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

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134

Mark J.I. Paine et al.

membrane-binding region^^^' ^^^. While the mem-

brane-bound form of

Z?5

participates in elongation

and desaturation of fatty acids^^'

^^^^ ^^^,

cholesterol

biosynthesis^^^, and drug metabohsm, soluble forms

Z?3

and

Z>3

reductase are also found in er5^hrocytes,

where they catalyze methemoglobin reduction^^^.

Mutations to either the b^ gene or the b^ reductase

gene can cause congenital methemoglobinemia^^^.

Soluble Z?5 has also been purified as a cytosolic

component in the NAD(P)H-reductive activation

pathway for porcine methionine synthase^^.

Like CPR, it is generally accepted that

charge-pair interactions drive the formation of

P450:Z?5 complexes^^^~^^^. This is supported by

site-directed mutagenesis of rabbit CYP2B4,

which has identified several positively charged

residues on the P450 surface (R122, R126, R133,

K139,

and K433) as being involved in binding

(ref [58]). However, the precise mechanism by

which b^ supports P450 catalysis is not yet clear.

As the redox potentials of cytochrome b^

and ferric P450 are approximately +25 mV and

-300 mV^^^ respectively, a role in the first elec-

tron transfer step of the P450 catalytic cycle is

unlikely, suggesting that b^ may only be involved

in the donation of the second electron''^^'

•'*^.

Since

electrons may be supplied to cytochrome b^ by

either NADH cytochrome b^ reductase or CPR^^,

the pathway of electron movement is difficult to

establish. Furthermore, studies that show that

apocytochrome b^ can stimulate the catalytic

activity of CYP3A4^^4' ^^^ 2C9, 4A7, and 17^^^

question a formal role in electron transfer. Thus,

over the years several different mechanisms have

been suggested by which

supports P450 cataly-

sis.

These include: electron transfer from NADPH

through CPR to cytochrome b^ and thence to

P450166,167. electron transfer from NADH through

b^ reductase to cytochrome b^ and thence to

P450'^^;

electron shuttling between P450 and b^

resulting in a tighter coupling between electron

flow and product formation^^^; reduction in the

uncoupled generation of superoxide or hydrogen

peroxide^''^; and an allosteric effect not dependent

on electron transfer^ ^'*' ^^^.

Much of the early data on cytochrome b^

interactions with P450 has come from in vitro

analysis using reconstituted systems. However, the

expression in

coli^^^,

insect cells^^^ or yeast^^^

of a complete mammalian P450 monooxygenase

complex comprising cytochrome b^, CPR, and

P450 has provided a means of studying the actions

of the individual components in a more physio-

logical context. Using yeast systems, Perret and

Pompon^^^ have shown that the redox properties

Z?5

were strictly required to enhance CYP3A4

activity, with results of rapid kinetic analysis of Z73

reduction following NADPH addition suggesting

that

Z>3

was reduced by the 3A4 ferrous-dioxygen

complex and reoxidized by subsequent P450 oxy-

genated intermediates ^^^. In E. coli, cytochrome

b^ significantly enhances the testosterone and

nifedipine activity of CYP3A4 and results in

the stabilization of the P450 during substrate

turnover'^^. This is consistent with the proposal

that b^ interaction reduces uncoupling and super-

oxide anion release'^^' '^^.

With the increasing sophistication in the

genetic tools now available it is becoming possible

to investigate the complex interrelationships of

the monooxygenase system in the correct context

of the whole organism. For instance, the absence

of any detectable P450 activity in mice where

CPR has been knocked out of the liver provides

compelling evidence that the alternative cyto-

chrome b^/b^ reductase electron transfer pathway

does not play any significant role in vivo^^. We

now await the construction ofb^ knockout mice to

provide further clues as to the precise role of

in the physiological function of P450s in higher

vertebrate organisms.

4. Iron-Sulfur Electron Donors:

Adrenodoxin, Putidaredoxin,

and their Reductases

4.1.

General

The Fe2S2-type ferredoxins can be arranged

into three distantly related classes based on amino

acid sequence homologies: bacterial-, plant-, and

vertebrate-type*^"*. Extensive information on the

function and mechanisms of this system has been

gained through work on the bacterial P450cam

system in Pseudomonas putida, which catalyzes

the conversion of (i-camphor to 5-exo-hydroxy-

camphor^^^. In

putida, the iron-sulfur protein

is putidaredoxin (Pdx), a 106 amino acid resi-

due ferredoxin. For catalysis, two reducing equiv-

alents are sequentially transferred from NADH

Electron Transfer Partners of Cytochrome P450

135

to P450cam via an FAD-containing enzyme,

putidaredoxin reductase (Pdr) and Pd. The puti-

daredoxin: P450cam binary complex forms with

a K^ that is dependent on the oxidation states of

the proteins and on whether substrate is bound to

the P450^^^. In the first electron transfer, reduced

Pd interacts with ferric (i-camphor-bound

P450cam and reduces it to the ferrous form. In the

second electron transfer, reduced Pd forms a com-

plex with ferrous dioxygen and (i-camphor-bound

P450cam to transfer the second electron and

reductively cleave bound dioxygen, producing

hydroxycamphor and water*^^. While most struc-

tural and biophysical studies of P450/ferredoxin

interactions have focused on the Fe2S2 putidare-

doxin from

putida (and its cognate P450cam),

other types of bacterial ferredoxins are recognized

to interact productively with P450s. For instance,

the Fe3S4 ferredoxin SoyB is a redox partner

for the Streptomyces griseus P450 Soy (SoyC)*^^

and the Fe^S^ ferredoxin from

subtilis (fer) has

been shown to undergo redox reactions with P450

Biol from the same bacterium*^^.

In animals, ferredoxins are primarily involved

in electron transfer interactions involving mito-

chondrial P450s. This fact is in agreement with

the theory of the prokaryotic origin of this

organelle. Much work has been done with adreno-

doxin (Adx) and the transfer of electrons from

NADPH-dependent adrenodoxin reductase (AdR)

to CYPUAl, which is involved in cholesterol

side chain cleavage, and to CYPUB, involved in

the formation of Cortisol and aldosterone*^"*.

Bovine adrenodoxin mRNA is translated as a 186

amino acid precursor molecule, which undergoes

proteolytic processing upon import into the mito-

chondria to remove the N-terminal 58 residues *^^.

The mature Adx comprises 128 amino acid

residues and has a molecular weight of 14.4 kDa.

In plants, [2Fe-2S] ferredoxins are involved in

conjunction with ferredoxin-reductase in photo-

synthesis reactions and the transfer of electrons

from photosystem I to NADP^*^*. However, evi-

dence for involvement of ferredoxins in electron

transfer to plant P450s awaits a more detailed char-

acterization of the multiple P450 systems present

in the biotechnologically important plant species.

Ferredoxins receive their electrons from

ferredoxin reductase, which represents the first

component of the electron transfer system. These

enzymes contain FAD and belong to a large group

of electron transferases that includes bacterial-

type putidaredoxin reductase (PdR)*^^, animal-type

adrenodoxin reductase (AdR)*^^ *^^, and plant-

type ferredoxin-NADP^ reductases'^. An AdR-

type reductase system (FprA) has recently been

characterized in the pathogen M. tuberculosis^^

and the structure reveals extensive similarity with

the mammalian enzyme*^^. In humans, AdR is

most abundant in the steroid-producing cells of

the adrenal cortex, the ovary, and the testis*^'.

4.2.

Interactions with P450

The structures and models of structures of

several bacterial, animal, and plant ferredoxins are

available (e.g., putidaredoxin^^^ and the Fe4S4

ferredoxin from Bacillus thermoproteolyticus^^^).

In particular, details of the electron transfer mecha-

nisms have developed through kinetic and muta-

tional analysis of the Pdx-P450cam system^'^.

These indicate that Pdx interacts with proximal sur-

face of P450cam through electrostatic interactions

involving the acidic Pdx residues Asp38 and Asp34

and the basic P450cam residue Arg 112^^^' ^^^

Additionally, Argl

appears to form the electron

transfer route in the P450cam-Pdx complex with

Cys39 and Asp38 of Pdx^^^'

^^^.

A role for the

C-terminal amino acid of Pdx (W106) in electron

transfer to P450cam was suggested on the basis of

diminished enzymatic activity in systems with

Pdx W106 mutants, but later studies of various

W106 mutants showed that this residue primarily

affects the K^ of Pdx for the P450i9^ Studies of

the interactions between cytochrome b^ and

P450cam show that b^ and Pdx compete for the

same binding site on the P450, suggesting a

common mode of charge-charge pairing that is

the major force driving interactions at the same

surface on the P450^^'^.

However, isothermal calorimetry experiments

show that the energetics of the P450cam/Pdx

association are most similar to those of binding

reactions of antibody to antigen complexes ^^^,

suggesting that van der Waals and hydrogen-bond-

ing interactions may predominate in the formation

of P450cam-Pdx complexes. This study also sug-

gested that binding between Pdr and Pdx might

be dominated by hydrophobic interactions. Not-

withstanding these data, a variety of mutagenesis

studies along with the fact that increased ionic

136 Mark J.I. Paine et al.

strength markedly increases the K^ of Pdx for the

P450,

highlight the important influence of electro-

statics on the docking process^^^' ^^^.

Catalytic interactions between Pdx and

P450cam are considered to follow a ping-pong

mechanism^^^. The flavin (FAD) of Pdr is reduced

directly to its hydroquinone by interaction with

the obligatory two-electron donor NADH, but

negligible formation of semiquinone is observed

during the successive single-electron transfer

reactions between Pdr and Pdx in steady-state

turnover of P450cam. This indicates destabiliza-

tion of the flavin semiquinone with respect to the

hydroquinone and oxidized forms ^^^. Rapid (laser

flash photolysis) kinetic studies indicate transient

formation of a blue semiquinone on the Pdr FAD,

which disproportionates rapidly ^^^.

Upon complex formation, Pdx induces confor-

mational changes in P450cam'^^ which convert

the high-spin state of ferric P450cam to low-spin

state and stretch the heme-axial ligand^^^.

NMR studies of the Pdx-P450cam complex also

show that Pdx binding perturbs several regions

involving the substrate access channel in

P450cam^^^ and induces a tilt in the heme plane,

leading to the movement of J-camphor and the

axial Cys to the heme-iron by 0.1-0.5 A and

facilitating heterolysis of the O-O bond^^^. It is

thus proposed that in addition to its redox

function, Pdx invokes structural changes that

facilitate the oxygen activation reaction^^^ Only

Pdx is capable of reducing the oxy form of

P450cam, in agreement with this hypothesis^^^.

The rate-limiting steps in the entire catalytic

cycle of P450cam are the electron transfers

between Pdx and the P450. The precise rate-

limiting step appears to change from the first

electron transfer to the second electron transfer as

the concentration of Pdx in the system is reduced

toward levels considered typical of the situation

in v/vo^^^.

With respect to the mammalian adrenodoxin/

adrenodoxin reductase system, crystal structures

of truncated and full-length oxidized bovine

Adx^^^'

^^"^

have been determined, along with

NMR structures of both oxidized and reduced

full-length forms of Adx^^^. Adrenodoxin, con-

tains a large hydrophobic core region that houses

a single Fe2S2 cluster, and an interaction domain

that contains acidic residues responsible for the

recognition of the redox partners, CYPllAl

and AdR206' 207 (pigure 4.11). The single Fe2S2

cluster is ligated by four cysteinyl thiolate ligands,

Cys46,

Cys52, Cys55, Cys92, replacement of any

Interaction

Domain

Core

Domain

Figure 4.11. Ribbon representation of bovine Adx (pdb codelAYF). The Fe2S2 cluster is shown in black ligated

to cysteines 46, 52, 55, and 92. The Fe and S atoms are represented as small and large spheres respectively

Electron Transfer Partners of Cytochrome P450

137

which,

produces a nonfunctional apoprotein^^^.

Sites involved in redox partner binding are also

present in the core domain centered around an

acidic patch at position Asp59^^''.

Two different models have been described to

explain the electron delivery system mediated by

Adx: (a) an organized cluster model comprising

AdR: Adx: P450 ternary/quaternary complexes^^^

and (b) a shuttle system where Adx sequentially

transports one electron at a time from AdR to

P45Q210 Qf these^ t^g shuttle hypothesis is most

favorable since the formation of a ternary com-

plex formed by AdR, Adx, and P450 is unlikely

from a structural perspective due to overlapping

mteraction regions^

The sequential model

is also that favored for the well-characterized

P450cam system. Following the resolution of

the bovine AdR crystal structure it has become

possible to fully investigate the structural mecha-

nisms driving the protein complex formation^ ^^.

The distinguishing feature of AdR is a large

groove between a9 and pi2 at the surface (Figure

4.12), which can accommodate the C-terminal

chain end of adrenodoxin^^^. Both Adx and AdR

contain highly asymmetric surface charge distri-

butions, which indicates that interactions between

these molecules and P450 are mediated by elec-

trostatic interactions ^^'^. The structural data are

largely confirmatory of the earlier data from

mutagenesis and modification studies, showing

the importance of charge pairing in the binary

complex between AdR and Ad^^^. Most recently,

the structure of the AdR/Ad complex was solved

at 2.3 A resolution^^^. Interactions sites were con-

firmed as involving predominantly acidic residues

of Adx (D76, 79, 72, and 39) with basic side

chains of Adr

(R211,

240, 244, and K27).

In the P450cam system, our understanding of

the nature of the interactions between the Pdr and

Pdx components is not fully developed. However,

Cys73 of Pdx has been reported as important in

the docking interaction between Pdx and Pdr^^^.

AdR

Adx

,..

^^::,

Figure 4.12. Surface representations of Adx and AdR (pdb codes lAYF and ICJC respectively). The molecules

are oriented according to their proposed docking positions^^"^j which involves charge-pair interactions between acidic

residues at the C-terminal end of Adx and basic residues around the docking groove of AdR.

138

Mark J.I. Paine ef a/.

Mutations to the adjacent Pdx surface residue

Glu72 also affect electron transfer reactions in

the P450cam redox system^^^. The generation of

catalytically efficient putidaredoxin reductase-

putidaredoxin-P450cam triple fusion protein

demonstrates that a much more simple single

component camphor hydroxylase system can be

produced, with obvious biotechnological implica-

tions.

The addition of short peptide linkers

between the respective polypeptides was compati-

ble with strong expression of fusion proteins in

an E. coli system and efficient electron transfer

reactions between the redox centers^^'^. Although

much structure/function work remains to be done

on the Pdr component of the P450cam system, a

novel finding regarding its activity was made

recently with the demonstration that Pdr has

NAD(H)-dependent dithiol/disulfide oxidoreduc-

tase activity^^2. However, much structural and

kinetic work remains to be done to provide a clear

description of the nature of interactions between

the Pdr and Pdx components of the P450cam sys-

tem, and how these relate to those in the analogous

Adr/Adx mitochondrial system.

5. Novel Redox Systems

The identification of the P450 BM3 system led

to the realization that alternative types of P450

redox systems (beyond those typified by mam-

malian hepatic class and P450cam) exist in nature.

In recent years, a variety of alternative modes of

P450 reduction have been reported—including

P450s that require no electron transfer at all (i.e.,

catalyze molecular rearrangements without oxy-

gen chemistry) such as the dehydratase allene

oxide synthase^^^ and a P450 that appears to inter-

act directly with hydrogen peroxide to facilitate

fatty acid oxidation in

subtilis^^^.

Various types

of ferredoxins have been shown to be competent

redox partners for selected P450 enzymes^'^

and fiavodoxins may also be physiologically

relevant mediators of electron transfer to P450s

in bacterial (and possibly plant) systems^^^' ^^^.

More recent studies suggest more exotic types

of P450 fusion enzymes may exist—including

fusions of P450s to ferredoxin and phthalate

dioxygenase reductase-like domains^^^'

•^^K

is highly likely that further insights into bio-

diversity of P450 redox systems will be gained as

ongoing genome sequencing projects reveal ever

more diverse means by which this intriguing

class of enzymes can operate and obtain reducing

power.

Acknowledgments

The authors gratefully acknowledge the sup-

port of the UK Medical Research Council, Cancer

Research UK, the Biotechnology and Biological

Sciences Research Council, the Wellcome Trust,

the Lister Institute of Preventative Medicine, and

the European Union, EPSRC (Engineering and

Physical Sciences Research Council) and the

Royal Society.

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