Ortiz de Montellano Paul R.(Ed.) Cytochrome P450. Structure, Mechanism, and Biochemistry

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124

Mark J.I. Paine et al.

in relation to hydride transfer. Non-conservative

substitutions of

Ser457,

Asp675, and Cys630 pro-

duce large decreases in cytochrome c reductase

activity^^' ^^' ^^' ^^. These parallel changes in the

rate of flavin reduction are associated with large

NADPD isotope effects^ ^ thus pointing to

impaired hydride transfer from NADPH. Based on

spectral analysis of flavin reduction of mutants

at residues Ser457, Asp675, and Cys630 and

analysis of the kinetics and pH dependence of

cytochrome c reduction, it was proposed that

Cys630 acts a proton donor/acceptor to FAD, and

Ser457 and Asp675 interact to stabilize both the

transition state and the FAD semiquinone^^. This

is supported by crystallographic analysis of

CPR mutants, which shows that both Ser457 and

Asp675 are directly involved in interaction with

the nicotinamide group of NADP(H) and likely to

orient the C-4 atom of NADP(H) into an optimal

position for hydride transfer^^. The catalytic triad

of Ser457, Asp675, and Cys630 is highly con-

served in all diflavin oxidoreductases apart from

NRl, which has a nonconservative Ala549 corre-

sponding to Cys630^^ and a greatly decreased

(~ 100-fold) rate of reduction relative to CPR.

2.6. The Electron Transfer

Mechanism

CPR catalyzes the transfer of electrons along

the pathway NADPH-> FAD-> FMN-> P450,

and there are two striking features of its involve-

ment in the monooxygenase system. First, the

flavin redox potentials are such that electron trans-

fer from NADPH to FAD is moderately unfavor-

able (the physiological -direction of electron

transfer in the structurally related ferredoxin

reductase is in the direction of NADPH forma-

tion).

Second, NADPH is an obligate two-electron

donor, but these two electrons must be delivered to

P450s individually at the appropriate points in the

catalytic cycle. CPR orchestrates the electron sup-

ply from NADPH to the P450 cytochromes by sta-

bilizing the one-electron reduced form of the flavin

cofactors FAD and FMN. This stabilization gives

rise to the blue semiquinone species (FADH7

FMNH*), which is observed in both kinetic and

equilibrium studies of CPR^^' ^^-84 cPR and the

aforementioned related structural isoforms of

nitric oxide synthase^^, methionine synthase reduc-

tase^"^,

and protein NRl^^ can accept a maximum

of four electrons. The different redox states of

these enzymes have distinct spectral characteris-

tics.

However, the fact that the oxidised, semi-

quinone and reduced states of both FAD and FMN

have virtually indistinguishable spectra means that

kinetic and equilibrium studies of the diflavin

reductases is complicated. This is alleviated, how-

ever, by using the genetic approaches discussed

previously to dissect the enzymes into their com-

ponent domains^^'

^^' ^^^ ^^.

This strategy has been

used successfiilly with human CPR, human

methionine synthase reductase, and human oxi-

doreductase NRl. Molecular dissection of these

diflavin enzymes removes ambiguity in spectral

assignment during redox titration, potentiometry,

and kinetic analysis^^'

^'^' ^^-^^.

With CPR, the do-

mains have also formed a basis for more detailed

study of the kinetic and thermodynamic proper-

ties of the frill-length enzymes^^'

^^' ^^.

The use of

FMN-depleted frill-length enzyme has also facili-

tated assignment of reduction potentials and indi-

vidual kinetic phases in other mammalian CPRs^^.

Each isolated flavin-binding domain of human

CPR (and of related family members) is soluble,

binds flavin, and is redox active^^' ^^. The mid-

point reduction potentials of the flavin couples are

similar in the isolated domains and ftill-length

CPR^^, indicating that the isolated domains are

good mimics of domain properties in ftiU-length

CPR. The blue semiquinone on the FMN is both

kinetically and thermodynamically stabilized, and

the reduction potential of the oxidized-semi-

quinone redox couple is much more positive than

those of the other redox couples in CPR (Figure

4.8).

This accounts for the "blue/green" appear-

ance of purified CPR prior to chemical treatment

with exogenous oxidants. The midpoint reduction

potentials measured for human CPR^^ are in broad

agreement with earlier studies performed with

rabbit CPR^^. The reductive half-reaction of the

isolated FAD domain has been studied by

stopped-flow methods^^. Hydride transfer is

reversible, and reduction of NADP^ by FADH2 is

more rapid (8 s ') than FAD reduction by

NADPH (3 s~^), consistent with the relative val-

ues of the midpoint reduction potentials for the

2-electron couples for FAD/FADH2 and NADPH/

NADP^. The more rapid reduction of NADP^

probably reflects the physiological role of the

ancestral FNR-like protein, which evolved to

catalyze NADP^ reduction in photosynthetic

electron transfer. The isolated FAD domain trans-

fers electrons to the isolated FMN domain, but the

Electron Transfer Partners of Cytochrome P450

125

-100 i

-200'

-300 i

-400 i

n^m^li

jMf^'^s&^ki^-

mm mm

mmmtm

FMN sq/hq

fMUQ^'Di^liil

iFlffNIl^tiNi

'W0^mm

fmmm

FMN sq/hq

FMIi^i^

FAD sq/hq FAD sq/hq

•PM^mm

NAE^PP

FAD sq/hq

CPR NR1 MSR NOS BM3

Figure 4.8. Midpoint reduction potentials of the various flavin couples in CPR and related diflavin enzymes.

Values are taken from Munro^^ (CPR), Finn^^ (NRl), Wolthers^^ (MSR), Noble^i (NOS), and Daff«9 (BM3).

observed rate is slow and shows a second-order

dependence (second-order rate constant 9.5 X 10"^

M"^ s~^) on domain concentration, reflecting

a bimolecular reaction. This indicates the impor-

tance of covalently tethering the two flavin-

binding domains of CPR to enable rapid

interdomain electron transfer.

Reduction of the isolated FAD domain by

NADPH occurs in a series of discrete kinetic

steps including at least two different charge-

transfer complexes involving NADPH and FAD

(Figure 4.9). Partial flavin reduction occurs in the

initial phase (—200 s~^) and this displays a kinetic

isotope effect with ^-side NADPD. FAD is then

further reduced in the slower phase (~20s~^).

NADP+ release is thought to gate complete reduc-

tion of the flavin in the slower phase, consistent

with a reversible scheme for flavin reduction in

the isolated FAD domain and full-length CPR

(Figure 4.9). Similarly, it is proposed that the rate

of flavin reduction is regulated by NADP^ release

in rat neuronal nitric oxide synthase^

^' ^^

and the

adrenodoxin reductase homolog FprA from

Mycobacterium tuberculosis^^. A number of the

early kinetic phases are obscured in studies with

full-length CPR^^, thus emphasizing the impor-

tance of conducting detailed studies of electron

transfer mechanism in single domains of CPR.

The relatively high midpoint reduction poten-

tial of the FMN oxidized-semiquinone couple

(—66mV) provides the driving force for inter-

domain electron transfer in full-length CPR^^.

This enables electron flow from NADPH to the

CPR FMN. Electron transfer then occurs from the

FMN to the cytochrome P450 enzymes (or other

redox acceptors) and is a key aspect of the elec-

tron transfer mechanism in virtually all diflavin

reductase enzymes^^'

^^' ^^.

Kinetic studies show

that the transient accumulation of the blue dis-

emiquinoid species of CPR occurs at a rate identi-

cal to hydride transfer from NADPH to FAD^^.

This indicates that interdomain electron transfer is

relatively fast. The blue disemiquinoid species

subsequently decays following a second hydride

transfer from NADPH (Figure 4.9), as the flavins

are reduced to their hydroquinone states.

126

Mark J.I. Paine et al.

k k k k

E + NADPH ., "* ^ E-NADPHCT1 ^ ^

E-NADPHCT2

^ \ EHgNADP^ ^ ^ EH2 + NADP*

'^-l

'^-2 ^^-3 '^-l

k^\k.

4'^

k k

E-NADPHCT2 ^i^ EH2NADP .^i

(NADPH) ^-^ (NADPH) ^-8

.i«

EH2 + NADP^

(NADPH)

(I)

FAD

E + NADPH '

FMN

FAD

E NADPH.

FMN

FAD

E NADPH .^

FMN (NADPH)

FADHo

E NADPH**

FMN

[Refer to Scheme I]

FADH

E NADP*

FMN

-50%

FAD

E NADP*

FMNH2

-50%

FADH2

' FMNHo

[Refer

Scheme I].,

-NADP*

+NADPH

FAD

E NADPH

FMNHo

FAD

E NADPH

FMNH (NADPH)

(II)

Figure 4.9. Kinetic schemes for the isolated FAD domain (upper scheme) and full-length CPR (lower scheme)

derived from stopped-flow studies (taken from Gutierrez^^). The reversible nature of the reaction is indicated,

as is the presence of the second nicotinamide coenzyme-binding site (indicated in parenthesis for the FAD domain).

For clarity, only enzyme intermediates bound with a single coenzyme are shown in the lower scheme for CPR,

but reference to the upper scheme indicates that a second molecule of NADPH has the capacity to bind to the

enzyme.

2.6.1.

Trp676 and FAD Reduction

As discussed above, the C-terminal aromatic

residue (Trp676 in human CPR) is positioned over

the re-face of the FAD isoalloxazine ring in such a

way that it would sterically prevent hydride trans-

fer from NADPH to FAD in the absence of side

chain movement (Figure 4.6). In human CPR,

rapid changes in tryptophan fluorescence emis-

sion accompany hydride transfer in the isolated

FAD-binding domain and in CPR^^. The complex

fluorescence transients observed in CPR as two

hydride equivalents that are transferred to the

enzyme are largely absent in a W676H mutant

CPR, suggesting that Trp676 is the origin of the

fluorescence signal in wild-type enzyme^^ and

that the observed transients may reflect the postu-

lated movements of this side chain. Substitution of

Trp676 by alanine substantially compromises the

rate of hydride transfer in CPR, revealing a key

role for the side chain of Trp676 in enzyme reduc-

tion. In Trp676His CPR the rate of FAD reduction

is modestly affected, but importantly the enzyme

is reduced only to the two-electron level in rapid-

mixing experiments. Reduction beyond the two-

electron level is prevented owing to the slow

release of NADP^, indicating a role for Trp676 in

the release of oxidized coenzyme from the FAD-

binding domain. A double-mixing stopped-flow

method in which Trp676His CPR is reduced ini-

tially with stoichiometric NADPH, and following

a suitable delay (100 ms) mixed with excess

NADPH, effects reduction to the four-electron

leveF^.

In the delay period, NADP^ can escape,

albeit at a relatively slow rate, allowing binding of

the second NADPH to the catalytic site.

Electron Transfer Partners of Cytochrome P450

127

2.6.2.

Binding of Two Coenzyme

IVIolecules

Rapid-mixing studies of the kinetics of

hydride transfer in both the isolated FAD-binding

domain and in CPR have led to a kinetic model

(Figure 4.9) that invokes the existence of two

kinetically distinct binding sites for NADPH^^.

Only one site is catalytic; binding to the second

site attenuates FAD reduction, probably by inter-

fering with NADP^ release from the catalytic site.

In wild-type CPR, NADP+ release from the cat-

alytic site is sufficiently rapid that binding to the

second noncatalytic site does not prevent reduc-

tion to the four-electron level in direct-mixing

stopped-flow experiments with excess NADPH.

Following the identification of two kinetically

distinct coenzyme binding sites in CPR and the

unusual mechanism for electron transfer to FAD,

similar dual binding-site models have been pro-

posed for nitric oxide synthase^^ and the adreno-

doxin reductase homolog FprA isolated from M

tuberculosis^^.

The presence of two coenzyme-binding sites is

unexpected since they cannot be inferred solely

from the crystal structure of

CPR^^.

Kinetic stud-

ies with wild type and W676H CPR at different

concentrations of NADPH have, however, pro-

vided further support for the existence of two

sites^^'

^^.

The rate of flavin reduction in the iso-

lated FAD domain and CPR increases as NADPH

is decreased from molar excess to stoichiometric

concentrations. At stoichiometric concentration,

the second noncatalytic site is predominantly

vacant and the partial inhibition on the rate of

flavin reduction from the catalytic site is therefore

relieved (Figure 4.9). Occupation of the noncat-

alytic site occurs at NADPH concentrations in

excess of the enzyme concentration, and impairs

NADP^ release from the catalytic site. This in

turn partially inhibits flavin reduction, the rate of

which is gated by NADP^ release. Preincubation

of the enzyme with a stoichiometric amount of

adenosine 2',5'-diphosphate does not lead to

inhibition of the flavin reduction rate^^. We infer

that the binding of adenosine 2',5'-diphosphate

prevents NADPH from binding to the noncatalytic

site.

This observation also suggests that it is the

nicotinamide-ribose-phosphate portion of NADPH

bound at the second site that hinders NADP^

release from the catalytic site. Clearly, these new

data indicate that the coenzyme plays a role in the

control of CPR catalytic activity as well as acting

as a substrate. Models that assume a single

coenzyme-binding site for steady-state turnover

to explain competitive inhibition by coenz>ine

fragments might be inappropriate. The effect of

the second coenzyme-binding site on steady-state

turn-over and inhibition might lead to revised, and

by necessity more complex, steady-state kinetic

models.

2.6.3. Internal Electron Transfer

Although a thermodynamic and kinetic frame-

work for electron transfer in CPR has emerged

from rapid-mixing kinetic and potentiometry

studies^^' ^^, alternative approaches have been

required to access the rate of internal electron

transfer between the flavins. Tollin and coworkers

have used flash photolysis studies employing pho-

toexcited 5-deazariboflavin to investigate the

kinetics of inter-flavin electron transfer in rabbit

CPR^^. CPR is reduced rapidly by photoexcited

5-deazariboflavin (6.8 X lO^M-^s"^) and this

phase is followed by a slower (70 s~^) partial loss

of blue semiquinone signal that reports on inter-

flavin electron transfer from FAD to FMN^^.

Pre-reduction of rabbit CPR prior to flash photol-

ysis yields a rate constant for internal electron

transfer of

This slower rate for pre-reduced

enzyme (FAD^^/FMN ) is consistent with the

smaller driving force for electron transfer from

FAD^

to FMN following electron donation by

5-deazariboflavin. Studies with human CPR have

used temperature-jump relaxation kinetic methods

to follow inter-flavin electron transfer^^' ^^. As

with flash photolysis, relaxation kinetic methods

bypass preceding steps that might limit the rate of

inter-flavin electron transfer in rapid-mixing

approaches (e.g., the preceding FAD reduction

step).

Application of relaxation kinetic methods to

CPR has allowed direct measurement of the rate

of inter-flavin electron transfer in enzyme reduced

at the two- and three-electron level, and also

opened up studies of conformational gating and

ligand effects on this reaction^^. A key require-

ment of the temperature-jump approach is to poise

the equilibrium of a reaction, such that perturba-

tion of the equilibrium by rapid heating, effects

an absorption change that can be assigned to an

128

Mark

J.I.

Paine

ef a/.

individual reaction step (i.e., internal electron

transfer from FAD to FMN in the case of CPR).

This is especially difficult with CPR where slow

thermodynamic relaxation would lead to an equi-

librium mixture of several different redox forms

of the enzyme. The experimental conditions were

selected carefiilly to minimize any uncertainty

arising from the identity of

the

enzyme species in

the final equilibrium distribution. Potentiometry

studies indicate that in two-electron-reduced

enzyme the electrons are distributed as an equilib-

rium between the two enzyme species FAD^^/

FMN^^ and FAD^/FMN. , and that -50% of each

sq ox nq'

enzyme species is populated^^. A rapid tempera-

ture increase shifts this equilibrium toward the

FAD^^/FMNH2 form, and the kinetics of this

process report on the rate of inter-flavin electron

transfer^^. Inter-flavin electron transfer in human

CPR is relatively slow. For CPR reduced at the

two-electron level with NADPH, two kinetic

phases are seen in temperature-jump experiments.

Fluorescence and absorbance studies with

dithionite-reduced CPR indicate that the fast

phase

(1/T

= 2,200 ± 300 s~^) is not associated

with electron transfer, but is attributed to local

conformational change in the vicinity of

the

FAD

when CPR is reduced with NADPH. The slow

phase

(1/T

55±2S~^)

reports an internal elec-

tron transfer, FAD^^-FMN^^ ^ FAD^^-FMN^^q.

An intrinsic electron transfer rate of

—10^^

s"^ is

predicted based on the separation distance of the

two flavins in rat CPR^^' ^^. The modest transfer

rates observed in the temperature-jump studies

indicate that electron transfer is gated. Possible

origins of the gating reaction have been consid-

ered. For example, the reaction involves the

deprotonation of the FAD blue semiquinone, but

solvent isotope effects do not accompany the reac-

tion, ruling out a rate-limiting deprotonation reac-

tion. Electron transfer is compromised in the

presence of glycerol (75% w/v glycerol), suggest-

ing conformational gating^^ and a conformational

search for domain orientations that maximize

electronic coupling between the flavins is consis-

tent with the atomic structure of CPR (and related

diflavin enzymes), which are consistent with a

high degree of interdomain mobility^^' ^^' ^^ The

equilibrium distribution of enzyme species is

poised differently by reduction of CPR at the

three-electron level^^. Relaxation experiments

with three-electron reduced CPR give access to

the kinetics

(1/T

= 20 ± 0.2 s"^) of electron

transfer in the nonphysiological direction

(FAD,^/FMN,^ ^ FAD^^/FMN^^).

In the W676H mutant CPR, this rate is substan-

tially increased

(1/T

= 263 ± 3 s~^), but the rate

of electron transfer in the physiological direction

(FAD^qFMN^q ^ FAD^^FMNj^q) is decreased by

only a factor of ~2

(1/T

= 27 ± 1 s~^). This there-

fore points to another important role for Trp676 in

controlling electron transfer in CPR in favoring the

transfer of electrons in the physiological direction

(NADPH -^ FAD -^ FMN -^ heme).

Although the initial equilibrium is the same,

inter-flavin electron transfer rates for CPR

reduced by NADH

(1/T

= 18 ± 0.7 s"') are -3-

fold less than those for CPR reduced by NADPH

(1/T

= 55 ± 0.5 s~^). This cannot be attributed to

thermodynamic differences since the reduction

potentials of the reducing coenzymes are essen-

tially identical. This suggests a role for coenzyme

binding in modulating inter-flavin electron trans-

fgj.95,

97 jjjjg jg supported by the observation

that the rate of inter-flavin electron transfer in

dithionite-reduced CPR

(1/T

= 11 ± 0.5 s"^ is

clearly less than that for CPR reduced with

NADPH

(1/T

= 55 ±0.5s-^)^^ Furthermore,

addition of 2',5'-ADP to dithionite-reduced

enzyme effects a ~

3-fold

increase in the rate of

inter-flavin electron transfer

(1/T

= 35 ± 0.2 s~^),

and this is accompanied by an increase in the

amplitude of the absorption signal change^^. Thus,

the binding of the adenosine moiety of NADPH,

and in particular the 2'-phosphate group, is a

major factor in enhancing the rate of inter-flavin

electron transfer. Potentiometry measurements

have shown that the binding of 2',5'-ADP does not

perturb the midpoint reduction potentials of the

four flavin couples.

propose that ligand binding

in the coenzyme-binding pocket effects a confor-

mational change involving domain movement, the

rate of which controls inter-flavin electron transfer.

2.6.4. Interaction with and Electron

Transfer to P450

The microsomal P450 monooxygenase

complex is localized to the endoplasmic reticulum

membrane. Like P450, CPR contains an N-

terminal hydrophobic region that spans the lipid

membrane and anchors it to the surface. In yeast,

Electron Transfer Partners of Cytochrome P450 129

the soluble form of the enzyme can support P450

activity"^^, but soluble mammalian CPRs that have

had the hydrophobic anchoring peptide removed

are incapable of coupling with cytochrome P450.

The anchoring of these enzymes to the membrane

surface thus appears to be the principal factor

required for correct spatial orientation of the

redox centers for effective electron transfer.

Studying the architecture and topology of

membrane proteins on the phospholipid bilayer is

experimentally extremely challenging. However,

new developments are being applied to analyze

the topography of the monooxygenase complex on

a phospholipid bilayer. Sligar and coworkers have

used 10-nm scale phospholipid bilayer disk struc-

tures to orient CPR and cytochrome P450 2B4 on

a surface for visualization by AFM^^' ^^^. The

height of CYP2B4 protruding above the surface is

estimated at 3.5 nm, which is consistent with a

hydrophobic tip of the molecule being partially

inserted into the membrane. The exact spatial rela-

tionship with CPR is not yet clear, but it is thought

that CPR lies in an orientation such that both the

FMN and FAD/NADPH domains lie close to the

membrane surface, which would allow close

communication between the FMN and the P450

heme22'

ioo_

Transient monooxygenase complexes are

formed on the membrane surface as a result of

collisions between P450s and CPR as each

dif-

fuses laterally within the membrane of the endo-

plasmic reticulum. The role of the membrane in

mediating these interactions is poorly understood.

However, early work has shown that the phospho-

lipid component of the membrane can affect

intermolecular interactions of the monooxygenase

complex^^^'

^^^

and influence substrate binding^^'

^^^

and may therefore be important in maintaining

efficient electron transfer from CPR to P450.

The structure and sequence of rat and human

CPR^^' ^^ show some interesting features that

might influence the orientation of the protein at

the membrane surface. One is a cluster of basic

residues R'^^KKK, which lies at the membrane

anchor: FMN domain junction. Their functional

significance is unclear, but it can be speculated

that the basic Arg and Lys side groups might

interact with anionic phospholipid head groups,

possibly to restrict the movement of CPR on the

membrane surface. The FMN-binding domain

has clusters of positive and negative charges at

opposite ends, leading to a strong electric dipole

moment (677 Debye)^^, which may influence the

orientation of the reductase at the membrane sur-

face.

The patch of positively charged residues

exposed at the surface of the human FMN-binding

domain (K72, K74, K75, R78, R97, KlOO, H103,

and R108) in particular could form an additional

membrane-binding site.

Protein-protein interactions are essential to

enable electron transfer from the reduced FMN

of the flavoprotein to the substrate-bound ferric

form of the P450. Since P450 is present in a

10-25-fold molar excess over CPR in the liver

microsome^^'^' ^^^, rapid association and dissocia-

tion of

P450:

CPR complexes is important for the

system to work effectively. A number of lines of

evidence point to electrostatic interactions being

the driving force behind the binding of P450 with

CPR. For instance, it has been demonstrated that

specific positively charged lysine and arginine

residues on the rat P450, CYPlAl, are involved

in forming an electron transfer complex with

CPR^^6.

It has also been reported that CYP2B1

and CYP2B4 interact with CPR through comple-

mentary charge interactions^^^' ^^^. In the case of

CYP2B4, site-directed mutagenesis has identified

a series of lysine and arginine residues on the

proximal surface near the heme ligand that inter-

act with CPR^^^. Neutralization of carboxylate

groups on CPR by chemical modification inhibits

both cytochrome c reductase activity and P450-

dependent monooxygenation"^^'

^^^.

Chemical cross-

linking studies indicated that a cluster of acidic

amino acids on CPR were involved in the interac-

tion with rat CYPlAl i^^.

The FMN domain may be assumed to provide a

major portion of the docking surface for P450s,

although as discussed above some reorientation

of this domain from its position in the crystal struc-

ture may be required. Electrostatic potential meas-

urement of the surface of the human CPR-

FMN-binding domain shows three distinct clusters

of acidic residues that could form ion-pair inter-

actions with the electron transfer partners^^' ^^^

Cluster

contains Asp207,Asp208,Asp209; cluster

Glu213, Glu214, Asp215; and cluster 3, Glul42,

Asp

144,

Asp

147.

The first two clusters correspond

to the region of CPR that was cross-linked to a

lysine residue in cytochrome c^^^, and have been

investigated by site-directed mutagenesis in rat^^^

and human^^ CPR. The results of these experiments

130

Mark

J.I.

Paine

ef a/.

show that while cluster 1 mutations do not afifect

C3^ochrome c binding, removing the charge from

Asp208 alters the binding of cytochrome P450. In

contrast, cluster 2 mutations affected the binding of

cytochrome c, but not P450. Mutations of residues

in cluster 3 produce a modest decrease in P450

activity with no effect on cytochrome c reductase

activity^^. Thus, discrimination is apparent between

residues involved with the interaction of CPR with

cytochrome c and with P450.

The finding that mutation of residues in clus-

ters

and

on either side of the FMN-binding site

affect P450 activity, suggests that c3^ochromes

P450 bind at the tip of

the

FMN domain in such a

way as to cover the FMN cofactor^^. Examination

of the crystal structure of rat CPR indicates that

structural rearrangements during catalysis of the

kind discussed above would be required to bring

the redox centers of CPR and P450 into an

appropriate configuration for efficient electron

transfer^^ (Figure 4.10). A similar structural

rearrangement has also been suggested for the

FMN and FAD domains of

BM3'

While it is generally considered that charge-

pair interactions are important in docking and

electron transfer reactions, this is not always

reflected in the ionic-strength dependence of

P450 reduction in vitro with reconstituted mono-

oxygenase systems. For instance, it has been

shown that in the presence of high concentrations

salt,

which should neutralize electrostatic inter-

actions, the reduction rate of P450 by CPR may

increase^

^'^~^^^.

It is possible that the observed

effects of ionic strength reflect the combined

result of perturbations of CPR-P450 interactions

and of domain-domain interactions within CPR.

Most recently Davydov et

al}^''

have compared

the role of electrostatic interactions in the associa-

tion of rabbit CPR with CYP2B4 and the heme

domain of P450 BM3, which is normally fused

with a reductase domain'

'^.

They found an increase

in K^ (10-90 nM) for the CPR-CYP2B4 complex

with increased ionic strength (50-500

JJLM),

con-

sistent with the involvement of charge pairing.

Interestingly a reverse relationship was observed

with BM3-heme and BM3-CPR domains, which

showed low affinity for each other. It is postulated

that this may reflect the fact that P450 BM3 is a

tethered complex, where there is no strong require-

ment for charge pairing.

Conformation in crystal

appropriate for inter-

flavin electron transfer

"Open"

conformation:

appropriate for electron

transfer to P450

Figure 4.10. Schematic model for the domain movement required for opening CPR for efficient electron transfer

to P450.

Electron Transfer Partners of Cytochrome P450

131

2.7.

Cytochrome P450 BM3

Since its discovery in the 1980s, flavocy-

tochrome P450 BM3 (CYP102A1) has undergone

intensive biochemical study and has established

itself as a model P450 system to rival P450cam.

The key aspect of

the

structure of P450 BM3 that

has led to this rise to prominence is the fact that it

was the first prokaryotic P450 enzyme recognized

to interact with a eukaryotic-like P450 reductase

redox partner (i.e., a FAD- and FMN-containing

NADPH-cytochrome P450 reductase) rather

than with an iron-sulfur ferredoxin and FAD-

containing ferredoxin reductase, long considered

to represent the "typical" bacterial P450 reductase

system^^l Thus P450 BM3, a soluble P450,

off-

ered great promise as a tool for understanding

redox partner interactions and electron transfer

in an experimentally more tractable system than

was the case for the membrane-bound eukaryotic

P450s and P450 reductase^. An even more attrac-

tive feature of P450 BM3 was that it was a natural

fusion protein—^with the reductase linked to the

P450 in a single 119 kDa pol5^eptide. Structural,

spectroscopic, and enzymological studies of P450

BM3 in the two decades since its discovery have

been extremely informative as regards P450 struc-

ture and mechanism, establishing P450 BM3

as a vitally important "model" system for the

P450 superfamily^.

P450 BM3 was discovered as a result of

studies by Armand Fulco's group on fatty acid

hydroxylase systems from the soil bacterium

megaterium^^^.

As implied by the name, BM3

was the third enzyme with fatty acid hydroxylase

activity identified in the bacterium. P450 BM3

became a major focus of Fulco's studies following

the finding that the gene was strongly induced by

addition of phenobarbital to the growth medium^^,

suggesting that the gene was under transcriptional

control akin to the types of regulation seen previ-

ously for mammalian drug-metabolizing P450

isoforms^^^. Subsequent studies have shown the

importance of

repressor protein (BM3R1) in the

control of CYP102A1 expression, with barbitu-

rate drugs and a range of other hydrophobic mol-

ecules being able to displace the repressor to

promote production of P450 BM312I' ^22. patty

acids are likely to perform the same role in vivo,

and have also been shown to facilitate expression

of a homolog of P450 BM3 in Bacillus subtilis.

The overproduction of P450 BM3 facilitated by

addition of phenobarbital to B. megaterium

growth medium simplified purification of the

enzyme, and cloning of the gene allowed its

expression and purification from E. coli^^^. It

quickly became apparent from analysis of both

gene and protein that P450 BM3 was an unusual

fusion protein comprised of a fatty acid-binding

P450 (homologous to the eukaryotic CYP4 fatty

acid hydroxylase family) fused to a CPR^^"^.

Analysis of the catalytic properties of the purified

enzyme showed it to have a very high catalytic

center activity relative to its eukaryotic fatty acid

hydroxylase relatives—with turnover numbers of

several thousand per minute with various long-

chain fatty acids^^' ^^^. This was rationalized as

being due to the efficiency of the electron transfer

system covalently associated with the P450, and

opened the door to mechanistic and kinetic studies

on the enzyme^^' ^^^' ^^^. In the BM3 system,

NADPH reduces the FAD flavin transiently to the

hydroquinone form. Electron transfer to the heme

is mediated by the FMN cofactor, which shuttles

electrons one at a time between the FAD and

heme^.

Genetic dissection provided further proof for

the multidomain nature of the enzyme-producing

first sub-genes encoding stable P450 (first —472

amino acids of P450 BM3) and CPR domain^^' ^^\

and then constructs encoding the individual

FAD/NADPH-binding and FMN-binding domains

of the CPR^^^. Domain dissection also enabled the

determination of the atomic structures of the heme

(P450) domain of the enzyme and also the struc-

ture of a nonstoichiometric complex of the heme

and FMN domains of the enzyme, arising from

proteolysis of a construct containing the heme

domain and the FMN-binding domain^^^.

The atomic structure of the heme domain has

been solved in substrate-bound and substrate-free

forms,

showing that a large structural change in

the protein accompanies the binding of substrate

(palmitoleic acid). Binding of fatty acid substrates

to the heme domain causes a shift in the heme iron

spin-state equilibrium toward the high-spin form,

which is accompanied by a large increase in

reduction potential for the ferric/ferrous transition

of the heme iron^^' ^^' ^^^. Binding of fatty acid

thus facilitates electron transfer to the heme iron

and triggers catalysis. In the absence of bound

substrate there is negligible electron transfer

132

Mark J.I. Paine et al.

between the FMN and heme cofactors. When

P450 BM3 binds its most efficient substrates, that

is those fatty acids, such as arachidonic acid, that

induce the greatest degrees of spin-state transition

and promote the fastest turnover^ ^^' ^^^, FMN-to-

heme electron transfer is rapid (c. 250 s^^ with

lauric acid) and catalysis occurs^^. The extent of

the increase in reduction potential accompanying

saturation with fatty acid is approximately 130 mV,

a change which elevates the heme reduction

potential above that of the ultimate reductant in

the system (NADPH: E^ = -320 mV cf -427

for BM3 heme in absence of substrate and -289

in presence of arachidonate) and within range of

that of the FMN cofactor (E^ = -203 mV for its

oxidized/hydroquinone couple)^^'

^^K

2.7.1.

Electron Transfer Properties of

BM3 Reductase

Interestingly, the BM3 reductase is the only

member of the diflavin reductase class of enzymes

which does not stabilize a neutral, blue semi-

quinone on its FMN. Formation of a blue semi-

quinone during reductive titration is a feature

typical of flavodoxins, and retained in other mem-

bers of the diflavin reductase family (e.g., nitric

oxide synthase, methionine synthase reductase,

and other eukaryotic CPRs). The fact that the

semiquinone is destabilized with respect to the

hydroquinone in BM3's CPR and FMN domains

has been rationalized on the basis of the structure

of the FMN domain. The presence of basic

residues (particularly Lys572 and Lys580) may

stabilize the anionic hydroquinone form and

destabilize the semiquinone^'^. The redox state of

the flavins is of great importance to the catalytic

properties of BM3, since early studies on the

enzyme showed that there was strong inhibition of

fatty acid hydroxylase activity following preincu-

bation of the enzyme with NADPH^^. It appears

that an unfavorable three-electron (and possibly

four-electron) reduced form of the enzyme accu-

mulates within a few minutes of incubation of the

enzyme with NADPH, and that electron transfer

in this species is diminished and results in a cat-

alytic rate of fatty acid hydroxylation less than

20%

of the maximal rate. Analysis of the redox

state of the flavins during turnover suggests that

both may exist predominantly in a semiquinone

state as catalysis progresses (a neutral semi-

quinone in the case of the FAD and a red, anionic

semiquinone in the case of the FMN), suggesting

that the CPR domain of the enzyme cycles favor-

ably between 0-2-1-0 electron-reduced states

during catalysis'^^. The situation appears similar

for the housefly CPR, but contrasts with that

reported for many eukaryotic CPRs, where the

cycle appears to be

1-3-2-1

'^^.

However, the pre-

cise reason for the apparent inactivation of BM3

in an "over-reduced" form remains obscure. Since

electron transfer to exogenous electron acceptors

via the flavins remains largely unaflected'^^, it

appears to be the case that the catalytic step per-

turbed is that of first or second electron transfer to

the heme iron. This may indicate that the reduc-

tion state and/or coenzyme-binding affects the

conformational status of the reductase domain,

in particular the movement of the FMN-binding

domain relative to the FAD-binding domain

discussed above.

Stopped-flow absorption spectroscopy has

provided information on some of the internal elec-

tron transfer processes in P450

BM3^^.

In particu-

lar, the rate of electron transfer between NADPH

and the FAD (in itself a composite of rates for

NADPH association, orientation and the actual

hydride transfer event) is very fast (500 s~' at

5°C) relative to those measured for mammalian

CPRs,

nitric oxide synthase, methionine synthase

reductase, and novel reductase 1 (NRl)^^' ^'^' ^^.

The rate of internal (inter-flavin) electron transfer

remains to be determined, but should be greater

than 250 s~' (the turnover rate with arachidonate).

The rapid electron transfer from NADPH to

flavin is diminished considerably for the NADH-

dependent reaction, indicating the importance of

the 2' phosphate group of NADPH in both recog-

nition and binding of

the

pyridine nucleotide, and

in the actual electron transfer event. Comparison

of the thermodynamic properties of the BM3

reductase with those of the other members of the

diflavin reductase family (Figure 4.8) indicates

that increased driving force does not underlie the

enhanced rate of NADPH-to-FAD electron trans-

fer observed for this isoform^^. For instance, in

human CPR there is a more favorable thermody-

namic balance for electron transfer between

NADPH (E^' = -320 mV) and the FAD cofactor

(E^

= -328 mV for the two-electron reduction to

the hydroquinone form) and an apparent rate of

Electron Transfer Partners of Cytochrome P450

133

electron transfer of ~3 s~^ at 25°C with excess

NADPH^^' ^l For BM3, the midpoint reduction

potential for the FAD cofactor is virtually identi-

cal to that for human CPR (-332 mV), and yet the

rate of electron transfer is apparently —200-300-

fold that of human CPR under similar conditions.

Obviously, structural differences in BM3 reduc-

tase underlie its capacity to drive electron transfer

so efficiently with respect to other diflavin reduc-

tases.

However, these differences await clarifica-

tion through structural elucidation of the BM3

reductase and/or its FAD/NADPH domain. In

view of the proximity of the redox couples of

NADPH and the FAD flavin in BM3 (and other

diflavin reductases), reversibility of the electron

transfer process (i.e., hydride transfer from FAD

hydroquinone to NAD(P)^) might be expected.

This has been shown to occur for both mammalian

CPR and for BM3 reductase^^'

^^'

1^4

Many aspects of the electron transfer reactions

in P450 BM3 are now reasonably well understood,

and the first mutagenesis experiments on the

enzyme ruled out the involvement of

tryptophan

residue adjacent to the heme in the flavin-to-heme

electron transport process^^^. However, even after

two decades of study of this enzyme, several fun-

damental issues remain unresolved. Key among

these are the reasons why the electron transfer

reactions in the reductase domain of the enzyme

(and between FMN and heme) are so efficient by

comparison with the eukaryotic P450 and CPR

systems. Thus, further detailed rapid kinetic and

structural studies are critical to gain a complete

understanding of this efficient electron transfer

system.

2.8.

Artificial CPR-P450 Fusion

Constructs

Many attempts have been made to increase

the catalytic efficiency of mammalian P450s to the

level of BM3 through artificial fusions with

CPR. The first such artificial fusion between rat

P450 CYPlAl and rat CPR was constructed by

Murakami et alP^. Since then, several fusions

with rat CPR have been constructed including

bovine CYP17a (ref [137]), human CYP3A4

(ref [138]), human CYPlAl (ref [139]), CYP4A1

(ref [140]), and rat CYP2C11 (ref [141]). In

addition, yeast CPR has been fused to rat CYPlAl

(ref [142]), and human CYP3A4 (ref [143]).

Most recently, a fusion comprising entirely human

genes was constructed between CYP2D6 and

human CPR^44

As yet none of these artificial systems

approach P450 BM3 in terms of its high catalytic

activity. Indeed, most artificial mammalian

fusions achieve no greater turnover levels than

their physiological counterparts in which the

reductase and P450 form noncovalent complexes.

This may indicate that the exceptional catalytic

rate exhibited by P450 BM3 arises from the

details of its mechanism rather than simply

from the fact that the reductase and P450 are

covalently linked. Nevertheless, such catalytically

self-sufficient P450-CPR fusion enzymes are

extremely useful for probing the biochemical

mechanisms involved in catalysis and electron

transfer between P450 and CPR. They also have

important practical applications in the develop-

ment of efficient P450-based biocatalysts and

high throughput screening systems.

3. Electron Transfer to P450s

from Cytochrome

Cytochrome b^ was originally discovered in

silkworm larvae

^"^^

and has since been most exten-

sively studied in mammals^'

^^^.

Like cytochromes

P450 and CPR, cytochrome

is an integral mem-

brane protein located on the C5^osolic side of the

endoplasmic reticulum where it is principally

involved in lipid biosynthesis. It functions along

with cytochrome b^ reductase as the electron

donor to microsomal desaturases that synthe-

size unsaturated fatty acids, plasmalogens, and

sterols^'^^. Indications of the involvement of

cytochrome b^ in P450 reactions date from early

studies that showed that NADH stimulates

NADPH-supported metabolism of drugs in micro-

somes^"^^' ^^^. Since then, evidence has accumu-

lated that c)^ochrome b^ has a significant role in a

number of P450 systems^^^, most significantly

CYP3A4 (ref. [151]), which is involved in the

majority of human drug oxidations.

Cytochrome b^ is a small polypeptide

(~17kDa) containing 134 residues, which are

divided into an amino-terminal hydrophilic heme

domain and a carboxyl-terminal hydrophobic