Organization of multiple cytochrome P450s with NADPH-cytochrome P450 reductase in membranes

www.elsevier.com/locate/pharmthera

Pharmacology & Therapeutics 98 (2003) 221–233

Associate editor: D. Kupfer

Organization of multiple cytochrome P450s with NADPH-cytochrome

P450 reductase in membranes

Wayne L. Backes*, Rusty W. Kelley

Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center,

533 Bolivar Street, New Orleans, LA 70112, USA

Abstract

Microsomal P450-mediated monooxygenase activity supported by NADPH requires an interaction between flavoprotein NADPH-

cytochrome P450 reductase and cytochrome P450. These proteins have been identified as the simplest system (with the inclusion of a

phospholipid (PL) component) that possesses monooxygenase function; however, little is known about the organization of these proteins in

the microsomal membrane. Although reductase and P450 are known to form a 1:1 functional complex, there exists a 10- to 20-fold excess of

P450 over the reductase. This raises several questions including ‘‘How are the enzymes of the P450 system organized in the microsomal

membrane?’’ and ‘‘Can one P450 enzyme affect the functional characteristics of another P450?’’ This review summarizes evidence

supporting the potential for enzymes involved in the P450 system to interact, focusing on the interactions between reductase and P450 and

interactions between multiple P450 enzymes. Studies on the aggregation characteristics of P450 as well as on rotational diffusion are

detailed, with a special emphasis on the potential for P450 enzymes to produce oligomeric complexes and to suggest the environment in

which P450 exists in the endoplasmic reticulum. Finally, more recent studies describing the potential for multiple P450s to exist as

complexes and their effect on P450 function are presented, including studies using reconstituted systems as well as systems where two

P450s are coexpressed in the presence of reductase. An understanding of the interactions among reductase and multiple P450s is important

for predicting conditions where the drug disposition may be altered by the direct effects of P450-P450 complex formation. Furthermore, the

potential for one P450 enzyme to affect the behavior of another P450 may be extremely important for drug screening and development,

requiring metabolic screening of a drug with reconstituted systems containing multiple P450s rather than simpler systems containing only a

single form.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: NADPH-cytochrome P450 reductase-P450 interactions; P450-P450 interactions; P450 reduction; Functional complex formation; Complementary

charge pairing; Aggregation state of P450

Abbreviations: CYP2B4, a rabbit P450 either purified from PB-treated rabbit liver or purified from an Escherichia coli expression system; CYP2C10,

CYP2D6, CYP2E1, and CYP3A4, recombinant human P450 enzymes expressed with either a baculovirus expression system or an Escherichia coli expression

system; DLPC, dilauroylphosphatidylcholine; 7-ER, 7-ethoxyresorufin; EROD, 7-ethoxyresorufin-O-dealkylation; human CYP1A2, purified from an

Escherichia coli expression system; kfast, fast-phase rate constant; kslow, slow-phase rate constant; LM2, CYP2B4 (also named as P450 2B4); LM4, CYP1A2

(also named as P450 1A2); bNF, b-naphthoflavone; P420, cytochrome P420; P450, cytochrome P450; PB, phenobarbital; PL, phospholipid; 7-PR, 7-

pentoxyresorufin; PROD, 7-pentoxyresorufin-O-dealkylation; rabbit CYP1A2, purified from bNF-treated rabbit liver; reductase, NADPH-cytochrome P450

reductase

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

2. Organization of proteins in the endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . 222

2.1. P450 reduction and its use in measuring reductase-P450 complex formation . . . . . . . 222

2.2. Interactions between P450 reductase and P450 . . . . . . . . . . . . . . . . . . . . . . 223

2.3. The role of phospholipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

0163-7258/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0163-7258(03)00031-7

* Corresponding author. Tel.: 504-568-5148; fax: 504-568-6888.

E-mail address: [email protected] (W.L. Backes).

W.L. Backes, R.W. Kelley / Pharmacology & Therapeutics 98 (2003) 221–233222

3. Cytochrome P450 aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

3.1. Effect of detergent on the aggregation state of cytochrome P450 . . . . . . . . . . . . . 224

3.2. Role of the N-terminal region on the aggregation characteristics of P450. . . . . . . . . 225

3.3. Interactions among P450 enzymes in membranes: rotational diffusion studies . . . . . . 225

4. Interactions between multiple P450 enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

4.1. Evidence for specific interactions between different P450 enzymes . . . . . . . . . . . . 227

4.2. Demonstration that interactions between CYP2B4 and CYP1A2 occur in microsomes . . 229

4.3. Possible interactions among multiple P450s and reductase . . . . . . . . . . . . . . . . 230

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231

1. Introduction

Cytochrome P450, the terminal component of an electron

transport chain found in the endoplasmic reticulum, has been

shown to be important in the metabolism of drugs, pollutants,

solvents, and other foreign compounds as well as of endo-

genous lipid-soluble substrates. The enzyme system takes

lipid-soluble substrates and converts them to more water-

soluble products by insertion of an oxygen atom into the

substrate molecule. Since the identification and reconstitution

of the components of this electron transport chain, there has

been an interest in the organization of constituent proteins in

the endoplasmic reticulum. The focus of this review is to

discuss the characteristics of the interaction of P450 not only

with the flavoprotein NADPH-cytochrome P450 reductase

(reductase) but also with other P450 enzymes.

2. Organization of proteins in the endoplasmic reticulum

The primary components, cytochrome P450, flavoprotein

NADPH-cytochrome P450 reductase, and phospholipid

(PL), were first identified in the seminal studies by Lu et

al. (1969). NADPH-supported catalytic activity requires

both protein components, with P450 and reductase forming

a 1:1 (M/M) functional complex (Miwa & Lu, 1984; Miwa

et al., 1979). Other protein components, such as cytochrome

b5 and NADH-cytochrome b5 reductase, also interact with

the P450 system (Hildebrandt & Estabrook, 1971), where

they can influence the rate of catalysis (Bernhardt, 1996;

Tamburini et al., 1986). PL also plays an important role in

monooxygenase activities. PL composition and concentra-

tion not only influence the rate, but also can govern whether

a catalytic activity will even be expressed (Causey et al.,

1990; Imaoka et al., 1988, 1992; Ingelman-Sundberg, 1977;

Ingelman-Sundberg et al., 1983; Taniguchi et al., 1979).

One of the major factors that confound our ability to

understand the interactions of the microsomal monooxy-

genase is the multiplicity of cytochrome P450s. It has been

known for over 25 years that there are multiple forms of

cytochrome P450 in the endoplasmic reticulum. In fact,

studies have identified hundreds of P450 enzymes (Nelson

et al., 1996). The presence of multiple P450s, each with their

own substrate selectivities, causes a dramatic increase in the

possible number of discrete protein-protein interactions,

some of which having the potential to alter catalytic function.

When present in microsomes, cytochrome P450 is in a

large excess over NADPH-cytochrome P450 reductase.

Although this ratio varies depending on induction status,

P450 levels exceed those of the reductase by a 10:1–20:1

ratio (Estabrook et al., 1971) in liver. Since P450 has been

shown to form a 1:1 functional reductase-P450 complex

(Miwa & Lu, 1984; Miwa et al., 1979), the reductase must be

capable of supplying electrons to each of the different P450

enzymes. In the event that particular P450 enzymes have a

higher affinity for association with reductase, then electrons

would preferentially flow to those P450s. Consequently,

those P450s less able to compete for limiting reductase must

have mechanisms that would permit them to receive elec-

trons, otherwise they would be metabolically silent.

2.1. P450 reduction and its use in

measuring reductase-P450 complex formation

The first step of the P450 cycle after formation of the

P450-substrate complex is the transfer of the first electron.

This process (also known as P450 reduction) is generally

considered to be rapid, but has the unique characteristic in

that it exhibits biphasic kinetics (Gigon et al., 1969). Simply

stated, this means that when an electron is being transferred

to P450, more than one process is occurring—a rapid

process followed by a slower process (Gigon et al., 1969).

Many investigators have tried to explain the biphasic nature

of P450 reduction, attributing it to the multiplicity of P450

enzymes in the microsomal membrane, the organization of

proteins in the microsomal membrane, the amount of high

spin content, and the ability of reductase to form a func-

tional complex with cytochrome P450 (Backes & Eyer,

1989; Eyer & Backes, 1992; Peterson et al., 1976; Tamburini

et al., 1984). Regardless of the mechanistic details, the rapid

rate of reduction (particularly of the fast phase) allows us to

use P450 reduction as a tool to examine formation of a

functional complex between reductase and P450 (Backes &

Eyer, 1989; Eyer & Backes, 1992).

Peterson and colleagues (1976) were among the first to

bring together the concept of how reductase can supply

W.L. Backes, R.W. Kelley / Pharmacology & Therapeutics 98 (2003) 221–233 223

electrons to the large excess of P450 molecules with an

explanation of why P450 reduction behaved as a biphasic

process. In their studies, the investigators examined P450

reduction using microsomal preparations and found that

� 70% of the P450 was reducible in the fast phase. They

postulated that the microsomal membrane is organized with

several P450 molecules clustered around each reductase.

The remaining P450 is not directly associated with the

reductase, and can only associate after lateral motion within

the membrane. These investigators reasoned that the fast

phase of reduction was due to reduction of those P450s in

the cluster, followed by a slower phase caused by reduction

of those P450s not in the cluster. Subsequent experiments

have shown that biphasic kinetics are obtained with a single

purified P450 enzyme at saturating reductase, conditions

where clusters would not be expected. These results indicate

that the organization of P450s in the microsomal membrane

cannot solely account for the biphasic nature of P450

reduction.

Despite the difficulties with this model in its strictest

sense, it has been extremely valuable as a working hypo-

thesis, having features that describe the potential interac-

tions between P450 and limiting levels of reductase in the

microsomal membrane. Furthermore, the idea that some

P450s can only associate with reductase after lateral motion

through the membrane is also a tenet of their hypothesis.

More recently, investigators have used first electron

transfer to P450 as a tool to examine interactions between

reductase and P450. The reaction has the advantage that the

majority of the reaction is complete within a few seconds,

permitting a rapid reduction of P450 molecules already in a

complex with the reductase (Backes & Eyer, 1989; Eyer &

Backes, 1992; Taniguchi & Pyerin, 1988; Taniguchi et al.,

1979, 1987). In one of these studies, the effect of alteration

of reductase:P450 ratio on P450 reduction was examined.

The results were subjected to kinetic analysis, treating the

data as a simple biphasic process, and demonstrated that

both kfast and kslow were increased, whereas the amount of

P450 reduced in the slow phase was essentially unaffected

as the reductase level was increased (Taniguchi et al., 1979).

These results demonstrate that a large amount of P450 that

is not already in a complex with reductase has thus far

escaped investigation in many studies. This ‘‘more slowly

reducible P450’’ may account for 50–90% of the P450

present in microsomes (Peterson et al., 1976). An under-

standing of the factors that control electron transfer to those

uncomplexed P450 enzymes is required to begin to explain

the behavior of P450s at limiting levels of reductase.

2.2. Interactions between P450 reductase and P450

There has been a continual interest in the factors con-

trolling the interaction between reductase and P450. When

reconstituted into liposomes, these proteins form a func-

tional complex with an apparent Km (for reductase) of � 0.2

mM (Voznesensky & Schenkman, 1992). Several factors

influence the characteristics of this complex, including the

P450 enzymes involved (Taniguchi & Pyerin, 1988; Vozne-

sensky & Schenkman, 1992, 1994) and the ionic strength of

the medium (Voznesensky & Schenkman, 1992, 1994).

Substrate also has a significant influence on the binding of

several P450 enzymes, increasing both the affinity of the

reductase-P450 complex (French et al., 1980) and the rate at

which the two proteins associate (Backes & Eyer, 1989;

Eyer & Backes, 1992). Complex formation has been

reported to occur via two different processes: complement-

ary charge pairing and hydrophobic interactions. Several

groups have presented evidence supporting the view that

reductase and P450 are held together by complementary

charged residues (Bernhardt et al., 1988; Nadler & Strobel,

1988; Shimizu et al., 1991), localizing the interaction to

particular regions of the reductase molecule. Bernhardt and

colleagues chemically modified lysine residues on P450

2B4 with 2-methoxy-5-nitrotropone (Bernhardt et al.,

1989) and fluorescein isothiocyanate (Bernhardt et al.,

1984). In each case, decreases in reductase-supported mono-

oxygenase activity and percent of aerobic reduction were

noted without an effect on cumene hydroperoxide- or

hydrogen peroxide-supported activities. The involvement

of lysine residues in the interaction between these proteins

has also been recently supported by site-directed muta-

genesis of LYS271 and LYS279 on CYP1A1 (Cvrk &

Strobel, 2001). Modification of LYS279 caused a decrease

in reductase-supported 7-ethoxycoumarin deethylation,

whereas the cumene hydroperoxide-supported reaction was

unaffected.

Interestingly, Voznesensky and Schenkman (1992)

reported that an increase in ionic strength actually facilitates

the rate of first electron transfer to P450 2B4. This increase

in reaction rate was reported to be due not only to an

increase in the fast-phase rate constant, but also to an

increase in the fraction of the P450 reduced in the fast

phase. These results are opposite of those expected if charge

pairing stabilizes the reductase-P450 2B4 complex. More

recently, Davydov et al. (2000a) have put forward a poten-

tial explanation for this discrepancy. Using fluorescence

energy transfer to more directly measure the interaction

between reductase and CYP2B4, these investigators dem-

onstrated that the Kd for the complex was increased as the

ionic strength was increased, consistent with charge pairing

between the proteins. These investigators provided the

explanation that although charge pairing governs complex

formation between reductase and CYP2B4 (leading to a

decrease in affinity with increasing ionic strength), the

results reported by Voznesensky and Schenkman (1992)

were due to an increase in the rate of electron flow through

those complexes that are formed (Davydov et al., 2000a).

Therefore, despite decreased affinity of the reductase-

CYP2B4 complexes as ionic strength is increased, the ionic

strength-dependent stimulation of electron flow leads to the

overall elevation in CYP2B4 reduction observed in the

earlier study.


Whereas modification of lysine residues on P450 inhib-

ited the interaction between reductase and P450, modifica-

tion of carboxyl groups on the reductase similarly inhibited

this interaction (Bernhardt et al., 1987; Nadler & Strobel,

1988, 1991; Tamburini & Schenkman, 1986). Modification

of the reductase with 1-ethyl-3-(dimethylaminopropyl) car-

bodiimide hydrochloride (EDC) caused up to an 85%

inhibition of P450 LM2 (CYP2B4)-supported NADPH

oxidase activity (Bernhardt et al., 1987) and an 80%

decrease in the kcat/Km (Nadler & Strobel, 1991). Interest-

ingly, P450 LM2 was able to protect the reductase from

EDC modification, further supporting the involvement of

electrostatic interactions in the interaction between reduc-

tase and P450 (Bernhardt et al., 1987; Nadler & Strobel,

1991). Shen and Kasper (1995) modified several acidic

residues on the reductase molecule by site-directed muta-

genesis. They examined two acidic clusters and determined

that modification of one of the clusters, 207Asp-Asp-Asp209

(specifically D208N), decreased the kcat for P450, but not

reduction of cytochrome c. Mutation of the other cluster (at

residues 213–215) decreased the specific activity for cyto-

chrome c reduction without affecting P450-dependent benz-

phetamine demethylation.

Some early evidence pointing to the role of hydropho-

bicity in complex formation comes from studies using

reductase molecules where the hydrophobic N-terminal

region has been removed. This truncated reductase is

incapable of accepting electrons from NADPH and trans-

ferring them to P450 (Johnson & Muller-Eberhard, 1977).

Furthermore, the hydrophobic reductase fragment is capable

of inhibiting the association of native reductase to P450

(Black et al., 1979). Recently, electron transfer from the

truncated reductase to P450 has been successfully accomp-

lished using an electrochemical technique (Estabrook et al.,

1996). These results suggest that the hydrophobic region

may be important for the transfer of electrons from NADPH

to the reductase, but may not be required for the transfer of

electrons from reductase to P450. Furthermore, the hydro-

phobic N-terminal region of CYP2E1 is not required for

functional complex formation. Removal of the N-terminal

region did not inhibit the reduction of this P450 in a

reconstituted system or affect its responsiveness to changes

in ionic strength (Voznesensky et al., 1994). These studies

illustrate the complexity of the interaction between these

proteins.

2.3. The role of phospholipid

The importance of the PL component for reconstituting

P450-dependent monooxygenase activities has been known

for 30 years (Lu et al., 1969; Strobel et al., 1970). PL

appears to influence monooxygenase activities by at least

two different process. First, it can serve as an effector where

PL can modulate enzymatic activities even at low PL:P450

ratios. This stimulation has been reported even with NaIO4-

supported reactions, reactions that do not involve an inter-

action with P450 reductase (Ingelman-Sundberg, 1977).

Second, PL constitutes a major component of the micro-

somal membrane, which provides a matrix for the inter-

action of these proteins. These effects are generally

observed at higher lipid levels where the interaction between

reductase and P450 can be affected (Causey et al., 1990;

Ingelman-Sundberg, 1977; Ingelman-Sundberg et al., 1983;

Taniguchi et al., 1979). In both the effector and the matrix

roles for Pls, different degrees of stimulation have been

observed as a function of PL composition (Ingelman-Sund-

berg, 1977; Ingelman-Sundberg et al., 1981). Taken

together, the data suggest that the PL dramatically influen-

ces the catalytic activity of P450-dependent reactions,

exhibiting both an effector role and a membrane matrix

function (Causey et al., 1990; Ingelman-Sundberg, 1977).

3. Cytochrome P450 aggregation

Because of their presence in the microsomal membrane

and their hydrophobic character, there is a potential for P450

enzymes to form P450-P450 complexes. However, it is

important to be able to differentiate between P450s forming

functionally important complexes and nonspecific com-

plexes that occur due to the hydrophobic nature of the

proteins. One of the difficulties in assessing the importance

of such interactions is that P450s tend to aggregate in

solution.

3.1. Effect of detergent on the

aggregation state of cytochrome P450

There are numerous citations reporting the tendency

toward aggregation of these enzymes. Studies by Gray

and colleagues and Berndt and colleagues (Dean & Gray,

1982; Myasoedova & Berndt, 1990; Tsuprun et al., 1986;

Wagner et al., 1984, 1987) demonstrated that CYP2B4

(LM2) and CYP1A2 (LM4) exist in solution as hexamers

or heptamers, respectively. The aggregation state was

decreased by addition of the detergent n-octylglucoside.

Optimal reductase-supported monooxygenase activities

were observed at detergent concentrations where P450

remained in an aggregated state. When sufficient detergent

was added to produce monomeric P450s, monooxygenase

activity was not found (Dean & Gray, 1982; Wagner et al.,

1984). The results suggest that oligomeric P450 is required

for functional activity.

The aggregation states of CYP2B4 and CYP1A2 were

also examined by Archakov and colleagues (Kanaeva et al.,

1992a, 1992b; Sevrukova et al., 1994). CYP2B4 was made

monomeric using the detergent Emulgen 913. Monomeric

CYP2B4 was shown to be less thermostable than oligomeric

P450 and had a lower affinity for binding of type I

substrates (Kanaeva et al., 1992a). In contrast to the loss

of monooxygenase activity observed by Dean and Gray

(1982), the Vmax for monooxygenase activity of monomeric


CYP2B4 was reported to be either equal to or greater than

that observed for the microsomal system. The Km for these

reactions were increased 2.5- and 20-fold compared with

that found using microsomes. Both of these parameters were

highly dependent on the substrate employed (Kanaeva et al.,

1992a, 1992b). In these studies, the aggregation state of

CYP2B4 was estimated by laser correlation spectroscopy,

which uses the Stokes-Einstein equation to estimate particle

size. It is unclear from this study whether there is a

distribution of particles of different sizes in the presence

of detergent.

Sevrukova and colleagues (1994) examined the effect of

aggregation state on CYP1A2 function. CYP1A2 was less

likely than CYP2B4 to form monomers on the addition of

Emulgen 913. At 0.25 g/L Emulgen 913, where CYP2B4

was reported to be monomeric (Kanaeva et al., 1992a),

CYP1A2 was shown to exist as a mixture of tetramers and

40-mers (Sevrukova et al., 1994). Interestingly, CYP1A2-

dependent monooxygenase activity was stimulated by the

addition of Emulgen 913, reaching a maximum at a con-

centration of 0.1 g/L, where smaller aggregates (possibly

pentamers) exist. However, further addition of Emulgen 913

to concentrations up to 8 g/L led to disaggregation to the

monomeric CYP1A2, with a concomitant decrease in mono-

oxygenase activity (Sevrukova et al., 1994). Taken together,

these results demonstrate that the addition of nonionic

detergents leads to disaggregation of P450 and that the

degree of disaggregation is dependent not only on the

detergent used, but also on the P450 enzyme examined.

Although alterations in monooxygenase function have been

reported as a function of detergent addition, it is difficult to

distinguish between those effects mediated by alterations in

the aggregation state of these proteins and direct inhibitory

effects of the detergents.

3.2. Role of the N-terminal region

on the aggregation characteristics of P450

The N-terminal region of P450 has been suggested to

play a role in association with the membrane, as well as its

tendency to aggregate in solution. There have been several

reports illustrating the importance of the N-terminal region

for both characteristics. Removal of the N-terminal region

had a significant influence on the aggregation characteristics

of P450 2E1, but did not affect the ability of the protein to

attach to the inner cell membrane using an Escherichia coli

expression system (Pernecky et al., 1993). Full-length

CYP2E1 was shown to exist as a 10-mer in solution. It

could not be dissociated by addition of up to 1% sodium

cholate (Pernecky et al., 1995). In contrast, truncated

CYP2E1 (residues 3–29) was pentameric in 0.1% sodium

cholate, and could be dissociated to form monomers at 0.5%

sodium cholate (Pernecky et al., 1995).

The aggregation characteristics of CYP2B4 were sim-

ilarly affected by truncation of the N-terminal region (res-

idues 2–27). Full-length CYP2B4 was shown to exist as a

hexamer, both in the absence and in the presence of varying

amounts of sodium cholate. The N-terminal deletion

reduced the amount of CYP2B4 that was incorporated into

inner bacterial membranes in the E. coli expression system,

but did not completely block membrane binding of the

protein (Pernecky et al., 1993). Although truncated

CYP2B4 appeared to be octameric in the absence of

detergent, it readily formed monomers in the presence of

0.25% sodium cholate (Pernecky et al., 1995). The truncated

protein was a functionally active monooxygenase when

reconstituted with reductase and PL, with an activity ran-

ging from 35% to 75% of the activity of full-length

CYP2B4, depending on the substrate employed. The activ-

ity of truncated CYP2B4 was not examined in the presence

of detergent, where it exists as monomers (Pernecky et al.,

1995).

Similar results were found with other P450 enzymes. The

N-terminal sequences were removed from both CYP2C3

and CYP2C5 (Cosme & Johnson, 2000; von Wachenfeldt et

al., 1997). Although full-length CYP2C3 is octameric in the

absence of detergent, the truncated form exists as a dimer.

Addition of 0.5% sodium cholate causes the dissociation of

truncated CYP2C3 to a monomeric state. Under these

conditions, the full-length enzyme exists as a 5,6-mer (von

Wachenfeldt et al., 1997).

Truncated CYP2C5 exists predominantly as a tetramer in

the absence of detergent. The complex can be dissociated to

a monomeric form by the addition of 0.5% sodium cholate

(von Wachenfeldt et al., 1997). Interestingly, significant

alterations in the aggregation state of this P450 are caused

by mutation of specific internal residues (particularly

N202H, I207L, S209G, and S210T) (Cosme & Johnson,

2000).

The N-terminal region of human CYP1A2 has also been

removed (residues 1–12 and 1–31). Unlike CYP2B4 and

CYP2E1, the truncated forms of CYP1A2 are highly aggre-

gated in solution, even in the presence of different deter-

gents (Dong et al., 1996).

These results indicate that the N-terminal region of P450

not only can affect membrane anchoring of the protein, but

also can significantly influence its aggregation character-

istics. Either removal of the N-terminus or association of the

N-terminal segment of P450 with a membrane environment

is likely to alter the aggregation characteristics of the

protein.

3.3. Interactions among P450 enzymes

in membranes: rotational diffusion studies

Although it is apparent that P450 aggregates when in

solution, the physical state of the enzyme incorporated into

membranes is less clear. Examination of P450 rotation has

provided information as to the functional state of P450 in

the membrane. In early rotational mobility studies by Gut et

al. (1982) and Kawato et al. (1982), a portion of P450 was

found to be immobilized, both in microsomes and in


reconstituted systems. This immobilized P450 is likely due

to the formation of large P450 aggregates. The percentage of

immobilized P450 was affected by lipid composition, tem-

perature, and relative lipid/protein ratio. At a 1:1 (w/w)

lipid:P450 ratio (a molar ratio of � 60:1), � 35% of the

P450 was immobile. As the lipid:P450 ratio increased to

10:1 and 30:1, the amount of immobile P450 decreased to

7% and 0%, respectively. The remainder of the P450 in

these reconstituted systems was mobile and existed either as

monomers or as small complexes (possibly dimers). The

large amount of immobile P450 at lipid:P450 = 1 is probably

due to the very high concentration of membrane proteins

and likely resulting from nonspecific, low-affinity, protein

aggregation. The decline in the fraction of immobile P450 as

the lipid:P450 is increased is consistent with the concept

that the immobile P450 exists as low-affinity complexes

(Kawato et al., 1982).

The presence of both immobile and mobile fractions of

P450 has also been reported in microsomes (Kawato et al.,

1982, 1991). Microsomes from control rats contained

� 40% immobilized P450. This value was increased after

induction by phenobarbital (PB), 3-methylcholanthrene, and

polychlorinated biphenyl to 54%, 52%, and 59%, respect-

ively.

Interestingly, the degree of immobilization is decreased

by the addition of NADPH-cytochrome P450 reductase (Gut

et al., 1982) or cytochrome b5 (Yamada et al., 1995).

Addition of reductase to a 1:5 reductase:P450 ratio led to

a small amount of mobilization (decreasing immobile P450

from 35% to 26%). However, addition of reductase to an

equimolar level resulted in only 5% of the P450 remaining

in the immobilized state (Gut et al., 1982). These results

suggest that the reductase (and cytochrome b5) can disrupt

the aggregation state of P450 when in a membrane.

Gut et al. (1983) provided additional evidence that the

disruption of P450 aggregates was caused by the formation

of complexes between reductase and P450. P450 was shown

to be rotationally mobile when in reconstituted systems

containing equimolar reductase. However, when the reduc-

tase is cross-linked using antireductase IgG, there is a

complete immobilization of the P450 in the complex,

suggesting that the reductase-P450 complexes form a net-

work in the presence of the antireductase antibody.

These results are consistent with the idea that despite the

large degree of aggregation found with P450 enzymes when

examined in solution, significant amounts of the enzymes

exist in a mobile state within lipid membranes. In both

microsomal membranes and membranes where high

amounts of protein are present, the fraction of immobilized

P450 can increase to � 50%. The immobile P450 is

consistent with the formation of nonspecific interactions

between P450 molecules, and does not appear to be high-

affinity complexes. Finally, the results support the concept

that complexes between reductase and P450 can disrupt the

aggregation state of P450. Based on the above information,

alterations in aggregation of the P450 system could have a

significant influence on the amount of reductase-P450

complex (or P450-b5 complex) formation, and consequently

could be a factor that governs monooxygenase function.

4. Interactions between multiple P450 enzymes

The potential for P450 enzymes to interact both in

solution and in the lipid membrane raises the possibility

that both homomeric and heteromeric P450-P450 interac-

tions can influence the function of these enzymes. Despite

the interest in how the proteins comprising the P450 system

are organized, very few studies have addressed the question

of whether one P450 can influence the catalytic character-

istics of another P450. Early studies by West and Lu (1972)

examined the effect of addition of P450 (rat CYP2B) on

P448 (rat CYP1A)-dependent 3,4-benzo[a]pyrene hydroxy-

lation. These studies were performed at P448 and reductase

concentrations of 0.11 mM and 0.03 mg (possibly � 0.4

mM), respectively, causing an inhibition of the rate of 3,4-

benzo[a]pyrene hydroxylation after addition of 1.6 mMP450 (CYP2B) to the reconstituted system. This inhibition

was relieved by supplementation of the reconstituted system

with additional reductase. These results are consistent with a

simple competition between these two P450 enzymes for the

reductase (West & Lu, 1972).

Kaminsky and Guengerich (1985) also examined the

potential for multiple P450s to interact in mixed reconsti-

tuted systems. They observed generalized inhibition of

P450-dependent warfarin metabolism with binary mixtures

of eight different P450s in reconstituted systems with

NADPH-cytochrome P450 reductase. Interestingly, there

are differences in the magnitude of inhibition among differ-

ent P450 enzymes, suggesting some specificity in the

response. They also observed that warfarin metabolism in

microsomal preparations is lower than that predicted from

the activities of the constituent P450 enzymes in reconsti-

tuted systems. The inhibition was not attributed to competi-

tion between the P450s for reductase as reported by West

and Lu (1972), but was ascribed to P450 aggregation. The

authors concluded that the inhibitory effects found in mixed

reconstituted systems are analogous to the interactions

found in microsomes.

Dutton et al. (1987), using similar P450 enzymes, but

different substrates, obtained contrasting results to those of

Kaminsky and Guengerich (1985). They reported that

testosterone metabolism catalyzed by several P450

enzymes was not inhibited by the presence of other

P450s in complex reconstituted systems. This lack of

effect was attributed to the different substrates used in

the study (Dutton et al., 1987), but could also be the result

of different reconstitution conditions. Whereas Dutton and

colleagues (1987) used higher lipid concentrations and

short preincubation times after combining the constituents

of the reconstituted systems, Kaminsky used lower lipid

levels and presumably longer preincubation times (Causey

Table 1

Interaction among CYP1A2, CYP2B4, and reductase in complex

reconstituted systems

System

components

[FpT]/

[total P450]

Benzphetamine1

(nmol/min)

PROD1

(pmol/min)

EROD

(pmol/min)

2B4 and

reductase

1.5:1 5.2 76 4.4 ± 1.1

1A2 and

reductase

1.5:1 0.17 1.4 25.9 ± 2.8

2B4, 1A2,

and reductase

1.5:1 6.2 69 29.9 ± 2.8

2B4 and

reductase

0.5:1 2.7 58 2.7 ± 0.1

1A2 and

reductase

0.5:1 0.09 0 11.5 ± 0.2

2B4, 1A2, and

reductase

0.5:1 3.1 12.7 22.6 ± 1.12

Rabbit liver CYP2B4 and CYP1A2 were combined with reductase in

DLPC as a reconstituted system containing either one or both P450

enzymes. The reconstituted systems contained CYP2B4 or CYP1A2 (0.1

mM) and reductase at concentrations of 0.05 or 0.15 mM. The metabolism of

benzphetamine, 7-pentoxyresorufin, and 7-ethoxyresorufin were examined

in complex reconstituted systems containing both P450s and comparing the

results to those found in simple reconstituted systems containing only a

single P450. The reductase concentration in the complex reconstituted

system was twice the concentration in the systems containing only a single

P450 enzyme in order to maintain [reductase]/[total P450] ratio. The EROD

data are the means ± SE for four determinations.1 Benzphetamine and PROD data are taken from the study of Cawley et

al. (1995).2 P < 0.05.


et al., 1990; Kaminsky & Guengerich, 1985; Muller-

Enoch et al., 1984).

Elegant studies by Alston et al. (1991) provided evidence

that some P450 enzymes in the microsomal membrane

formed complexes. In these studies, the potential for the

formation of P450-P450 complexes was examined by using

a chemical cross-linking approach. Microsomes from 3-

methylcholanthrene-treated rats were treated with the

bifunctional cross-linking agent sulfo-SADP (sulfosuccini-

midyl(4-azidophenyldithio)propionate) to cross-link any

closely associated proteins. The microsomes were then

solubilized and immunoprecipitated with antibody to

P450c (CYP1A1). The immunoprecipitate was then sub-

jected to immunoblot analysis using antibodies specific to

different P450 enzymes. This method detects any antibodies

capable of cross-linking to CYP1A1. The investigators

found that CYP3A2 coprecipitated with the cross-linked

CYP1A1, which is consistent with complex formation

between these two P450s. Interestingly, other P450s (i.e.,

CYP1A2 and CYP2E1) did not cross-link to CYP1A1.

These results indicate that different P450 enzymes interact

to the extent that cross-linked products can be obtained,

strongly pointing to the existence of heteromeric complexes.

Furthermore, specificity in the formation of these complexes

is apparent from the results showing that not all P450s could

be cross-linked under the conditions of their assay (Alston et

al., 1991).

4.1. Evidence for specific

interactions between different P450 enzymes

Although previous studies have suggested different

potential modes of interaction among these microsomal

enzymes, the functional consequences of these interactions

are less clear. Therefore, we designed experiments to

determine whether the presence of one P450 enzyme could

influence the functional characteristics of another P450

(Cawley et al., 1995). To test this hypothesis, different

reconstituted systems, simple binary systems (containing a

single P450 and reductase), and a mixed reconstituted

system (containing reductase and two different P450

enzymes) were prepared. The general protocol is shown in

Table 1. Interactions among P450s and reductase can be

detected by comparing the sum of the rates of the binary

systems with that obtained in the mixed reconstituted

system. The P450s used in these experiments were CYP1A2

and CYP2B4, which were purified from b-naphthoflavone(bNF)- and PB-treated rabbit liver, respectively. Reductase

also was purified from PB-treated rabbit liver. When exam-

ined in simple reconstituted systems, benzphetamine deme-

thylation was catalyzed � 35 times more effectively by

CYP2B4 than by CYP1A2. This selectivity was observed at

both saturating (1.5:1) and subsaturating (0.5:1) reducta-

se:P450 ratios. In order to determine if the function of a

P450 enzyme is modulated by the presence of a second

P450, benzphetamine metabolism was examined in a mixed

reconstituted system containing reductase, CYP1A2, and

CYP2B4. If the interactions between reductase and P450s

were unaffected by their presence in a complex reconstituted

system, then the rate of benzphetamine metabolism in the

mixed reconstituted system would be expected to be the sum

of the rates from the separate reconstituted systems. These

results show a slight increase (� 20%) from that expected

rate, suggesting only minimal changes in the interactions of

these proteins. Interestingly, 7-pentoxyresorufin-O-dealky-

lation (PROD) produced a completely different pattern. This

substrate was also more effectively dealkylated by CYP2B4

than by CYP1A2. PROD exhibited a small amount of

inhibition in the mixed reconstituted system at saturating

reductase. However, the magnitude of the inhibitory action

of CYP1A2 was much more pronounced at subsaturating

reductase (Table 1). These results clearly demonstrate that

P450 enzymes in complex reconstituted systems interact

differently when compared with systems containing only a

single P450 enzyme, and that the effect is dependent on the

substrate examined.

Results from the initial study (Table 1) show that

CYP1A2 dramatically inhibits CYP2B4-dependent PROD

by influencing the metabolic behavior of CYP2B4. The

ability of CYP1A2 to produce this effect is dependent on

which substrate is present. Finally, the results are consistent

with CYP1A2 (in the presence of 7-pentoxyresorufin) being

capable of forming a high-affinity complex with reductase,

which effectively draws reductase away from CYP2B4.

Table 2

Interaction between different P450 enzymes and NADPH-cytochrome P450 reductase in complex reconstituted systems

Substrate Enzyme system P450 (mM) Subsaturating reductase Saturating reductase

7-Ethoxyresorufin CYP1A1 0.1 99 ± 21 227 ± 37

(pmol/min) CYP2B4 0.1 2.7 ± 0.3 2.9 ± 0.5

Mixed system 0.1 + 0.1 157 ± 251 synergism 302 ± 42 additive

Sum of 1A1 + 2B4 102 ± 21 230 ± 37

7-Pentoxyresorufin CYP1A1 0.05 1.1 ± 0.05 2.7 ± 0.3

(pmol/min) CYP2B4 0.05 21.8 ± 2.2 28.8 ± 4.2

Mixed system 0.05 + 0.05 16.6 ± 1.92 inhibition 31.9 ± 4.9 additive

Sum of 1A1 + 2B4 22.9 ± 2.2 31.4 ± 4.0

Benzphetamine CYP1A1 0.1 0.28 ± 0.09 0.53 ± 0.11

(nmol/min) CYP2B4 0.1 2.85 ± 0.54 6.38 ± 1.11

Mixed system 0.1 + 0.1 4.55 ± 0.862 synergism 6.91 ± 1.17 additive

Sum of 1A1 + 2B4 3.13 ± 0.59 6.91 ± 1.17

p-Nitroanisole CYP1A1 0.05 30.8 ± 6.7 47 ± 14

(pmol/min) CYP2B4 0.05 75 ± 10 136 ± 16

Mixed system 0.05 + 0.05 137 ± 16 additive 235 ± 173synergism

Sum of 1A1 + 2B4 112 ± 11 192 ± 25

Interactions among reductase and two P450s were examined using CYP1A1-CYP2B4-reductase. If the interactions among different P450 enzymes follow

simple mass action phenomena, the results should be additive. Substrates not exhibiting interactions are not shown in this table. However, we have found

interactions among these proteins with � 70% of the substrates tested (rabbit liver CYP1A1 was the generous gift of Dr. Dennis Koop). Significant differences

in the mixed reconstituted system were determined by comparing the results of the ‘‘Mixed system’’ group with the ‘‘Sum of 1A1 + 2B4’’ group.1 P< 0.001.2 P< 0.05.3 P< 0.01.


We have also examined the interactions among reduc-

tase/CYP1A1/CYP2B4 and reductase/CYP1A1/CYP1A2

reconstituted systems (Table 2). These data indicate that

the interactions among P450 enzymes are not restricted to

the CYP2B4/CYP1A2 system. Generally, the effect is most

readily observed at subsaturating reductase concentrations.

Fig. 1. Models describing the potential modes of interaction among multiple P450

single P450 and reductase) are shown above the horizontal lines. Possible models f

P450 reductase.

However, there is evidence that CYP2B4-predominant p-

nitroanisole demethylation is synergistically activated by the

addition of CYP1A2 at saturating reductase levels. These

data demonstrate that functional interactions among P450

enzymes are not uncommon, and can be found with differ-

ent combinations of P450 proteins and substrates.

enzymes and reductase in a membrane. Simple binary systems (containing a

or the ternary systems are shown below the lines. FpT, NADPH_cytochrome

Fig. 2. Comparison of benzphetamine and 7_pentoxyresorufin metabolism

in reconstituted systems containing reductase, CYP1A2, and CYP2B4 with

microsomal preparations enriched in these P450 enzymes. (A) Reconsti-

tuted systems were prepared containing reductase, CYP2B4, and CYP1A2,

as described in Table 1, except that the concentrations of the P450 enzymes

were 0.05 mM. The data demonstrate the differential response of these

substrates in mixed reconstituted systems. Whereas benzphetamine

demethylation was found to be elevated in the mixed reconstituted system,

as compared with the simple binary systems, PROD was dramatically

inhibited in the presence of both P450 enzymes. (B) We are able to enhance

the expression of CYP2B4, CYP1A2, and both P450 enzymes by

pretreating rabbits with PL, bNF, and both inducers, respectively. This

pretreatment mimicked the conditions used in the reconstituted systems

described in (A). The goal of these experiments was to determine if the

differential response to benzphetamine and 7-PR in the mixed reconstituted

systems could be reproduced in the microsomal systems. Note that in both

systems, PROD was inhibited, whereas benzphetamine metabolism was not

inhibited. Data from Cawley et al. (2001).


The next series of experiments were designed to distin-

guish between three possible models that can describe the

behavior of these proteins within a membrane (illustrated in

Fig. 1). In the first mode of interaction, the proteins would

behave in the same manner in mixed reconstituted systems

as in simple binary systems (Fig. 1, option 1). No unusual

interactions would be expected from this system, and the

ability of reductase to associate with a particular P450

would be governed by mass action. The second mode

(option 2) involves two binary complexes (reductase-

CYP1A2 and reductase-CYP2B4). This is similar to option

1, except that a particular substrate causes a change in the

affinity of one P450 (in this case, CYP1A2) for the

reductase. In the third mode of interaction (Fig. 1, option

3), substrate causes the formation of a CYP1A2-CYP2B4

complex that has a high affinity for reductase. At limiting

reductase, the reductase binds only to the CYP1A2 moiety

of the CYP1A2-CYP2B4 complex.

To distinguish between these models, PROD was com-

pared in simple reconstituted systems containing a single

P450 and reductase and in mixed reconstituted systems

containing both CYP2B4 and CYP1A2 with reductase.

Each of these systems was measured as a function of

reductase:P450 ratio. When CYP1A2 and CYP2B4 were

present together in the ternary reconstituted systems, a

dramatic inhibition of PROD was observed that was more

pronounced at subsaturating reductase. As the reductase

concentration (i.e., the reductase:dilauroylphosphatidylcho-

line (DLPC) ratio) was increased, less inhibition was

observed, producing ‘‘s’’-shaped curves (Backes et al.,

1998). These results are consistent with the formation of a

high-affinity reductase-CYP1A2 complex that was more

effective at competing for reductase than CYP2B4. By

using a mathematical model, we simulated the expected

data, assuming either binary (reductase-P450) complexes

(option 2) or formation of higher-order complexes (option 3)

(Fig. 1). These results are consistent with the formation of a

ternary (reductase-CYP1A2-CYP2B4) complex (option 3)

that binds reductase with high affinity at the CYP1A2-

reductase site (Backes et al., 1998).

4.2. Demonstration that interactions

between CYP2B4 and CYP1A2 occur in microsomes

Although interactions among P450s have been docu-

mented in reconstituted systems, it was unclear whether

these effects could also be observed in microsomal prepa-

rations. In an effort to detect these interactions in micro-

somes, we took advantage of the large inhibition of

CYP2B4-dependent PROD in the presence of CYP1A2,

conditions where benzphetamine demethylation would not

be inhibited (Cawley et al., 2001). When examined in

reconstituted systems, benzphetamine demethylation was

not inhibited by the addition of CYP1A2 at subsaturating

reductase but appeared to be stimulated (Fig. 2A). In

contrast, rabbits were treated with PB, bNF, and PB+ bNF,

NF, conditions that enrich the microsomes with CYP2B4,

CYP1A2, and both enzymes, respectively. Benzphetamine

demethylation activity was equivalently elevated in both the

PB and the PB+ bNF groups, consistent with the induction

of CYP2B4 in both groups. In contrast, PROD activity in

the PB + bNF group was < 25% of that found in the PB-

treated rabbits (Fig. 2). This large inhibition of PROD

would be expected based on the inhibition obtained from

the reconstituted systems. These data demonstrate that the

interactions observed in reconstituted systems are not an

artifact of the reconstitution process, but are observed under

the more natural conditions of the microsomal membrane.

Taken together, these results demonstrate that interactions


among P450 enzymes can have a dramatic influence on the

disposition of foreign compounds and can be influenced by

the induction status of the P450s and the substrates present.

Finally, these interactions have been demonstrated to occur

both in reconstituted systems and in the endoplasmic

reticulum, attesting to the importance of understanding

how these proteins interact and the metabolic consequences

of these interactions.

Demonstration of interactions among P450 enzymes is not

restricted to rabbit forms, but has also been reported for

human P450s. Yamazaki et al. (1997b) examined the poten-

tial interactions among several human recombinant P450s,

including CYP2C10, CYP2D6, CYP2E1, CYP1A2, and

CYP3A4. CYP3A4-dependent testosterone 6b-hydroxyla-tion was not affected by the addition of CYP1A1, CYP2C10,

CYP2D6, or CYP2E1. However, CYP1A2 caused a syn-

ergistic stimulation (2- to 3-fold) of this CYP3A4-dependent

activity (Yamazaki et al., 1997b). Similar effects on

CYP3A4-dependent testosterone 6b-hydroxylation were alsoobserved when CYP1A2 from rat and rabbit were used.

These results suggest that the interactions among different

P450 enzymes are specific and not simply the result of

generalized aggregation. Furthermore, their results indicate

that these interactions are observed with P450s from different

species and that CYP1A2, in particular, is capable of pro-

ducing this effect.

Tan et al. (1997) used a baculovirus expression system to

co-express CYP2A6, CYP2E1, and reductase in a micro-

somal membrane. With this system, the investigators were

able to maintain the expression of CYP2A6 and CYP2E1 at

1:1 molar ratios and vary expression of reductase. They

found that the rate of CYP2A6-dependent coumarin hydrox-

ylation was lower in the presence of CYP2E1, as compared

with the simple binary system. This inhibition of coumarin

hydroxylation was relieved at higher reductase:P450 ratios.

These results are consistent with CYP2A6 and CYP2E1

competing for NADPH-cytochrome P450 reductase. This is

an example of a simple competition being governed by mass

action, and is similar to that reported by West and Lu

(1972).

Li et al. (1999) obtained similar results co-expressing

reductase, CYP2D6, and CYP3A4 in E. coli. They found

that CYP3A4-dependent testosterone metabolism was inhib-

ited in the presence of CYP2D6 when the CYP2D6 sub-

strate bufuralol was also present. The amount of inhibition

was attenuated at higher reductase:P450 ratios. These results

also are consistent with multiple P450 enzymes competing

for reductase, similar to the early studies of West and Lu

(1972) and those of Tan and colleagues (1997) mentioned

above.

Davydov et al. (2000b) examined the effect of the

addition of rabbit CYP1A2 on the stability of CYP2B4 in

solution. CYP2B4 was shown to be very sensitive to

changes in hydrostatic pressure, leading to a P450 to P420

conversion at relatively low pressures. In contrast, CYP1A2

was much more stable. Mixing of the two proteins led to a

stabilization of CYP2B4, protecting CYP2B4 from pres-

sure-mediated inactivation. This stabilization process was

slow, with the mixture being more stable after 9 hr than was

found immediately after mixing of the proteins. These

results support the idea that P450 enzymes behave differ-

ently when in the presence of a second P450 (Davydov et

al., 2000b).

4.3. Possible interactions

among multiple P450s and reductase

Data demonstrating interactions among these multiple

proteins could take several forms. Potential interactions

among reductase, CYP3A4, and CYP1A2 are illustrated in

Fig. 3. Depending on the mechanism of interaction, we

anticipate that the results will exhibit one of the following

patterns:

1. No altered interactions among the proteins (Fig. 3, only

Species A and B are present). In this instance, the results

of the ternary reconstituted systems would be the sum of

the binary systems at all reductase concentrations, similar

to the results obtained with benzphetamine in Table 1

(Cawley et al., 1995), as well as those obtained by

several other groups (Li et al., 1999; Tan et al., 1997;

West & Lu, 1972).

2. Substrate increases the affinity of a particular P450

enzyme for the reductase (Fig. 3, only Species A and B

are present). There are only two catalytically competent

complexes, CYP1A2-reductase and CYP3A4-reductase,

with the affinity of either one or both complexes altered

in the presence of substrate. Under these conditions, a

greater than additive effect would be observed at

subsaturating reductase concentrations. If the substrate

enhances reductase binding to the less-active P450, then

a less than additive effect would be observed (similar to

PROD data in Table 1).

3. Substrate causes formation of a heteromeric P450-P450

complex that has an altered catalytic rate. The overall

rate would be the sum of the activities of the five

catalytically competent complexes described in Fig. 3.

Formation of an CYP1A2-CYP3A4 complex could lead

to an enhanced (or diminished) catalytic activity for one

or both enzymes. Elevated catalytic activities would be

expected at both saturating and subsaturating reductase

concentrations. If the heteromeric P450 complex is less

active than the single enzymes (not in P450-P450

complexes), then a less than additive effect would be

expected. The results obtained by Yamazaki et al.

(1997a) are consistent with this model.

4. Substrate promotes formation of a heteromeric P450

complex that has an altered affinity for the reductase.

Each of the five catalytically competent complexes

contributes to the overall reaction rate (see Fig. 3). The

CYP1A2-CYP3A4 complex could have an altered

affinity for the reductase. Under these conditions, we

Fig. 3. Possible catalytic complexes involved in reconstituted systems containing reductase and multiple P450 enzymes. The figure illustrates the potential

complexes that could lead to product formation in mixed reconstituted systems containing multiple P450 enzymes. The diagram shows the complexes possible

from mixed reconstituted systems of reductase, CYP3A4, and CYP1A2. The initial rate of metabolism from these complexes would equal the sum of the rates

from each of the complexes.


would expect to observe either a less than additive effect

or a greater than additive effect at subsaturating

reductase. These possibilities are consistent with the data

observed with CYP1A2 and CYP2B4 with the substrate

7-pentoxyresorufin (Table 1) and our other reports

(Backes et al., 1998; Cawley et al., 1995).

5. Combinations of Items 3 and 4. It is possible that

formation of a heteromeric P450 complex can affect both

the affinity of a P450 for the reductase, as well as its

catalytic activity.

5. Conclusions

The above data clearly demonstrate that P450s can

function differently in a complex reconstituted system when

compared with the simple binary systems. Furthermore, the

results demonstrate that at least some of the interactions

among these proteins involve the formation of heteromeric

P450 complexes that can have a significant influence on the

catalytic function of these microsomal electron transport

proteins, having definite implications for drug metabolism

and the bioactivation of compounds in vivo. These data

point to the importance of considering P450s not as mono-

meric polypeptides that can interact with the reductase, but

as proteins that may readily interact to form P450-P450

complexes. The tendency for these proteins to aggregate in

solution may actually be more specific than once believed,

and may be a significant factor influencing monooxygenase

function. More recently, expression systems for human

P450s have been developed and are being used to estimate

the disposition of drug candidates in humans. Although

there are expression systems for many different P450s, each

system generally contains only reductase and a single

human P450. The interactions between multiple P450s

described in this review may lead to significant errors in

estimates of the disposition of these drugs. The clinical

importance of such interactions on drug disposition will

depend on the magnitude of these interactions with human

P450s, and will require additional study.

Acknowledgments

This work was funded, in part, by grants from the

National Institute of Environmental Health Sciences (R01

ES04344), institutional support from the Research Enhance-

ment Fund program at LSU Health Sciences Center School

of Medicine, and support from the Stanley S. Scott Cancer

Center. R.W.K. was supported in part by the Stanley S. Scott

Cancer Center Fellows Program.

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Organization of multiple cytochrome P450s with NADPH-cytochrome P450 reductase in membranes