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

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114 Thomas L. Poulos and Eric F. Johnson

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Electron Transfer Partners of

Cytochrome P450

Mark J.l. Paine, Nigel S. Scrutton, Andrew W. Munro, Aldo Gutierrez,

Gordon C.K. Roberts, and C. Roland Wolf

Introduction

Cytochromes P450 contain a heme center

where the activation of molecular oxygen occurs,

resulting in the insertion of a single atom of

oxygen into an organic substrate with the con-

comitant reduction of the other atom to water. The

monooxygenation reaction requires a coupled and

stepwise supply of electrons, which are derived

from NAD(P)H and supplied via a redox partner.

P450s are generally divided into two major classes

(Class I and Class II) according to the different

types of electron transfer systems they use. P450s

in the Class I family include bacterial and mito-

chondrial P450s, which use a two-component

shuttle system consisting of an iron-sulfur protein

(ferredoxin) and ferredoxin reductase (Figure 4.1).

The Class II enzymes are the microsomal P450s,

which receive electrons from a single mem-

brane-bound enzyme, NADPH cytochrome P450

reductase (CPR), which contains FAD and FMN

cofactors (Figure 4.1). Cytochrome b^ may also

couple with some members of the Class II P450s

family, notably CYP3A4, to enhance the rate of

catalysis ^

Although P450 redox partners are usually

expressed independently, "self-sufficient" P450

monooxygenase systems have also evolved through

the fusion of P450 and CPR genes. These fusion

molecules are found in bacteria and fungi, the best-

known example being P450 BM3, a fatty acid

(0-2 hydroxylase from Bacillus megaterium, which

comprises a soluble P450 with a fiised carboxyl-

terminal CPR module (recently reviewed by

Munro^). BM3 has the highest catalytic activity

known for a P450 monooxygenase^ and was for

many years the only naturally occurring ftised sys-

tem known until the identification of a eukaryotic

membrane-bound equivalent fatty acid hydroxy-

lase,

CYP505A1, from the phytopathogenic fungus

Fusarium

oxysporurrP.

A number of novel P450 sys-

tems are starting to emerge from the large numbers

of genome sequencing projects now underway^.

In this chapter, we review the most recent

advances being made in understanding the func-

tion of P450 redox partners and the electron trans-

fer process. Special attention is paid to CPR,

which occupies a particularly important position

because of its central involvement in human drug

metabolism.

Mark J.l. Paine and C. Roland Wolf

Hospital and Medical School, Dundee, UK.

Nigel S. Scrutton Andrew W. Munro, Gordon C.K. Roberts and Aldo Gutierrez

Biochemistry, University of

Leicester,

Leicester, UK.

Biomedical Research Centre, University of

Dundee,

Ninewells

Department of

Cytochrome

P450:

Structure,

Mechanism,

and

Biochemistry,

3e, edited by Paul R. Ortiz de Montellano

Kluwer

Academic

/ Plenum Publishers,

New

York,

2005.

115

116

Mark J.I. Paine

al.

Class I: Iron-sulfur partners

Adx/AdR Pdx/PdR

NAD(P)H NAD(P)*

NADPH NADP

i FADp^\mQ)

Mitochondrial membrane

Class II: Diflavin reductase partners

CPR CPR fusion

NADPH NADP

Novel Systems

Fe2S2 fusion

NAD*

NADH

Figure

4.1.

Electron transfer partners

cytochrome P450.

Class

systems, electrons

are

shuttled from

NAD(P)H through

FAD-containing ferredoxin reductase

and an

iron-sulfur containing ferredoxin

P450;

prokaryotes these

are

typified

putidoredoxin reductase (PdR)

and

putidoredoxin (Pdx),

and in

eukaryotes

mitochondrial membrane associated adrenodoxin reductase (AdR) and adrenodoxin (Adx). Class II systems are driven

by electrons delivered from NADPH through diflavin (FMN- and FAD-containing) reductases. In eukaryotes these are

bound to the endoplasmic reticulum, while fused systems such as P450 BM3 exist in bacteria and fungi. Novel systems

now include P450RhF, which contains

FMN-containing reductase fused with

ferredoxin-like center and

P450.

NADPH-Cytochrome P450

Reductase and the Diflavin

Reductase Family

2.1.

Background

In view of the key role that cytochrome c

has recently been found to play in regulating

apoptosis and cellular homeostasis^' ^, it is inter-

esting that CPR was first isolated from yeast as an

FMN containing NADPH-dependent cytochrome

c reductase^. A mammalian equivalent was later

isolated from pig liver and reported to contain an

FAD cofactor^. By 1962, the enzyme was shown

to be localized at the endoplasmic reticulum^, and

the flavin cofactors to be involved in the reduction

Electron Transfer Partners of Cytochrome P450

117

of cytochrome c^^. The true physiological redox

partner was eventually discovered as P450 through

the reconstitution of laurate co-hydroxylase activ-

ity from a detergent solubilized preparation

of cytochrome P450, NADPH cytochrome c

reductase^

^' ^^,

and a heat stable component, which

was later identified as the phospholipid phos-

phatidylcholine^^.

The initial difficulties in identifying a physio-

logical role for CPR were due to the purification

methods used, which incorporated trypsin or lipase

treatment^' ^. These resulted in the cleavage of

the amino-terminal membrane anchoring domain,

which constitutes the first 60 or so amino acid

residues and is responsible for interactions with the

phospholipid bilayer and P450^'^. Thus, while

proteolytically cleaved CPR is fully functional and

capable of reducing c3^ochrome c and a range of

artificial electron accepting compounds, it is unable

to reconstitute with P450s. The intact form of the

reductase was eventually purified from liver micro-

somes using detergent solubilization procedures

and found to have a molecular weight of 76-80 kDa

and to support P450-dependent reactions^^' ^^.

In the early 1970s, the enzyme was shown to

contain one molecule each of FMN and

FAD^^'

^^.

A useful feature of flavins is that their absorption

spectra are altered by changes in their reduction

state.

Thus, the reduction state can be examined

by measuring changes in the visible absorbance

range (Figure 4.2). This unique property stimu-

lated research into the redox properties'^' '^, of the

enzyme and the complex processes of hydride/

electron transfer from NADPH, across the flavins

and on to P450, discussed later in this chapter.

The cDNA and corresponding primary amino

acid sequences of several CPRs including rat'^,

rabbit^^, and human^' were obtained by the

mid-1980s, and the development of Escherichia

coli expression systems paved the way for detailed

molecular characterization of the polypeptide

through site-directed mutagenesis. The three-

dimensional structure of rat CPR was determined

by X-ray crystallography in 1997 by Kim and

coworkers^^, providing the structural prototype for

dual flavin oxidoreductases.

2.2.

The Diflavin Reductase

Family

CPR is the prototype for a small family of

diflavin reductases which are believed to have

300 400

500

600 700

Wavelength (nm)

Figure 4.2. Absorbance spectra of oxidized and reduced human CPR. The absorption maxima for the oxidized

enzyme are located at 380 nm and 454 nm. The direction of the arrow shows the absorption changes that occur upon

reducton of the flavins, in this case using a 2-fold and 20-fold excess of NADPH.

118

Mark J.I. Paine et al.

ferredoxin reductase |

flavodoxin I

FMN

CPR

IMS

BM3

iPSgf^:

NOS[

':^1^^''-

Figure 4.3. Schematic outline of the diflavin reductase family. Members contain an N-terminal FMN-binding

flavodoxin-like domain and

C-terminal FAD/NADPH-binding ferredoxin reductase-like domain, which contains an

additional linker

region.

Shown

are:

CPR, which has an amino-terminal membrane anchor region

(Anc);

NRl (Novel

reductase

1);

MSR (methionine synthase reductase), which contains an additional interdomain

sequence;

P450

BM3,

which is fused to a P450 domain; and NOS (nitric oxide synthases), which has linked to a heme-containing

oxygenase domain that is structurally distinct from the P450s.

evolved as a result of

gene fusion event between

ancestral FMN- and FAD-containing flavopro-

teins^^.

Indeed, CPR is one of only four mam-

malian enzymes that contain both FMN- and

FAD-binding domains, the other three being

methionine synthase reductase^"*, NRl^^, and the

nitric oxide synthases^^ (Figure 4.3). Bacterial

members of the family include the

megaterium

cytochrome P450

BM3^^

and the reductase subunit

of sulfite reductase^^. While these are all struc-

turally related, there exist some key differences.

For instance, methionine synthase reductase and

NRl both contain the same domain organization as

P450 reductase but lack the membrane anchoring

sequence, and are thus located in the cytosol.

Methionine synthase also has a much larger inter-

domain linker than CPR, although the functional

significance of this is unclear. In nitric oxide syn-

thase, the reductase region is fused with a heme

domain, and has acquired an extra calmodulin-

binding domain to regulate electron transfer.

Experimental evidence for the gene fusion

concept comes from work of Smith et

al?"^,

who

dissected the FMN and FAD/NADH-binding

domains of human CPR and expressed them as

discrete functional units in E. colfl^. The individ-

ual domains not only folded correctly but also

could be reconstituted to form an active complex

capable of reducing cytochrome c and donating

electrons to the P450 monooxygenase complex,

albeit with greatly reduced efficiency^^. A num-

ber of other diflavin reductases have been dis-

sected into functional units including: rat CPR^^,

BM3^''

^^,

NRl^^, and methionine synthase reduc-

tase^"^.

The ability to separate CPR and other

diflavin reductases into their component parts has

greatly facilitated structural and functional studies

of these enzymes.

2.3.

CPR Genes

Apart from plants, which contain multiple

CPR genes^^'^^, most organisms contain a single

CPR gene. At the time of writing, a relatively

modest total of 20 CPR genes have been identi-

fied, which will doubtlessly increase as a result of

the many genome sequencing projects currently

underway. New sequences can be monitored using

the website www.icgeb.trieste.it, which provides

sequence updates on P450s and related enzymes.

The rat gene was cloned in 1990^^ and comprises

16 exons, with the first exon being non-coding.

Amongst the coding exons there is a general

correspondence with the structural domains, with

exons 2 and 3 encoding the membrane anchor

region, exons 4-7 the FMN-binding domain, and

exons 8-16 the FAD/NADPH-binding region.

Electron Transfer Partners of Cytochrome P450

119

A similar intronic arrangement is found for the

mouse gene located on chromosome 6^^' ^^. The

human gene is located on chromosome 7^^

Although the regulation of expression of

individual P450s in response to exogenous and

endogenous factors has been studied extensively,

the factors controlling the expression of the

CPR gene are less well understood. Reductase

expression may be induced by a number of

cytochrome P450 inducers such as phenobarbi-

tal'^^

but the regulation of reductase gene expres-

sion is independent of that of cytochrome P450'^^.

The upstream regulatory region of the rat

CPR gene has a number of interesting features"^^.

Unlike many drug-metabolizing enzymes, the

promoter does not contain either a TATA or

CCAAT box, it is GC rich, contains multiple

Spl consensus sequences, and utilizes two major

transcription start sites.

The CPR gene is regulated by thyroid hor-

mone, which stimulates CPR expression via the

thyroid response element (TRE) in the upstream

promoter'*^'

^^.

Most recently, peroxisome prolifer-

ators,

which regulate a battery of rodent P450

genes,

have also been shown to regulate CPR

levels"^^,

possibly through a putative DNA bind-

ing site for PPARa that exists in the mouse

CPR promoter between -568 and -556 bp^^

Interestingly, following exposure to peroxisome

proliferators CPR gene transcript levels appear

to increase, while levels of CPR protein are

decreased"^^. Thus, opposing transcriptional and

translational or post-translational mechanisms

appear to play a role in the regulation of CPR by

peroxisome proliferators"^^.

2.4. Probing the Physiological

Role of CPR

The reductase gene has been knocked out in

mice^^'

^^;

this leads to embryonic lethality, thus

demonstrating that CPR is essential for normal

development in higher organisms, presumably

because it plays a key role in the biosynthesis of

signaling factors such as retinoic acid, sterols,

prostaglandins, and steroids that are dependent

on P450 catalysis. The precise role of CPR in

embryogenesis is still unclear and the effects of

gene deletion on the developing embryos are com-

plex. Embryos lacking CPR do not survive beyond

8-9 days and display a number of defects includ-

ing neural tube, cardiac, eye, and limb abnormali-

ties,

and generalized defects in cell adhesion"^^.

Kasper's group has examined the effects of

CPR gene disruption by removing the natural

translation initiation site and deleting the

membrane-binding domain of CPR"^^. However,

some homozygous null embryos (CPR~^~) were

found to produce a truncated 66 kDa c5^oplasmic

form of CPR, presumably through initiation at an

alternative start site. That these still produced the

spectrum of embryonic defects leading to mid-

gestational lethality provides strong evidence that

the microsomal location of CPR is essential for the

physiologically important functions of

the

P450s.

The problem of the lethality of a CPR knock-

out has been resolved by applying the regulatable

Cre/loxP system^^ to generate mice in which

CPR can be deleted post-natally in the liver^^. Such

"conditional knockout" mice, in which all the

hepatic P450s are essentially inactivated, provide

the means of evaluating P450 function in normal

homeostasis, drug pharmacology, and chemical

toxicity in vivo. Hepatic CPR-null mice exhibit

many intriguing phenotypes^^. Due to an inability

to produce bile acids, they no longer break down

cholesterol, and whereas hepatic lipid levels were

significantly increased, circulating levels of cho-

lesterol and triglycerides are severely reduced.

There are also profound changes in the in vivo

metabolism of pentobarbital and acetaminophen,

demonstrating the predominant role of the hepatic

P450s in the pharmacology and toxicology of

these compounds, and illustrating the power of

transgenic models in understanding P450 function.

One of the remarkable aspects of the hepatic

CPR deletion in mice was the fact that they live

and reproduce normally. This means that in adult

mice at least, the hepatic P450 system is not essen-

tial for life, and indicates most strongly a funda-

mental role in providing protection against toxic

environmental agents. The data from these trans-

genic models^^'

also demonstrate that potential

alternative electron transfer pathways for P450s,

such as the cytochrome b^/b^ reductase systems

that are discussed later, play only a minor role, if

any, in vivo.

The earliest CPR gene deletions were of

course carried out with yeast, and the data offer

some interesting contrasts with mammalian

deletions"^^'

'^^.

The deletion of the CPR gene in

120 Mark J.I. Paine et al.

Saccharomyces cerevisae is not lethal but instead

produces a viable mutant with an increased sensi-

tivity (>200-fold) to ketoconazole, an azole

inhibitor of the ftingal P450

CYP51,

a lanosterol

14a-demethylase involved in the essential ergo-

sterol biosynthesis pathway"*^. Although this

hypersensitivity is suggestive of inefficient sterol

biosynthesis, it has been observed that the

cpr~ strains still accumulate significant amounts

of ergosterol, around 25% of those in the wild-

type parent"^^. This indicates that, unlike mice, an

alternative electron donor is effective in delivering

two electrons for P450 activity^^. In contrast to

Shen's observations with cpr-deleted mice"^^, a

cytosolic truncated version of native CPR with the

N-terminal membrane anchor removed effectively

reversed the phenotypes associated with cpr

deletion in yeast^^. Thus, there appear to be

fundamental differences in the mechanisms of P450

interactions between yeast and mammalian CPRs.

While the major role of CPR is associated with

cytochrome P450 and the phase I metabolism of

xenobiotic compounds, the enzyme also has the

ability to reduce other electron acceptors such as

cytochrome c^, cytochrome

(ref [50]), and heme

oxygenase^ ^ In addition, it has recently been impli-

cated, along with cytochrome

Z?^,

in the bioreduc-

tive activation of methionine synthase^^, thus

pointing to a possible role in the regulation of

methionine synthesis. CPR has also been shown to

be involved in the regulation of oxidative response

genes such as hypoxia-inducible factor

(ref [53]).

P450 reductase plays a role in the bioactivation

of therapeutic prodrugs through its ability to

reduce a range of one-electron acceptors such

as the quinone drugs doxorubicin^^ and mito-

mycin

c^^

and aromatic TV-oxides such as the novel

benzotriazine, tirapazamine^^. Evidence is also

emerging that other members of the CPR family

including nitric oxide synthases^^ and NRl^^ may

play a similarly important role in the metabolism

of chemotherapeutic drugs.

Overall, the biological relationships of CPR

with proteins other than P450 are not especially

well established. However, attention is starting

to shift toward understanding the role of CPR in

other redox pathways. In particular, data is accu-

mulating on CPR interactions with cytochrome

(ref [58]) and heme oxygenase^^. Consistent with

the findings for cytochrome P450 enzymes,

molecular investigations on the binding of CPR to

human heme oxygenase-1 (hHO-1) establish that

ionic interactions contribute to the binding of these

proteins^^. Like P450, positively charged hHO-1

surface residues contribute to the binding of this

molecule to CPR, most likely via the negatively

charged residues in the FMN domain of CPR.

2-5.

Structure

of CPR

The crystal structure of rat CPR lacking the

N-terminal membrane-binding sequence^^ shows

that the catalytic region comprises three distinct

domains: an FMN-binding domain structurally

similar to flavodoxins; an NADPH/FAD-binding

domain similar to ferredoxin-NADP^ reductase;

and a "linker" domain, present as an insert in the

sequence of the NADPH/FAD-binding domain

(Figure 4.4). The "linker" sequence is the major

structural feature that distinguishes these mole-

cules from the single-domain ferredoxin reduc-

tases,

and is likely to play a structural role in

positioning the FMN- and FAD-binding domains

correctly for direct electron transfer. This is sup-

ported by mutagenesis studies in which compari-

son of wild-type and mutant structures shows

significant differences in the relative position of

the two flavin domains of rat CPR^^. Furthermore,

there is increasing evidence from structural stud-

ies with other dual flavin reductases that the inter-

play between NADPH, flavin cofactors, and heme

is directed by conformational changes. This is

exemplified by nNOS, where structural relaxation

appears to be an important part of the calmodulin-

dependent activation mechanism^ *.

2.5.1.

The FMN-Binding Domain

The FMN-binding domain interacts with

cytochrome P450 to transfer electrons during

catalysis. In addition to rat CPR, detailed struc-

tural studies of the isolated human FMN domain

have been carried out using NMR^^, and X-ray

crystallography^^. The isoalloxazine ring of FMN

is sandwiched between two aromatic side chains,

Tyrl40 and TyrlTS^^'

62,63

jyj.173 ij^s parallel to

the 5/-side of the FMN ring, while the second

aromatic, Tyrl40, located on the re-side, lies at

an angle of ~40° to the isoalloxazine ring

(Figure 4.5). A similar arrangement is found in

the FMN-binding domain of P450 BM3 and in

Electron Transfer Partners of Cytochrome P450

121

most flavodoxins, apart from Clostridium flavo-

doxin where the r^-side aromatic side chain

residue is replaced by methionine^"^. Removal of

the aromatic side chains of Tyrl40 and/or Tyr 178

from rat liver CPR by site-directed mutagenesis

results in decreased affinity for FMN^^.

In addition to these two key residues, a third

aromatic side chain, PhelSl, lies close to the FMN

isoalloxazine ring on the ^/-side at the pyrimidine

end, although not in contact with it (Figure 4.5).

This residue is highly conserved in P450 reductase

and related enzymes, indicating a possible role in

FMN binding. Mutations that remove the aromatic

side chain result in a 50-fold reduction in affinity

for FMN, and NMR spectra show significant

changes in amide chemical shifts of residues in

_ 'linker'

Figure 4.4. Schematic representation of rat CPR domains (protein data base [pdb] code lAMO). In the crystal,

the two flavins, FMN and FAD, are 4 A apart.

FMN

Y178

Y140

F181

Figure 4.5. Close up of the FMN isoalloxazine ring showing the aromatic residues, Tyrl40 (Y140), Tyrl78

(Y178), and PhelSl (F181) associated with FMN binding in human CPR (pdb code IBIC).

122

Mark J.I. Paine et aL

the p4-a6 loop. This loop includes PhelSl, the

key FMN-binding residue Tyrl78, and three

other residues that form hydrogen bonds to the

isoalloxazine ring, as well as residues in the adja-

cent p5-a7 loop^^. These are consistent with local

changes in structure on substitution of PhelSl,

and with the idea that the packing of this con-

served residue plays a significant role maintaining

the orientation of residues which interact with the

isoalloxazine ring and hence in the formation of

the FMN-binding site.

2.5.2.

FAD/NADPH-Binding Domain

Like FNR, FAD is bound in an extended con-

formation in rat CPR^^. Key residues that serve to

regulate flavin binding and reactivity are Arg454,

Tyr456, Cys472, Gly488,

Thr491,

and Trp67722.

Site-directed mutagenesis of these residues has

been carried out to examine their role in FAD

binding and catalysis^^. Trp677 is stacked against

the re-face of

FAD,

while Tyr456 lies at its 5/-face.

Deleting Trp677 reduces catalytic activity 50-fold

but has little effect on FAD content. However,

mutations to Tyr456, which also hydrogen bonds

to the ribityl 4'-hydroxyl, decreases affinity for

FAD by around 8,000-fold. Side chains from

Thr491 and Arg454 stabilize the pyrophosphate

group of FAD. Substitution of Thr491 has

a relatively modest effect on FAD binding (~

100-

fold decrease) compared with mutation of Arg454,

which decreases affinity 25,000-fold.

NMR studies show that the pro-R-hydrogen

attached to the C-4 atom of NADPH is transferred

directly to FAD as a hydride ion^^. However, an

understanding of NADPH binding and the factors

involved in hydride transfer have been hampered by

the spatial disorder in this region of the molecule

in the crystal structure. While the electron density

of the adenosine moiety of the coenzyme is well

defined in the structure, that from the nicotinamide

ring is not, and is consistent with at least two

dif-

ferent positions for this ring, neither of which is in

contact with the isoalloxazine ring of FAD^^.

Similar observations have been made in enzymes

the

FNR family which are structurally similar to

the FAD/NADPH-binding domain of CPR^^-^i. An

aromatic amino acid residue is located at the car-

boxyl-terminus in all members of

the

FNR family

In rat and human CPR, this position is occupied by

Trp677 and Trp676, respectively. The planar side

chain of

the

tryptophan stacks against the isoallox-

azine ring of FAD, and structural rearrangements

are thought to be essential to enable access of the

nicotinamide ring to the isoalloxazine ring of

FAD

for hydride transfer (Figure 4.6). The proposed

mechanism for binding involves the tryptophan

residue flipping away from FAD to be replaced by

the nicotinamide region of

NADPtf^.

There has been extensive site-directed mutage-

nesis of amino acid residues in the vicinity of the

NADPH-binding region to examine their role in

cofactor binding and hydride transfer (Figure 4.7).

Removal of the aromatic side chain from Trp676

shows that it plays an essential role in the

discrimination between NADPH and NADH as

cofactors for CPR^^' ^^, notwithstanding the fact

that it does not interact with the 2'-phosphate

which is the sole difference between the two

cofactors. Investigation of the steady-state kinet-

ics shows that substitution of Trp676 with Ala

decreases the K^ for NADH from 48 to 0.3 mM,

and increases the

k^^^

to ~4,000 min~^ while the

^^NADPH ^Qps tQ 0.2

|ULM

and

k^^^

to lOmin^^

The Trp676Ala mutant therefore binds both

coenzyme molecules more tightly, the change in

cofactor specificity (expressed as a k^JK^ ratio)

being predominantly a reflection of changes in

k^^^.

A similar change in cofactor specificity also

occurs with pea FNR with the analogous mutation

Tyr308Ser^4' ^^ Both in pea FNR^^ and in CPR^^

substitution of the C-terminal aromatic residue

allows the nicotinamide ring of the cofactor to

bind close to the isoalloxazine ring, the thermody-

namically unfavorable flipping no longer being

required. Hence, removal of this physical rate-lim-

iting step produces enzymes that have lower

apparent K^ values for nicotinamide cofactors.

Further kinetic evidence for a critical role for this

Trp residue in regulating NADPH binding and

hydride transfer is detailed in Section

2.6.1.

Several conserved amino acids including ser-

ine 596, arginine 597, and lysine 602 have been

proposed to be involved in binding the adenosine-

ribose moiety of NADPH (Figure 4.7), and

specific charge-charge interactions between these

and the 2'-phosphate of NADPH are thought

to be responsible for discrimination against

NADtf^'

^^-^l Working on the hypothesis that

mutations to these residues might reduce the

affinity of the enzyme for NADP(H) and thus pre-

vent inhibition by NADP^, Elmore and Porter^^

Electron Transfer Partners of Cytochrome P450

123

298

Figure 4.6. Arrangement of

FMN,

FAD, and NADP+ in the rat CPR crystal structure (pdb code lAMO). The

Trp677 aromatic ring has

to move

away from the

FAD

isoalloxazine ring

allow access of nicotinamide ring

FAD

for hydride transfer.

-oJ.,,-^J Nicotinamide

Adenosine-ribose

RS^

S596

R597

R634

Figure 4.7. Schematic diagram of residues associated with NADPH binding in human CPR (taken from Dohr^^).

have coupled mutations to residues in the 2'-phos-

phate-binding site with the W677 substitution.

The mutant combinations R597MAV677A and

R597M/K602WAV677 A showed a significant 4-

fold reduction in inhibition by NADP^ relative to

W677A, while their catalytic efficiency with

NADH was increased at least 500-fold.

Three residues Ser457, Asp675, and Cys630,

lie in close proximity to the FAD isoalloxazine

ring in CPR, and have been closely examined