase in the rate of reduction was seen with CYP2C12 and reduction of CYP2C6 was increased only by 50% (Fig. 4). The fact that reduction of all four P450 enzymes was stimulated by ionic strength suggests that elec- trostatic forces inhibit all of them in their interaction with the reductase. The different extents of the stimulation supports the conclusion made for CYP2B4 and CYPlA2: the local distribu- tion of charged residues on the protein surfaces involved in the interaction with the reductase are different for the various forms of P450.

The Number of Interacting Charges-Different P450 forms have different values of K, for the reductase. The relationship between K, and vo is nonlinear and although qualitative con- clusions can be made after comparison of the reaction rates, quantitative analysis should be based on the equilibrium con- stants.

Interaction of P450 and reductase can be described by the scheme:

k+l t z R + P * R P + R + P *

k-1

SCHEME I

where R is the reductase, P and P* are oxidized and reduced P450, respectively. Reduction of P450 by the reductase is a relatively slow reaction proceeding under steady-state condi- tions. Changes in the concentration of the hemoprotein-fla- voprotein complex can be determined as:

d[RPydt = ~+,u,u, - k_,a, - k+,a,

SCHEME 2

- I -” I w

J

I O O r n M NaPO-3

0 “-7”

tion of four forms of rat P450. CYP2C6, CYP2C12, CYP2C13, or FIG. 4. Influence of ionic strength on the initial rate of reduc-

CYP2El were reconstituted with the reductase in DLPC (P450/ reductase = lO/l, DLPC/protein = 80/1 mol/mol). Reduction of P450 forms was monitored at 5 and 100 mM sodium phosphate, pH 7.4, 20% glycerol as described under “Materials and Methods.” Initial rate in 5 mM phosphate served as a control.

, : d , j /’ ..e;’ ’5rnh.i NaPOl 2C 13 2 E 1 2C6 2c 12

where uR, up, and uRp are activities of reductase, P450, and the reductase-P450 complex, respectively. Activities have to be used instead of concentrations because of the charges on the protein surface. The involvement of electrostatic forces re- quires us to treat the reaction between reductase and P450 as a reaction between “protein ions.” Substituting activities for the products of respective concentrations and activity coeffi- cients (aR = [Rly,, up = [Ply,, and am = [RPlym):

d[RPW = k+l[RI[Ply~y, - k-,[RPlyw - k+z[RPlyw

After using the steady-state treatment (d[RPl/dt = 0 ) and re- grouping:

(Eq. 3a)

Reduction of cytochrome P450 is a slow nondiffusion limited process and the rates of reductase-P450 interactions calculated from lateral diffusion measurements are several orders of mag- nitude higher than the rate of electron transfer within the flavoprotein-hemoprotein complex (Blanck et ul., 1984; Wu and Yang, 1984). This suggests that the equilibrium between free reductase, free P450, and reductase-P450 complex is reached in the system. Under these conditions K, would be a true equi- librium constant and the apparent association constant K4,app could be calculated as the reciprocal of K,. Since by definition the association constant of the two proteins is

K.,app = [RPY([RI[Pl) (Eq. 3b)

Equation 3a can be rewritten as:

k+l k,, + k - I

” YRP

- K.,.pp - (Eq. 4a) Y J P

At zero ionic strength y,J( y g p ) becomes unity and Equation 4a becomes an expression for the equilibrium constant at zero ionic strength, KO.

k+14k+2 + k-, 1 = KO (Eq. 4b)

KO can be determined from the measurements of the apparent association constant Ka,app at various salt concentrations by extrapolation to zero ionic strength. Now Equation 4a can be written as:

(Eq. 5a)

Quantifying Interacting P450 and Reductase Charges 15729

or

loglo(Ka,app/KO) = l0g10(Yp) + l0gIO(YR) - l0g1O(YRP) (Eq. 5b)

According to the Debye-Huckel theory if the charges of ions, z1 and z2, relate as z1 = Az,, where A is a constant, their activi- ties relate as log,,(y,,) = A210g,,(yz,). If

zR = mz, zp = nz, zRp = (m + n)z (Eq. 6a)

Where m, n, and (m+n) are the numbers of elementary charges, z, on the reductase (m), P450 (n), and their complex (m+n), respectively. The activity coefficients (yR, yp, and yRP) then will relate to the activity coefficient of the elementary charge ( y ) as log,,(Y,) = m2 log,,(Y), log,,(Y,) = n2 log,,(Y), 10glO@RP) = (m + n)’ log,,(Y) then

l o g l ~ ( ~ a , a p p ~ ~ , ) = m2 log,,@) + n2 log,,(Y) - (m + n)’ lw,,(Y) (Eq. 6b)

and, after simplification

logl,(Ka,app/K,) = - 2mn log,,@) (Eq. 7)

Note that m and n are not the total charges of the flavoprotein and the hemoprotein, but only the number of charges affecting the complex formation, because only their behavior will influ- ence Ka,app. When loglo(Ka,ap~Ko) is plotted uersus -log,,(y) (Equation 7) the line will have a slope of 2 mn, thereby allowing us to calculate the product of the numbers of interacting charges on the two proteins. When log,,(y) was determined as described under “Materials and Methods” and equilibrium con- stants for CYPlA2 and CYP2B4 were calculated and plotted in the above coordinates, the slopes of the plots were different for the two proteins (Fig. 5A). The plot gave a slope of 21.68 for CYP2B4 and a slope of 13.27 for CYPlA2, indicating that the product of the number of interacting charges is 10.84 for the reductase-CYP2B4 pair and 6.64 for the reductase-CYPlA2 pair. The values suggest that for the reductase and CYF’2B4 repulsion between 3 charges on one protein and 3-4 charges on the other may influence the complex formation. In the case of CYF’lA2, the value suggests the presence of 3 repulsing charges on one protein and 2 on the other. The numbers, of course, are approximate and reflect only the net effect of the electrostatic interactions of the cytochrome P450 forms and the reductase. Since both the reductase and P450 have a large number of differently charged residues on their surfaces it is quite possible that each protein has a mixture of differently charged residues at the site of interaction. The slope in the above mentioned plot depends upon the net values i.e. the number of repulsing charges minus the number of attracting charges on each protein. Positive slopes of the plots indicate that the net effect of the electrostatic interaction of the cyto- chrome P450 forms and the reductase is a repulsive one. At present it is not possible to determine which value corresponds to P450 and which can be assigned to the reductase, because activities of charges on the hemoprotein and the flavoprotein are equally affected.

To confirm the validity of our approach, previously obtained cytochrome b5-CYP2C11 association constants (Tamburini and Schenkman, 1986b) were plotted in the coordinates of Equation 7. Data points fit a straight line with a slope of -13.9 (Fig. 5B). In agreement with the established charge-pairing nature of the P450-cytochrome b, complex, the line had a negative slope. The slope suggests that there are 2-3 interacting charges on each CYP2Cll and cytochrome b,. This number agrees well with previous reports where 3-5 charges on the cytochrome 6, were suggested to participate in the formation of electrostatic com- plexes with the other redox proteins (Dailey and Strittmatter, 1979, 1980; Ng et al., 1977; Tamburini et al., 1985).

0 . 0 0 0.03 0.06 0.09 0.12 0.15

-1.0 ~

-1.5 -

-2.0 ~

i 0.0 0 . I 0.2

FIG. 5. Dependence of reductase-P450 and cytochrome b,-P450 association constant on the ionic activity coefficient. A, associa- tion constants obtained for CYF’2B4 (0) and CYPlA2 (A) were plotted in the coordinates of the Equation 7. B , previously obtained cytochrome b5-CYP2B1 association constants (Tamburini and Schenkman, 1986b) were plotted in the same coordinates. See text for details.

DISCUSSION

Electrostatic forces play an important role in the interactions between electron transfer proteins. Electron transfer proceeds within a protein-protein complex, and in many cases charge pairing interactions are responsible for the complex formation. Thus, a complex of P450 with cytochrome b, is formed by at- traction of carboxyl groups on cytochrome b, and amino groups on P450 (Tamburini et al., 1986, 1985). An electron transfer complex of NADH-cytochrome-b, reductase and cytochrome b, is also formed by attraction of oppositely charged amino acid residues (Dailey and Strittmatter, 1979; Strittmatter et al., 1990).

In contrast, repulsion between the charged residues of two proteins may interfere with complex formation. We showed

15730 Quantifying Interacting P450 and Reductase Charges

earlier that charge repulsion inhibits interaction between CYP2B4 and the reductase (Voznesensky and Schenkman, 1992a, 1992b). In the present study we examined the influence of electrostatic forces on the interaction of the reductase with several other forms of P450. The progressive increase in the salt content gradually neutralized electrostatic interaction by lowering the activity coefficients of the ions. If electrostatic forces interfere with complex formation, a decline in the activ- ity coefficients will facilitate protein-protein interactions by decreasing the interference. The decrease in the activity coef- ficients resulted in an increased fast phase rate constant of P450 reduction in the phenobarbital-induced rabbit liver mi- crosomes (Fig. l). The fast phase of CYPlA2 reduction was also facilitated both in the reconstituted system (Table 11) and in P-naphthoflavone-induced microsomes (Table 111), indicating that the results were not due to the nature of the reconstituted system. The data indicate that electrostatic forces also inhibit the interaction of CYPlA2 and the reductase. In agreement with this suggestion, when the ionic strength was changed from low (10 mM sodium phosphate) to high (100 mM phosphate) the K,, of CYPlA2 for the reductase decreased 7-fold, while the V,,, did not change. Under identical conditions the K,,, of CYP2B4 showed a 26-fold decrease while V,, was unaffected (Voznesen- sky and Schenkman, 1992a). Neutralization of charged resi- dues by increased ionic strength also facilitated reductase- P450 interactions in control and phenobarbital-induced rat liver microsomes, and in the reconstituted system with four purified forms of rat liver P450: CYP2C6, CYP2C12, CYP2C13, or CYP2E1 (Fig. 41, suggesting that charge repulsion probably inhibits interaction of the reductase with all microsomal P450 forms. As in the case of CYP2B4 and CYPlA2, the extent of stimulation differed considerably for the different rat enzymes, indicating that the number of charges involved in the protein- protein interaction varies from P450 to P450.

The conclusion that the observed effect is due to electrostatic protein-protein repulsion was supported by charge neutraliza- tion with short chain charged polypeptides. Polylysine and polyglutamic acid neutralize protein charges by binding to an- ionic and cationic regions on the protein surface, respectively. Although micromolar concentrations of the polypeptides did not change the ionic strength of the medium, their addition stimulated the electron transfer from the reductase to the cy- tochromes (Table IV). Addition of polylysine to the reconsti- tuted system stimulated both CYP2B4 and CYPlA2 reduction, while addition of polyglutamic acid facilitated electron transfer only to CYP2B4, suggesting that different P450 forms a have different distribution of charges at the reductase binding site. This conclusion also agrees with the different extent of de- crease in the K,,, of rabbit liver CYPlA2 and CYP2B4 for the reductase at higher salt levels (7- and 26-fold, respectively). NADPH-cytochrome P450 reductase is a highly charged pro- tein containing 95 glutamate + aspartate residues and 73 ar- ginine + lysine residues (Katagiri et al., 1986). CYP2B4 has 52 glutamate + aspartate residues and 58 arginine + lysine resi- dues (Gasser et al., 19881, and CYPlA2 contains 51 glutamate + aspartate residues and 63 arginine + lysine residues (Kagawa et at., 1987). In agreement with our observations analysis of the alignment of microsomal P450 forms (Nelson and Strobel, 1988) suggested that a map of surface charges on the reduc- tase-P450 interface may actually differ for different forms of P450.

In this paper we developed a method for the evaluation of the number of charges involved in protein-protein interactions based on the estimation of the activity coefficients. The analy- sis indicated that a different number of charges are involved in the interaction of different forms of P450 with the reductase. The product of the number of charges on the interacting pro-

teins is 10.84 for the CYP2B4-reductase pair, and 6.64 for the CYPlA2-reductase pair, suggesting 3-4 interacting residues on each protein in the former pair and 2-3 charges in the latter. The numbers are approximate because the equations were de- rived using a simplified model. The numbers also tell us noth- ing about the sign of interacting charges. That information would require a separate study. However, the fact that polyly- sine stimulated reduction of both CYPlA2 and CYP2B4 (Table IV) suggests charge repulsion between negatively charged resi- dues (Glu andor Asp) of the cytochromes and the reductase. In a similar manner charge repulsion may influence the interac- tion between cytochrome b, and NADPH-cytochrome P450 re- ductase. This conclusion is consistent with the observed stimu- lation of cytochrome b, reduction by the reductase after chemical neutralization of carboxyl residues of either cyto- chrome 6, or reductase (Tamburini and Schenkman, 1986a; Tamburini et al., 1985). Also in agreement is the observed stimulation of electron transfer from the microsomal reductase to P450, cytochrome b,, and cytochrome c by the divalent cat- ions, Ca2+ and MgZ' (Fouts and Pohl, 1971; Tamura et al., 1990). Divalent cations may form salt bridges between anionic resi- dues on the two proteins or neutralize an adjacent pair of negative charges on the protein surface. The same may also be true for the stimulation by poly-Lys and poly-Glu.

From the stimulation of CYPlA2 reduction by polyglutamic acid we conclude that positively charged residues (Lys and/or Arg) are also involved in the charge repulsion between the reductase and its redox partners. In support of this suggestion it was reported that amidination of lysine residues of the re- ductase facilitated reduction of cytochrome b, and cytochrome c (Tamburini and Schenkman, 1986a). Amidination does not neu- tralize cationic charges but may interfere with interactions by insertion of a bulky alkyl group. Amidination of the reductase also stimulated substrate turnover by three P450 enzymes, although slight inhibition of P450 reduction was observed (Tamburini and Schenkman, 1986a). Acetylation of lysine resi- dues of the reductase, which neutralizes cationic charges, de- creased the K,, of CYP2Bl and CYPlA2 for the flavoprotein (Nadler and Strobel, 1988). These observations suggest the presence of charges of different sign at the hemoprotein-fla- voprotein interface. Along with above mentioned stimulatory effects, inhibition of reductase-P450 interaction was reported upon chemical neutralization of P450 lysine residues (Bern- hardt et al., 1983, 1988). Modification of charged residues by site-directed mutagenesis also impaired reductase-P450 inter- actions (Shimizu et al., 1991). On the other hand, methylam- idination of the reductase carboxy groups decreased V,, with- out effect on the K,,, for the reductase (Tamburini and Schenkman, 1986a). Results of chemical modification and site- directed mutagenesis studies are difficult to interpret because of the potential alteration of the protein structure. Insertion of a bulky group of a chemical modifier exerts steric influence along with charge neutralization; substitution of charged amino acids by site-directed mutagenesis may interfere with protein folding. Thus even conservative mutations (Asp-Glu) were shown to disrupt the formation of the ferredoxin electron transfer complexes with P450,,, and ferredoxin reductase (Coghlan and Vickery, 1992).

If the interaction of charged residues pushes P450 and re- ductase apart, what force holds the two proteins in the electron transfer complex? We suggest that this force is the interaction of complementary hydrophobic patches on the two proteins. Interaction of complementary hydrophilic and hydrophobic patches has been shown to be the driving force in the associa- tion of some multisubunit proteins (Korn and Burnett, 1991). Although the reductase is a charged protein it still can partici- pate in hydrophobic interactions. Even for highly charged pro-

Quantifying Interacting P450 and Reductase Charges 15731

teins nonelectrostatic interactions were shown to take part in protein-protein complex formation (Zhou and Kostic, 1991). Our data indicate that the reductase-P450 electron transfer complex is not stabilized by electrostatic interactions. The pres- ence of as many as 11 conserved hydrophobic regions in the amino acid sequence of P450 forms (Nelson and Strobel, 1988) and the membrane binding of P450 forms with the truncated amino-terminal region (Pernecky et al., 1993; Sagara et al., 1993) also suggest the presence of hydrophobic patches on the hemoprotein surface that may be involved in the formation of the electron transfer complex with the reductase.

The concept of reductase-P450 complex formation by inter- actions of complementary hydrophilic and hydrophobic patches is very appealing because it agrees well with the flexible nature of the P450 monooxygenase system. One reductase supplies electrons to an array of functionally diverse and structurally different P450 forms as well as non-P450 proteins. The forma- tion of the complex by complementary charge pairing interac- tions would require charged amino acids to be positioned pre- cisely in the same orientation in a large number of different proteins, imposing considerable constraints on their structure. Protein-protein recognition is much easier to achieve in such a system through hydrophobic patch complementarity, because it allows high structural variability as long as the general patch motif is preserved. As indicated above, alignment of the P450 sequences supports the possibility of such conserved hydropho- bic domains (Nelson and Strobel, 1988).

Since electron transfer requires precise orientation of pro- teins within the complex, a high degree of complementarity would be expected. On the other hand, high hydropathy complementarity is often associated with static inflexible inter- actions (Korn and Burnett, 1991). Since reductase was shown to interact with P450 according to a free-diffusion model (Miwa et al., 1979; Muller-Enoch et al., 1984; Voznesensky, 1989), the role of the observed charge repulsion between P450 and reduc- tase may be to counterbalance the high degree of hydropathy complementarity needed for protein orientation and to allow proteins to stay in the complex only long enough for the elec- tron transfer to occur.

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