Báo cáo khoa học: Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding docx

Interflavin electron transfer in human cytochrome P450 reductase
is enhanced by coenzyme binding
Relaxation kinetic studies with coenzyme analogues
Aldo Gutierrez
1,2
, Andrew W. Munro
1
, Alex Grunau
1,2
, C. Roland Wolf
3
, Nigel S. Scrutton
1
and Gordon C. K. Roberts
1,2
1
Department of Biochemistry and
2
Biological NMR Centre, University of Leicester, UK;
3
Biomedical Research Centre,
University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
The role of coenzyme binding in regulating interflavin
electron transfer in human cytochrome P450 reductase
(CPR) has been studied using temperature-jump spectros-
copy. Previous studies [Gutierrez, A., Paine, M., Wolf,
C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry
(2002) 41, 4626–4637] have shown that the observed rate,
1/s, of interflavin electron transfer (FAD
sq
) FMN

sq
fi
FAD
ox
) FMN
hq
) in CPR reduced at the two-electron level
with NADPH is 55 ± 2 s
)1
, whereas with dithionite-
reduced enzyme the observed rate is 11 ± 0.5 s
)1
,sug-
gesting that NADPH (or NADP
+
) binding has an
important role in controlling the rate of internal electron
transfer. In relaxation experiments performed with CPR
reduced at the two-electron level with NADH, the observed
rate of internal electron transfer (1/s ¼ 18 ± 0.7 s
)1
)is
intermediate in value between those seen with dithionite-
reduced and NADPH-reduced enzyme, indicating that the
presence of the 2¢-phosphate is important for enhancing
internal electron transfer. To investigate this further, tem-
perature jump experiments were performed with dithionite-
reduced enzyme in the presence of 2¢,5¢-ADP and 2¢-AMP.
These two ligands increase the observed rate of interflavin
electron transfer in two-electron reduced CPR from

1/s ¼ 11 s
)1
to 35 ± 0.2 s
)1
and32±0.6s
)1
, respectively.
Reduction of CPR at the two-electron level by NADPH,
NADH or dithionite generates the same spectral species,
consistent with an electron distribution that is equivalent
regardless of reductant at the initiation of the temperature
jump. Spectroelectrochemical experiments establish that the
redox potentials of the flavins of CPR are unchanged on
binding 2¢,5¢-ADP, supporting the view that enhanced rates
of interdomain electron transfer have their origin in a con-
formational change produced by binding NADPH or its
fragments. Addition of 2¢,5¢-ADP either to the isolated
FAD-domain or to full-length CPR (in their oxidized and
reduced forms) leads to perturbation of the optical spectra
of both the flavins, consistent with a conformational change
that alters the environment of these redox cofactors. The
binding of 2¢,5¢-ADP eliminates the unusual dependence of
the observed flavin reduction rate on NADPH concentra-
tion (i.e. enhanced at low coenzyme concentration) ob-
served in stopped-flow studies. The data are discussed in the
context of previous kinetic studies and of the crystallo-
graphic structure of rat CPR.
Keywords: coenzyme binding; cytochrome P450 reductase;
electron transfer; flavoprotein; temperature-jump relaxation
spectroscopy.

Members of the cytochrome P450 mono-oxygenase super-
family catalyse the hydroxylation of a wide range of
physiological and xenobiotic compounds; in eukaryotes the
type II cytochromes P450, located in the endoplasmic
reticulum, play a key role in drug metabolism. NADPH-
cytochrome P450 reductase (CPR; EC 1.6.2.4) catalyses
electron transfer to these type II cytochromes P450 [1–5].
CPR is a 78-kDa enzyme containing one molecule each of
FAD and FMN [6]. Sequence analyses [5] and X-ray
crystallographic studies of rat CPR [7] have revealed that
CPR consists of an N-terminal membrane anchor, respon-
sible for its localization to the endoplasmic reticulum, and
three folded domains: an FAD- and NADPH-binding
domain related to ferredoxin-NADP
+
reductase, a flavo-
doxin-like FMN-binding domain and a connecting ÔlinkerÕ
domain. In addition to the cytochromes P450, CPR can
donate electrons to cytochrome b
5
[8], haem oxygenase [9],
the fatty acid hydroxylation system [10] and a number of
artificial redox acceptors [11,12] and drugs [13–17].
CPR is related to a number of mammalian diflavin
reductases including the isoforms of nitric oxide synthase
[18], methionine synthase reductase [19] and protein NR1
Correspondence to N. S. Scrutton, Department of Biochemistry,
University of Leicester, University Road, Leicester LE1 7RH, UK.
Fax: + 44 116 252 3369, Tel.: + 44 116 223 1337,
E-mail:,BiologicalNMR

Centre, University of Leicester, University Road, Leicester LE1 7RH,
UK. Fax: + 44 116 223 1503, Tel.: + 44 116 252 2978,
E-mail:
Abbreviations: CPR, NADPH-cytochrome P450 reductase; 2¢,5¢-ADP,
adenosine 2¢,5¢-bisphosphate; 2¢-AMP, adenosine 2¢-monophosphate.
Enzymes: NADPH-cytochrome P450 reductase (CPR; EC 1.6.2.4).
Note: In this paper the term ÔintactÕ CPR refers to soluble CPR,
containing the FMN-binding, FAD-binding and ÔlinkerÕ domains and
lacking only the N-terminal membrane-anchoring peptide sequence.
(Received 14 March 2003, revised 17 April 2003,
accepted 24 April 2003)
Eur. J. Biochem. 270, 2612–2621 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03633.x
[20]. It also shares some mechanistic and structural similar-
ity with prokaryotic diflavin reductases such as cytochrome
P450 BM3 [21] and sulfite reductase [22]. The mechanism of
electron transfer in mammalian CPRs has been studied in
detail by stopped-flow kinetic and potentiometric methods
[23–26]. The reduction potentials of the flavin couples have
been determined, and key intermediates in the reaction
sequence have been identified. Moreover, the roles of
specific residues in flavin reduction by NADPH have been
elucidated by mutagenesis methods [25,27,28]. The rate of
interflavin electron transfer in mammalian CPRs has been
determined by flash photolysis [29] and relaxation kinetic
methods [28]. We recently demonstrated [28] that this rate is
relatively slow (55 s
)1
) in human CPR, despite the very close
proximity (3.85 A
˚

) of the flavin cofactors observed in the
crystal structure of the rat enzyme [7], and is limited by
conformational change and regulated by coenzyme binding
[28]. This rate is lower (10 s
)1
) for dithionite-reduced
enzyme, indicating that nicotinamide coenzyme optimizes
interflavin electron transfer. We also showed that mutation
of W676, which is located over the re-face of the FAD in
CPR [7,30], has a major effect on the kinetics of interflavin
electron transfer in both the ÔforwardÕ and ÔreverseÕ direc-
tions. We have now extended our relaxation kinetic
experiments to include studies of internal electron transfer
in the presence of NADPH, NADH and fragments of
nicotinamide coenzymes. We show that binding of the
2¢-phosphate of NADPH, and to a lesser extent other
regions of the reducing coenzyme, specifically enhance the
rate of internal electron transfer. Our studies point to a
complex mode of regulation of electron transfer reactions in
human CPR, triggered by coenzyme binding and involving
conformational change over a relatively large distance, and
highlight further the key role of conformational gating in
biological electron transfer reactions.
Experimental procedures
Materials
NADPH, NADH, 2¢,5¢-ADP, 2¢-AMP, nicotinamide
1,N
6
-ethenoadenine dinucleotide phosphate and sodium
dithionite were from Sigma. 2¢(3¢)-O-(trinitrophenyl)adeno-

sine 5¢-monophosphate was from Molecular Probes. All
other chemicals were of analytical grade.
Protein purification
Human fibroblast CPR (lacking the N-terminal membrane-
anchoring region) and the functional FAD-binding domain
were expressed in Escherichia coli BL21(DE3)pLysS from
appropriate pET15b plasmid constructs, and purified as
described previously [24]. The FAD-binding domain con-
struct includes the so-called linker domain [7].
Temperature-jump and stopped-flow kinetic methods
Temperature-jump experiments, using a TJ-64 temperature-
jump instrument (Hi-Tech Scientific), were performed under
anaerobic conditions according to the method recently
described [28], and the heating time was estimated to be 4 ls
using a standard phenolphthalein-glycine buffer test
(T-jump users’ manual, Hi-Tech Scientific, Salisbury,
UK). The initial temperature was 20 °C. CPR samples
(140 l
M
) were prepared in an anaerobic glove box (Belle
Technology Ltd). Reduction of the enzyme to the two-
electron level by different electron donors (dithionite,
NADH or NADPH) was monitored optically as described
previously [28]. Typically, 20 transients were collected and
averaged for each reaction condition. Optical artefacts
caused by the high voltage discharge through the cell were
accounted for by control measurements with the oxidized
form of the enzyme, as described previously [28]. Relaxation
transients were fitted to a monophasic process using
Hi-Tech software dedicated to the T-jump instrument.

Stopped-flow experiments with fluorescence detection
were performed using an Applied Photophysics SX.18
M
V
stopped-flow instrument. Tryptophan fluorescence studies
were performed using an excitation wavelength of 295 nm.
The emission band was selected using a WG320 cut-off
emission filter (Coherent Optics) in combination with a
UG-11 filter (Coherent Optics) to block stray visible light.
The photomultiplier voltage was kept constant during the
measurements. The oxidized form of the CPR enzyme
(10 l
M
) was used in these experiments, which were
performed in 50 m
M
potassium phosphate buffer (pH 7.0)
at 25 °C.
Inhibition studies
The inhibition constant for 2¢,5¢-ADP was determined by
using steady-state kinetic assays with cytochrome c as
electron acceptor and NADPH as electron donor. Reaction
mixtures contained 7 n
M
CPR, 50 m
M
potassium phos-
phate buffer (pH 7.0), 50 l
M
cytochrome c and variable

concentrations of NADPH and of inhibitor 2¢,5¢-ADP;
reactions were initiated by making microlitre additions of
NADPH to the reaction mix. Assays were performed
at 25 °C and the initial velocity of the reaction was
measured by reduction of cytochrome c
3+
at 550 nm
(De ¼ 21.1 m
M
)1
Æcm
)1
) using a Cary-300 UV/visible spec-
trophotometer. Data were first analysed graphically using
double-reciprocal plots (1/v
i
vs. 1/[NADPH]) to determine
the type of inhibition. Initial velocity values were then fitted
to the equation describing competitive inhibition
v
i
¼
V
max
S
K
M
1 þ
I
K

i

þ S
hi
ð1Þ
by nonlinear regression analysis using the
GRAFIT
software
package [31].
Potentiometric titrations
Spectroelectrochemical titrations were performed within a
Belle technology glove box under a nitrogen atmosphere
(oxygen maintained at < 5 p.p.m.) in 100 m
M
potassium
phosphate buffer (pH 7.0) containing 10% (v/v) glycerol
(titration buffer), at 25 ± 2 °C, essentially as described
previously [26]. Anaerobic titration buffer was prepared by
flushing freshly prepared buffer with oxygen-free argon.
CPR protein samples (typically % 50–80 l
M
)admittedto
the glove box were de-oxygenated by passing through a Bio-
Rad EconoPack 10 DG desalting column pre-equilibrated
Ó FEBS 2003 Electron transfer in human cytochrome P450 reductase (Eur. J. Biochem. 270) 2613
in the anaerobic titration buffer. The final CPR concentra-
tion used for the redox titrations was 60 l
M
. Solutions of
benzyl viologen, methyl viologen, 2-hydroxy-1,4-naphtho-

quinone and phenazine methosulfate were added to a
final concentration of 0.5 l
M
as redox mediators for the
titrations. Absorption spectra (300–800 nm) were recorded
on a Cary UV50 Bio UV-visible spectrophotometer external
to the glove box, with absorption signals relayed to the
instrument from an absorption probe (Varian Inc.)
immersed in the protein sample, via a fibre optic cable.
The electrochemical potential was monitored using a Hanna
instruments pH/voltmeter coupled to a Russell Pt/calomel
electrode immersed in the CPR solution. The electrode
was calibrated using the Fe(II)/Fe(III)-EDTA couple
(+108 mV) as a standard. The enzyme solution was titrated
electrochemically using sodium dithionite as reductant and
potassium ferricyanide as oxidant, as described by Dutton
[32]. Duplicate titration data sets were collected for CPR in
the presence and in the absence of 2¢,5¢-ADP (60 l
M
).
Approximately 100 different spectra were recorded during
each redox titration, covering the range between around
)400 mV and +200 mV (vs. normal hydrogen electrode).
Reductive and oxidative titrations of the enzyme indicated
that there were no hysteretic effects, and that the enzyme did
not aggregate to any significant extent over the course of the
5–8 h required to complete the titrations.
Data analysis was performed using Origin (Microcal),
essentially as described previously [26]. Throughout the
titrations, the enzyme remained soluble and corrections for

turbidity were not required. The absorption vs. spectral data
were fitted by using Eqn (2), which describes a two-electron
redox process derived by extension to the Nernst equation
and the Beer–Lambert Law, or Eqn (3), which represents
the sum of two two-electron redox processes. Fitting
procedures are described in detail in our previous studies
[26,33].
A ¼
a10
ðEÀE
0
1
Þ=59
þ b þ c10
ðE
0
2
ÀEÞ=59
1 þ 10
ðEÀE
0
1
Þ=59
þ 10
ðE
0
2
ÀEÞ=59
ð2Þ
A ¼

a10
ðEÀE
0
1
Þ=59
þ b þ c10
ðE
0
2
ÀEÞ=59
1 þ 10
ðEÀE
0
1
Þ=59
þ 10
ðE
0
2
ÀEÞ=59
þ
d10
ðEÀE
0
3
Þ=59
þ e þ f10
ðE
0
4

ÀEÞ=59
1 þ 10
ðEÀE
0
3
Þ=59
þ 10
ðE
0
4
ÀEÞ=59
ð3Þ
In these equations, A is the total absorbance; a, b and c are
component absorbance values contributed by one flavin in
the oxidized, semiquinone and reduced states, respectively,
and d, e and f are the corresponding absorbance compo-
nents associated with the second flavin. E is the observed
potential in mV; E
1
¢ and E
2
¢ are the midpoint potentials for
oxidized/semiquinone and semiquinone/reduced couples,
respectively, for the first flavin, and E
3
¢ and E
4
¢ are the
corresponding midpoint potentials for the second flavin.
The complexity of the system (4-electron titration with

probable overlap of midpoint potentials) necessitated the
use of a two-stage fitting process. Our previous studies using
intact CPR and the FMN and FAD domain of human
CPR identified isosbestic points for the oxidized/semi-
quinone and semiquinone/reduced transitions for both the
FAD and FMN flavins at % 500 nm and 430 nm, respect-
ively. For intact CPR, data fitting at these wavelengths
yielded, within the uncertainty of the measurements, the
same midpoint potentials for the redox couple contributing
to the absorbance change whether Eqn (2) or Eqn (3) was
used. In using Eqn (2), pairs of variables were set as
constant and equal prior to fitting. Thus, to determine the
oxidized/semiquinone couples, b ¼ c and e ¼ f (the absorp-
tion values for the semiquinone and reduced forms of the
first and second flavins, respectively, at the isosbestic point),
and to determine the semiquinone/reduced couples, a ¼ b
and d ¼ e. Midpoint potential values obtained from the fits
at isosbestic points were then used as starting points to
enable accurate fitting of absorption vs. reduction potential
data for intact CPR at wavelengths near-maximal for the
oxidized flavins (455 nm) and for the neutral blue semi-
quinone species observed to accumulate during the redox
titration (585 nm). The oxidized flavins were assumed to
have equal absorbance coefficients, specifically a ¼ d.This
is known to be the case from previous studies with the
individual domains [26]. Reduction potentials for the system
were determined using A
455
and A
585

data and Eqn (2)
through an iterative process in which midpoint potentials
associated with one isosbestic point were held fixed (e.g.
FMN and FAD oxidized/semiquinone) while the other pair
were varied. The process was then repeated with the
resultant potentials determined for the second pair fixed and
the first pair allowed to vary in the second round of analysis.
This process was repeated iteratively until there was no
further change observed. Data derived in this way are
similar to those originally estimated from the fits at the
isosbestic points [26].
Results
Chemical relaxation reactions with NADH-reduced CPR
The optical spectrum of CPR reduced by stoichiometric
NADH is identical to that obtained with NADPH
(Fig. 1A), indicating that binding of the coenzyme
2¢-phosphate does not affect the redox potentials of the
flavins. Furthermore, the optical spectra obtained with
nicotinamide coenzymes are very similar to the spectrum of
CPR following reduction with stoichiometric sodium dithio-
nite [28], indicating that the potential of the flavin couples is
the sole determinant of the redox equilibrium within the
enzyme under these conditions, and hence of the nature of
the equilibrium perturbation following rapid elevation of
temperature in a temperature-jump experiment. A detailed
discussion of the electron distribution prior and subsequent
to capacitor discharge in the temperature-jump instrument
for NADPH- and dithionite-reduced enzyme can be found
in our previous publication [28]. Briefly, two different two-
electron reduced species of CPR are thought to predominate

prior to temperature elevation. One species contains the
high-potential blue semiquinone form of the FMN
(FMN
ox/sq
¼ )66 mV) and the semiquinone form of
FAD (FAD
ox/sq
¼ )283 mV). The second species contains
oxidized FAD and the hydroquinone form of FMN
(FMN
sq/hq
¼ )269 mV). These two species are present in
approximately equal concentration (Scheme 1), consistent
with our measured values for the relevant couples of the
FAD and FMN [26]. We have shown that perturbation of
this equilibrium by a temperature jump leads to further and
2614 A. Gutierrez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
transient oxidation of the FAD semiquinone form as it
transfers an electron to the FMN
sq
to produce more of the
FAD/FMNH
2
species. This is observed as a net loss of
absorbance at 600 nm (i.e. loss of blue semiquinone
signature). Transient oxidation of the FAD semiquinone
shifts the equilibrium towards the FAD/FMNH
2
species
(Scheme 1). The relaxation process therefore involves

electron transfer between the two flavin cofactors (i.e.
FAD
sq
–FMN
sq
(q)FAD
ox
–FMN
hq
). With NADPH-
reduced CPR, electron transfer occurs with an observed
rate, 1/s,of55±2s
)1
[28].
Although the initial equilibrium distribution is identical,
the observed rate of internal electron transfer in CPR
reduced with NADH (1/s ¼ 18 ± 0.7 s
)1)
is a factor of
three less than the corresponding value for NADPH-
reduced CPR (1/s ¼ 55 ± 0.5 s
)1
;Fig.1B),andmore
similar to that seen for dithionite-reduced CPR
(11 ± 0.5 s
)1
). The midpoint redox potential values for
the NADPH/NADP
+
and NADH/NAD

+
couples are the
same ()320mV,pH7.0,30°C), so that the altered rate of
interflavinelectrontransferisnotrelatedtoachangein
driving force. The presence of a phosphate group esterified
attheribose2¢ position in the redox-inactive adenosine
moiety of NADPH is the only chemical difference between
the two coenzymes. The observation of a threefold reduc-
tion in rate of interflavin electron transfer in NADH-
reduced CPR thus suggests a role for coenzyme binding,
and in particular for interactions made by the 2¢-phosphate
of NADPH, in modulating interdomain electron transfer in
CPR. Temperature-jump experiments performed with frag-
ments of NADPH were performed to test this hypothesis
and are described in the following sections.
Binding of adenosine 2¢,5¢-bisphosphate to CPR
and effects on interflavin electron transfer
In the crystal structure of rat CPR [7], NADP
+
appears to
be bound to the enzyme predominantly through inter-
actions involving its adenine end, whose binding site is
contained within the FAD-binding domain of the protein
(Fig. 2). The electron density for the NMN portion of the
coenzyme is poorly defined, and the position of the
nicotinamide ring is conjectured to be different in each of
the two independent molecules present in the crystal [7]. To
investigate any effect of binding 2¢,5¢-ADP on the flavin
environment, difference absorption spectra on binding 2¢,5¢-
ADP were measured with both the isolated FAD-binding

domain and the intact soluble CPR enzyme (see Note).
Binding of a stoichiometric amount of 2¢,5¢-ADP to the
isolated FAD-binding domain, in either the oxidized form
or the form reduced to the one-electron level with dithionite,
leads to clear changes in the absorption spectrum of the
FAD, consistent with a perturbation of the cofactor
environment (Fig. 3). Similar experiments performed with
intact CPR (oxidized and two-electron reduced) show that
the optical changes induced on binding 2¢,5¢-ADP
(Fig. 3C,D) are different from those observed in the isolated
FAD domain in both oxidized and reduced states. This is
especially evident for the reduced forms (Fig. 3B,D) in the
region 500–700 nm. Thus, in intact CPR the binding of
2¢,5¢-ADP to the FAD domain appears to modify not only
the environment of the FAD but also that of the FMN
Fig. 1. Relaxation transients observed with CPR reduced at the two-
electron level with nicotinamide coenzymes. (A and C) Absorption
spectra obtained after reduction of human CPR (140 l
M
)with
NADPH (140 l
M
). Conditions: 100 m
M
potassium phosphate buffer,
pH 7.0, 25 °C. Identical spectral changes to those shown in (A) were
also observed on adding 140 l
M
NADH to human CPR. (B) Tem-
perature-jump difference absorption transients obtained for CPR

reduced at the two-electron level with NADPH (upper transient) and
NADH (lower transient). Conditions: thermostat temperature, 20 °C;
temperature-jump, +7 °C; voltage discharge, 12.5 kV; 140 l
M
CPR,
100 m
M
potassium phosphate buffer, pH 7.0. For the difference
absorption transient measured at 450 nm for CPR reduced with
NADPH; 1/s ¼ 55 ± 2 s
)1
. For the difference absorption transient
measured at 450 nm for CPR reduced with NADH;
1/s ¼ 18 ± 0.7 s
)1
. Difference absorption transients were generated
by subtracting the transient obtained for the oxidized enzyme from
that obtained for reduced CPR as described previously [28].
Ó FEBS 2003 Electron transfer in human cytochrome P450 reductase (Eur. J. Biochem. 270) 2615
cofactor. This structural perturbation transmitted between
the two domains may play a role in the kinetics of internal
electron transfer (see below).
Titration with 2¢,5¢-ADP at protein levels sufficient for
good spectral signals gave linear plots, implying that the K
d
is quite low. Thus, it was necessary to measure the inhibition
constant for 2¢,5¢-ADP in steady-state CPR-catalysed
reactions as a guide to its strength of binding to CPR.
Steady-state kinetic studies with cytochrome c as electron
acceptor demonstrated that 2¢,5¢-ADP is a competitive

inhibitor with respect to NADPH. The K
m,app
for NADPH
increases as the 2¢,5¢-ADP concentration is increased,
without effect on V
max,app
(Fig. 4). The value of the
inhibition constant for 2¢,5¢-ADP (K
i
¼ 5.4 ± 1.3 l
M
)is,
within error, identical to the K
m,app
for NADPH
(6.7 ± 1.3 l
M
), suggesting that this moiety is the major
determinant of coenzyme binding to CPR.
The rate of interflavin electron transfer for CPR reduced
at the two-electron level by dithionite (1/s ¼ 11 ± 0.5 s
)1
)
is increased approximately threefold to 35 ± 0.2 s
)1
in the
presence of a stoichiometric amount of 2¢,5¢-ADP, and this
is accompanied by an increase in the amplitude of the
absorbance change. This is consistent with the notion that
the 2¢,5¢-ADP moiety of NADPH makes a major contribu-

tion to the enhanced rate of internal electron transfer in
NADPH-reduced CPR as compared to the dithionite-
reduced enzyme. The absence of a comparable rate
enhancement in NADH-reduced CPR suggests that the
2¢-phosphate of NADPH is a major factor in increasing the
rate of interdomain electron transfer. It should be noted,
however, that the binding of 2¢,5¢-ADP to dithionite-
reduced enzyme does not lead to as fast an electron transfer
rate as that observed in NADPH-reduced samples
(35 ± 0.2 s
)1
vs. 55 ± 0.5 s
)1)
. This suggests that other
interactions (perhaps with the pyrophosphate bond) can
also contribute to the enhancement of the rate of interflavin
electron transfer.
Similar temperature-jump experiments performed with
2¢-AMP are also consistent with this conclusion. The
observed rate of interflavin electron transfer obtained with
dithionite-reduced CPR in the presence of either 140 l
M
or
1800 l
M
2¢-AMP (K
i2¢-AMP
¼ 180 ± 11 l
M
) is identical,

within experimental uncertainty, to that obtained with
2¢,5¢-ADP (32 ± 0.6 s
)1
and 35 ± 0.2 s
)1
, respectively).
Spectroelectrochemical titration
Any perturbation in the redox potential of the relevant
flavin couples on binding 2¢,5¢-ADP might account for the
observed increase in the rate of internal electron transfer,
and perhaps for the changes in the flavin absorption spectra.
We have therefore determined the redox potentials of CPR
in the absence and presence of 2¢,5¢-ADP. Electrochemical
titration of CPR with dithionite has revealed that each of
the four flavin couples in CPR can be distinguished and
their reduction potentials determined [26]. Spectral changes
accompanying a typical titration are shown in Fig. 5A and
plots of absorbance at 455 nm vs. potential in Fig. 5B; in
these experiments CPR concentration was 60 l
M
and
2¢,5¢-ADP was present in equimolar amounts. The oxidized
enzyme has visible absorption maxima at 455 nm and
381 nm, with the semiquinone species having a maximum at
585 nm. Redox titrations performed in the presence of
2¢,5¢-ADP exhibited similar spectral changes to those
observed in its absence.
Plots of absorption vs. potential data for CPR in the
presence and absence of 2¢,5¢-ADP were essentially identical
(Fig. 5B), indicating that there was negligible difference in

redox behaviour of the flavins in the presence of this ligand.
While the midpoint reduction potential values for three of
the four couples, estimated as described in Experimental
procedures, are slightly more positive than those reported
previously, all values are within error of the previously
reported data set [26]. The redox potentials were not altered
to any significant extent by addition of 2¢,5¢-ADP. Thus, the
midpoint potentials for both the 2¢,5¢-ADP-bound and
Fig. 2. The crystal structure of NADP
9
-bound rat CPR in its oxidized
form [7]. (A) The overall polypeptide fold. The different structural
domains are indicated. The FAD-domain contains the binding site for
NADPH and is related to the FNR family of flavoproteins. For clarity,
the NADPH-binding subdomain is also indicated. (B) The coenzyme-
binding site. The main residues known to interact with the adenosine
2¢-phosphate group are shown (R298, R597 and K602). W677 (W676
in human CPR), which shields the Re-face of the FAD isoalloxazine
ring position, is also shown. The nicotinamide moiety of NADP(H) is
highly disordered in the crystal.
2616 A. Gutierrez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
ligand-free forms of CPR are )76 ± 5 mV (FMN ox/sq);
)248 ± 10 mV (FMN sq/hq); )260 ± 12 mV (FAD ox/
sq) and )361 ± 7 mV (FAD sq/hq). Our findings contrast
with published work on cytochrome P450 BM3
,
where
NADP(H) binding to the reductase domain is thought to
induce significant changes in the reduction potentials of the
FAD[34].WeconcludethatinthecaseofCPRthe

observed effects of the binding of coenzyme and coenzyme
fragments in increasing the rates of electron transfer result
largely from structural perturbations that affect the rate-
limiting step(s) for electron transfer ) probably inter–
domain interactions ) without altering the driving force.
Effects of binding adenosine 2¢,5¢-bisphosphate
on hydride ion transfer
In previous work with CPR and the isolated FAD-
domain we provided kinetic evidence for the existence of
a second kinetically distinct noncatalytic NADPH-bind-
ing site in the enzyme, occupation of which led to a two-
to fivefold decrease in the rate of hydride ion transfer
[24,25]. Similar observations have subsequently been
made with the structurally related enzymes neuronal
nitric oxide synthase [35] and the adrenodoxin reductase
homologue FprA from Mycobacterium tuberculosis [36].
A general model has emerged from these kinetic obser-
vations in which occupation of this second site by
NADPH hinders the release of NADP
+
.Because
electron transfer is reversible, hydride transfer leads to
an equilibrium distribution of enzyme species comprising
oxidized enzyme, oxidized enzyme bound to NADPH,
and reduced enzyme bound to NADP
+
.Releaseof
NADP
+
from the latter species will lead to further flavin

reduction as the equilibrium distribution is shifted toward
reduced enzyme (see [24,35] for a more detailed discus-
sion). Thus, if the binding of NADPH to the noncata-
lytic site hinders NADP
+
release from the catalytic site,
the observed rate of FAD reduction will be decreased at
Fig. 3. Difference absorption spectra for the binding of 2¢,5¢-ADP to the isolated FAD-domain and CPR. (A) FAD-domain. Difference absorption
spectra (spectrum in the presence of 100 l
M
2¢,5¢-ADP minus spectrum in the absence of 2¢,5¢-ADP) of oxidized FAD-domain (100 l
M
). (B) As for
panel A, but for the FAD-domain (100 l
M
) reduced at the one-electron level (blue semiquinone species) with sodium dithionite. (C) CPR.
Difference absorption spectra (spectrum in the presence of 50 l
M
2¢,5¢-ADP minus spectrum in the absence of 2¢,5¢-ADP)ofoxidizedCPR(50l
M
).
(D) As for (C) but for CPR (50 l
M
) reduced at the two-electron level (blue disemiquinone species) with sodium dithionite.
Fig. 4. The inhibition of cytochrome c reductase activity by 2¢,5¢-ADP.
Reaction mixtures contained 7 n
M
CPR, 50 m
M
potassium phosphate

buffer, pH 7.0, 50 l
M
cytochrome c and various concentrations of
NADPH and of the inhibitor 2¢,5¢-ADP. The assay temperature was
25 °C and the reaction rate was measured by reduction of cyto-
chrome c
3+
. Five different concentrations of 2¢,5¢-ADP were used: 5,
10, 20, 40 and 80 l
M
corresponding to curves a, b, c, d and e,
respectively.
Ó FEBS 2003 Electron transfer in human cytochrome P450 reductase (Eur. J. Biochem. 270) 2617
high NADPH concentrations, and this is in fact observed
[24,25]. By contrast, in stopped-flow experiments per-
formed with NADH a conventional saturable increase in
the rate of flavin reduction with increasing NADH
concentration is seen (Fig. 6B). This indicates that
NADH does not bind to the second coenzyme-binding
site and/or that NAD
+
dissociates too rapidly to limit
the rate of hydride transfer. In light of the major
importance of the 2¢-phosphate in coenzyme binding, the
latter seems a likely explanation.
In rapid-mixing stopped-flow experiments, preincubation
of the isolated FAD-domain with stoichiometric 2¢,5¢-ADP
eliminates this inhibitory effect observed at high NADPH
concentration, presumably by preventing the simultaneous
binding of two NADPH molecules (Fig. 6A and inset). In

temperature-jump experiments with dithionite-reduced
CPR in the presence of a 10-fold molar excess of 2¢,5¢-
ADP, the observed rate of interflavin electron transfer
(1/s ¼ 28 ± 0.5 s
)1
) is only slightly less than that observed
with stoichiometric 2¢,5¢-ADP (1/s ¼ 35 ± 0.2 s
)1
). Com-
bined, the stopped-flow and temperature-jump data indicate
that increasing the concentration of 2¢,5¢-ADP above
stoichiometric levels does not have any substantial addi-
tional effect on the rate of internal electron transfer. These
results suggest that the observed inhibitory effect of high
NADPH concentrations on the reductive half-reaction is
associated with the nicotinamide moiety of the coenzyme.
The role of W676 in interdomain electron transfer
Our previous studies have shown that microsecond tem-
perature perturbation of CPR reduced at the two-electron
level with NADPH yields two different relaxation processes
[28]. The initial fast relaxation (1/s ¼ 2200 ± 300 s
)1
)is
not associated with electron transfer, but is attributed to
local conformational changes in the vicinity of the cofactors
and induced by NADPH binding [28]. It is not observed in
the W676H mutant of CPR [28], suggesting that these
changes may involve W676, a residue that stacks against the
Re-face of the isoalloxazine ring of FAD in CPR (Fig. 1B)
and which is involved in a series of conformational changes

associated with the hydride transfer step [25]. This fast
relaxation process is not observed in temperature-jump
experiments with CPR reduced by NADH, nor in
Fig. 5. Redox potentiometry studies. (A)
Spectral changes during redox titration of
human CPR. Human CPR (60 l
M
)was
titrated electrochemically as described in
Experimental procedures. Progressive reduc-
tion of the enzyme leads to bleaching of the
oxidized flavin spectrum and accumulation of
neutral blue flavin semiquinones with
absorption maximum at 585 nm. Positions of
isosbestic points in the titration are indicated
at 501 nm (oxidized/semiquinone couple for
both flavins) and at 435 nm (semiquinone/
reduced couple). (B) Absorption vs. potential
data for 2¢,5¢-ADP-bound and ligand-free
forms of human CPR. Plots of absorption
data at 455 nm (at the flavin maximum for the
oxidized CPR) vs. applied potential are shown
for both ligand-free (h)and2¢,5¢-ADP-bound
(d) forms of human CPR. Enzyme concen-
trations in all titrations were 60 l
M
.
2618 A. Gutierrez et al. (Eur. J. Biochem. 270) Ó FEBS 2003
experiments with dithionite-reduced CPR incubated with a
stoichiometric amount of 2¢,5¢-ADP. That only NADPH

(and not NADH, or 2¢,5¢-ADP) can elicit this rapid
structural relaxation indicates additional complexity in
the interaction of coenzymes with CPR. This suggests that
the nicotinamide ring of NAD
+
interacts differently with
the enzyme than that of NADPH or that NAD
+
is bound
weakly to reduced CPR.
It is possible that the conformational changes produced
by occupation of the 2¢-phosphate binding site that increase
the rate of electron transfer also affect the nicotinamide ring
binding site and W676. Stopped-flow tryptophan fluores-
cence experiments indicated a rapid but small increase
(complete in about 2 ms after the mixing event; instrument
dead-time 1 ms) in fluorescence emission on mixing
oxidized CPR (10 l
M
) with a stoichiometric amount of
2¢,5¢-ADP (Fig. 7A). However a similar transient is
observed in control experiments involving rapid mixing of
Fig. 6. Stopped-flow kinetic studies of flavin reduction in the presence of
2¢,5¢-ADP. (A) Fluorescence stopped-flow transients (excitation at
340 nm; emission at 450 nm) for NADPH oxidation by the FAD-
domain. Conditions: 50 m
M
potassium phosphate buffer, pH 7.0,
25 °C, 10 l
M

FAD-domain, 200 l
M
NADPH. Transient a: mono-
phasic fluorescence transient (k
obs
,of3.2s
)1
) obtained for NADPH
oxidation by the FAD-domain. Transient b: fluorescence transient
obtained for NADPH oxidation by the FAD-domain preincubated
with a stoichiometric amount of 2¢,5¢-ADP (10 l
M
). Data are best
described by a double-exponential expression, yielding values k
obs1
and k
obs2
of 26 s
)1
and 2.5 s
)1
, respectively. Inset: Dependence of the
observed rates on NADPH concentration. s, n, Estimated values for
k
obs1
and k
obs2
, respectively, for FAD-domain preincubated with
stoichiometric 2¢,5¢-ADP. d, Estimated values for k
obs

in the
absence of 2¢,5¢-ADP preincubation. Note the overlapping of n (k
obs2
)
and d (k
obs
). (B) NADH concentration dependence of the rate of
flavin reduction in intact CPR. Absorption stopped-flow experiments.
Reaction monitored at 450 nm. Conditions: 50 m
M
potassium phos-
phatebuffer,pH7.0,25°C, 10 l
M
CPR.
Fig. 7. Stopped-flow tryptophan fluorescence studies with oxidized CPR
and 2¢,5¢-ADP. Conditions: 10 l
M
CPR, 10 l
M
2¢,5¢-ADP, 50 m
M
potassium phosphate buffer pH 7.0, 25 °C. Excitation wavelength
295 nm. (A) Transient observed upon rapid-mixing of oxidized CPR
and 2¢,5¢-ADP. (B) Control experiment. Fluorescence change observed
when CPR was mixed against buffer alone. The transients observed in
(A) and (B) are most likely to originate from fast pressure-pulse pro-
pagation after the initial mixing event (apparatus dead time %1ms).
(C) Transient a: same as in (B). Transient b: fluorescence kinetic
transient observed during reduction of CPR with 10-fold excess
NADPH (100 l

M
). Mutagenesis studies demonstrated that these
fluorescence fluctuations are related to changes in the environment of
W676 (see Fig. 2), accompanying hydride transfer from the nicotind-
amide nucleotide to the FAD cofactor [25].
Ó FEBS 2003 Electron transfer in human cytochrome P450 reductase (Eur. J. Biochem. 270) 2619
CPR against buffer alone (Fig. 7B), ruling out any specific
effect on the conformation of W676 induced by the binding
of 2¢,5¢-ADP. Thus, although coenzyme binding is driven
primarily by interaction of the 2¢,5¢-ADP moiety with CPR,
the larger tryptophan fluorescence changes on mixing
NADPH with CPR in stopped-flow experiments ([24,25];
Fig. 7C), attributed to environmental effects on W676,
must derive from additional interactions of the coenzyme.
We have previously rationalized the lack of a tryptophan
fluorescence change observed in temperature-jump experi-
ments with NADPH by suggesting that the equilibrium
position of NADP
+
in the two-electron reduced enzyme is
that observed in the reported crystal structure (i.e. the
nicotinamide ring is not tightly associated with the enzyme
and is distant from the isoalloxazine ring) [28].
Discussion
Our previous temperature-jump studies with human CPR
highlighted the importance of conformational change in
limiting the rate of internal electron transfer and pointed to
the role of the coenzyme in enhancing reaction rate [28]. We
have now extended our temperature-jump studies to identify
key interactions that are responsible for modulating the rate

of internal electron transfer between the two domains of this
enzyme. We have demonstrated that the role of the
2¢-phosphate of NADPH is to optimize electron transfer
between the flavin cofactors. Occupation of the 2¢-phos-
phate binding site by NADPH, 2¢,5¢-ADP or 2¢-AMP leads
to altered rates of conformational change. These conform-
ational changes are transmitted over a relatively large
distance through the protein to optimize electron transfer
betweentheflavins.Inthecaseof2¢,5¢-ADP binding, optical
spectroscopy provides further evidence for a Ôlong-rangeÕ
perturbation of the environments of the isoalloxazine rings
of the flavins. This conformational change appears to
involve significant domain movement, as it is sensitive to the
effects of solution viscosity [28]. Reduction of the enzyme
with NADH leads to a slower rate of internal electron
transfer owing to the absence of the phosphate group in the
2¢-phosphate-binding site. However, electron transfer in the
NADH reduced enzyme is faster than in dithionite-reduced
CPR, indicating that other interactions made by the
coenzyme play some role in optimizing electron transfer
between the flavins. This is further suggested by the fact that
the observed rate of internal electron transfer in the presence
of 2¢,5¢-ADP and 2¢-AMP for dithionite-reduced enzyme is
increased to % 60% the value seen for NADPH-reduced
enzyme.
In agreement with our kinetic work on human CPR,
crystallographic studies of mutant forms of rat CPR also
indicate a role for conformational change and domain
re-orientation [30]. The multidomain structure of CPR is
highly flexible in solution. The crystal structure strongly

suggests that the so-called linker domain plays a key role in
controlling the mutual orientation of the two flavin-
containing domains. Our kinetic [24] and NMR studies
(B. Hawkins, I. Barsukov, L Y. Lian, G. C. K. Roberts,
unpublished data) indicate that the two isolated flavin-
binding domains do not form a strong complex in solution,
and electron transfer between the isolated domains is much
slower than in the intact enzyme, with a second-order rate
constant of 9.5 · 10
4
M
)1
Æs
)1
[24]. By tethering the domains
with the linker region the electron transfer rate is optimized,
but more importantly new opportunities are presented for
regulating the rate of interflavin electron transfer by
coenzyme binding to the NADPH/FAD domain.
Multiple conformations of CPR in the absence of
coenzyme have been suggested from earlier steady-state
studies with the rat enzyme [37]. It was rationalized that
binding of NADPH would then induce conformational
change and that a unique conformation would be populated
compatible with hydride transfer. Our use of equilibrium-
perturbation methods with CPR has now provided evidence
that coenzyme binding energy is utilized to optimize
reaction steps other than the initial hydride transfer event.
Given the conserved multidomain structure of other mem-
bers of the diflavin reductase family, it seems reasonable to

propose that conformational gating of electron transfer may
also occur in these enzymes. The temperature-jump method
may be useful for probing these aspects in related enzymes
is currently in hand.
Acknowledgements
We thank C. Baxter and Dr Andrew Westlake for assistance in
preparing Fig. 2. The work was funded by the Medical Research
Council, the Wellcome Trust and the Lister Institute of Preventive
Medicine. N.S.S. is a Lister Institute Research Professor.
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