Báo cáo khoa học: Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1 doc

Determination of the redox potentials and electron transfer
properties of the FAD- and FMN-binding domains of the human
oxidoreductase NR1
Robert D. Finn
1
, Jaswir Basran
2
, Olivier Roitel
2
, C. Roland Wolf
1
, Andrew W. Munro
2
, Mark J. I. Paine
1
and Nigel S. Scrutton
2
1
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, UK;
2
Department of Biochemistry, University of Leicester, UK
Human novel reductase 1 (NR1) is an NADPH dependent
diflavin oxidoreductase related to cytochrome P450 reduc-
tase (CPR). The FAD/NADPH- and FMN-binding
domains of NR1 have been expressed and purified and their
redox properties studied by stopped-flow and steady-state
kinetic methods, and by potentiometry. The midpoint
reduction potentials of the oxidized/semiquinone ()315 ±
5 mV) and semiquinone/dihydroquinone ()365 ± 15 mV)
couples of the FAD/NADPH domain are similar to those
for the FAD/NADPH domain of human CPR, but the rate

of hydride transfer from NADPH to the FAD/NADPH
domain of NR1 is % 200-fold slower. Hydride transfer is
rate-limiting in steady-state reactions of the FAD/NADPH
domain with artificial redox acceptors. Stopped-flow studies
indicate that hydride transfer from the FAD/NADPH
domain of NR1 to NADP
+
is faster than hydride transfer in
the physiological direction (NADPH to FAD), consistent
with the measured reduction potentials of the FAD couples
[midpoint potential for FAD redox couples is )340 mV, cf
)320 mV for NAD(P)H]. The midpoint reduction potentials
for the flavin couples in the FMN domain are
)146 ± 5 mV (oxidized/semiquinone) and )305 ± 5 mV
(semiquinone/dihydroquinone). The FMN oxidized/semi-
quinone couple indicates stabilization of the FMN semiqui-
none, consistent with (a) a need to transfer electrons from the
FAD/NADPH domain to the FMN domain, and (b) the
thermodynamic properties of the FMN domain in CPR and
nitric oxide synthase. Despite overall structural resemblance
of NR1 and CPR, our studies reveal thermodynamic simi-
larities but major kinetic differences in the electron transfer
reactions catalysed by the flavin-binding domains.
Keywords: novel reductase 1; cytochrome P450 reductase;
flavoprotein; potentiometry; kinetics.
Human novel reductase 1 (NR1) is a new member of the
growing family of diflavin reductases that contain both
FAD and FMN prosthetic groups [1]. In mammalian
systems, cytochrome P450 reductase (CPR) was the first
diflavin reductase isolated [2,3], followed by the isoforms of

nitric oxide synthase (NOS [4,5]); and methionine synthase
reductase (MSR [6]). Bacterial members of the family
include flavocytochrome P450 BM3 (CYP102 [7]); and
sulfite reductase [8]. CPR is the most extensively character-
ized member of the mammalian diflavin reductases. In
eukaryotic cells, type II cytochromes P450 are located in the
endoplasmic reticulum, where they receive electrons from
CPR. Like all members of the diflavin reductase family,
CPR accepts electrons from NADPH, an obligatory
2-electron donor. These electrons are then transferred in a
finely coupled stepwise manner to various physiological
redox acceptor proteins, in the case of CPR to the P450
enzymes bound to the endoplasmic reticulum [9]. CPR is a
78-kDa membrane-bound flavoprotein and is likely to have
evolved by the fusion of two ancestral genes encoding
proteins related to ferredoxin-NADP
+
reductase (FNR)
and flavodoxin (Fld) [10], bringing the two flavins (FAD
and FMN) in close proximity for electron transfer. The
enzyme also transfers electrons to cytochrome b
5
[11], haem
oxygenase [12], and the fatty acid elongation system [13].
CPR can also reduce a number of artificial redox acceptors
[14,15] and drugs [16–20], and may also have a role in the
generation of reactive oxygen species in the cell.
The recent cloning and expression of a cDNA encoding
protein NR1 in insect cells has established functional
similarities with CPR [1]. As with CPR, human NR1

catalyses the NADPH-dependent reduction of cytochrome
c and various other electron accepting compounds. How-
ever, overall the enzymatic activities are substantially less
than those seen for CPR. NR1 also supports the NADPH-
dependent reduction of the quinone antineoplastic agent
Correspondence to N. S. Scrutton, Department of Biochemistry,
University of Leicester, University Road, Leicester LE1 7RH.
Fax: + 44 116 252 3369, Tel.: + 44 116 223 1337,
E-mail: or
M. J. I. Paine, Biomedical Research Centre, University of Dundee,
Ninewells Hospital and Medical School, Dundee, DD1 9SY.
Fax: + 44 1382 669993, Tel.: + 44 1382 496420,
E-mail:
Abbreviations: CPR, cytochrome P450 reductase; DCPIP, 2,6-dichlo-
rophenolindophenol; MSR, methionine synthase reductase; NOS,
nitric oxide synthase; NR1, novel reductase 1; FDR, ferredoxin
NADP
+
reductase; FLD, flavodoxin.
(Received 20 November 2002, revised 15 January 2003,
accepted 21 January 2003)
Eur. J. Biochem. 270, 1164–1175 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03474.x
doxorubicin, and menadione [1], a functional property also
shared by the NOS family of enzymes [21,22]. The biological
role of NR1 is unknown, but it is expressed at high levels in
a wide range of cancer cell lines suggesting a role in the
metabolic activation of bioreductive drugs. The lack of a
membrane anchor in NR1 suggests that reduction of the
P450 enzymes attached to the endoplasmic reticulum is an
unlikely physiological role.

In this paper we have characterized in detail the redox
and electron transfer properties of the component flavin-
binding domains of protein NR1. These studies have
enabled us to make detailed comparison with equivalent
studies performed on the flavin-binding domains of human
CPR [23,24]. Despite the structural similarity of NR1 and
CPR inferred from alignment of protein sequences, we
demonstrate major functional differences between the two
enzymes. Studies with the isolated FAD-domain reveal that
hydride transfer from NADPH to FAD is substantially
impaired in NR1, accounting for the poor catalytic rates in
steady-state studies with various redox acceptors. The
reduction potentials of the FAD and FMN redox couples
in NR1 are similar to those in CPR and NOS, suggesting
that impaired hydride transfer is attributed to less favour-
able alignment of the nicotinamide coenzyme with FAD
rather than a thermodynamic effect. Possible reasons for the
poor hydride transfer rates in NR1 are discussed.
Experimental procedures
Chemicals and reagents
All chemicals were purchased from Sigma (Poole, Dorset,
UK) and all enzymes from Life Technologies Inc. (Paisley,
UK), except where stated. The synthesis of A-side deuter-
ated NADPD was as described in our previous work with
NOS [25].
Expression constructs for the NR1 flavin-binding
domains
The cDNA encoding NR1 was cloned previously from
MCF7 cells by RT-PCR [1]. The construction of an
expression clone (pFAD-PET) suitable for production of

the NR1 FAD/NADPH domain has been described [1].
The NR1 FMN domain construct was generated by PCR
amplification using Pfu polymerase (Stratagene) and using
the oligonucleotides 5¢-TGGAATCCATATGCCGAG
CCCGCAGCTTCTG-3¢ and 5¢-GGAATTCCGGATCC
TTAGGGCAGGGGGACTCC-3¢ as forward and reverse
primers, respectively. Following amplification, the PCR
product was cloned into pCR Blunt (Invitrogen) and
sequenced to verify clone integrity. The NR1 FMN domain
coding sequence was subcloned into the unique NdeI/XhoI
sites of pHRT (patent pending). The resulting plasmid
termed pHRT-NR1 FMN was used for expression of the
FMN domain.
Recombinant protein expression and purification
For expression of the various domains in Escherichia coli,
strain BLR (DE3/pLysS) containing the appropriate
plasmid strains were grown overnight at 37 °CinLB
broth containing ampicillin (50 lgÆmL
)1
)andchloram-
phenicol (34 lgÆmL
)1
)toaD
600
of 0.4–0.8. Isopropyl
thio-b-
D
-galactoside was then added (0.5 m
M
and 1 m

M
for the FAD-domain and FMN-domain constructs,
respectively) to initiate protein expression, and cultures
were incubated for a further 12 h at 30 °C. Cells were
harvested by centrifugation (5000 g, 20 mins) and resus-
pended in binding buffer (20 m
M
sodium phosphate
buffer, pH 8.0, 500 m
M
NaCl, 5 m
M
imidazole and 10%
glycerol).
The NR1-FAD domain was purified over nickel agarose
and 2¢,5¢-ADP Sepharose as described previously [1]. For
purification of the FMN domain, cell suspensions were
lysed by incubating at 30 °C for 15 min in the presence of
100 lgÆmL
)1
lysozyme, followed by 30 min at 4 °Cinthe
presence of 0.1% Triton X-100. The lysates were sonicated
(MSE probe, several short bursts at high power) and
centrifuged (40 000 g,30min,4°C). The supernatants
were filtered through a 0.45-lm filter before being loaded
on a Hi-trap nickel column. The column was washed
sequentially with binding buffer and binding buffer
containing 20 m
M
imidazole. The bound protein was

eluted with binding buffer containing 350 m
M
imidazole.
The eluted NR1 FMN domain was exchanged into
Ôthrombin cleavage bufferÕ (20 m
M
Tris/HCl pH 8.4,
150 m
M
NaCl, 2.5 m
M
CaCl
2
), and rebound to the nickel
resin. Cleavage was performed at 4 °C overnight in the
presence of thrombin at a concentration of 0.5 UÆmg
)1
of
protein. Cleaved NR1-FMN domain was separated from
the nickel bound fusion tag by centrifugation (2000 g,
5 min). NR1-FMN domain was exchanged into 20 m
M
Tris/HCl buffer, pH 8.0, and loaded onto a Hi-Trap
Mono Q column equilibrated with 20 m
M
Tris/HCl buffer,
pH 8.0. The column was washed sequentially with 20 m
M
Tris/HCl buffer, pH 8.0, containing 50 m
M

NaCl and then
20 m
M
Tris/HCl buffer pH 8.0, containing 100 m
M
NaCl.
NR1-FMN domain was eluted from the column with
20 m
M
Tris/HCl buffer, pH 8.0 containing 200 m
M
NaCl.
Glycerol was added to 20% before the purified protein
was stored at )70 °C. During purification, protein con-
centrations were determined by Bradford analysis using
Bio-Rad reagents and bovine serum albumin as a protein
standard.
Steady-state enzyme assays
Reduction of prototypical cytochrome P450 reductase
substrates dichlorophenol indophenol [DCPIP, 0–100 l
M
,
e
600
¼ 22 000
M
)1
Æcm
)1
] and ferricyanide (0–250 l

M
,
e
420
¼ 1020
M
)1
Æcm
)1
) was carried out using NR1 or
CPR FAD/NADPH domain (700 pmol and 60 pmol
enzyme, respectively) in 50 m
M
potassium phosphate buf-
fer, pH 7.0, at 25 °C. The final assay volume was 1 mL.
Apparent K
m
values for NADPH for the various FAD/
NADPH domains were determined by measuring the rate
of potassium ferricyanide reduction at 25 °Cin50m
M
potassium phosphate buffer, pH 7.0, essentially as described
previously for CPR [26]. Ferricyanide concentration was
saturating at 250 l
M
. The NADPH concentration range
was 0.5 l
M
to 100 l
M

.
In attempts to reconstitute cytochrome c reductase
activity, 100 pmol of either NR1 or CPR FAD/NADPH
Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1165
domain was mixed with various amounts of the NR1 FMN
domain, ranging from 0 to 2 nmol, in 50 m
M
potassium
phosphate buffer, pH 7.0. The rate of reduction of
cytochrome c in the presence of 200 l
M
NADPH and
100 l
M
horse heart cytochrome c at 25 °Cwasthen
determined at 550 nm (e
550
¼ 22 640
M
)1
Æcm
)1
)ona
Varian Cary UV50 BioI scanning spectrophotometer.
Stopped-flow kinetic studies
Single turnover stopped-flow kinetic studies were per-
formed using an Applied Photophysics SX.18 MV
stopped-flow spectrophotometer. Measurements were car-
ried out at 25 °Cin50m
M

potassium phosphate buffer,
pH 7.0. Protein concentration (NR1 FAD domain) was
13 l
M
(reaction cell concentration) for photodiode array
experiments and 4 l
M
(reaction cell concentration) for
measurements in single wavelength mode. The sample-
handling unit of the stopped-flow instrument was con-
tained within a Belle Technology glove-box to maintain
anaerobic conditions. All buffers were made oxygen-free
by evacuation and extensive bubbling with argon before
use. Prior to stopped-flow studies, protein samples were
treated with potassium hexacyanoferrate to effect com-
plete oxidation of the domains, and excess cyanoferrate
was removed by rapid gel filtration (Sephadex G25).
Treatment with hexacyanoferrate did not affect the kinetic
behaviour of the domains.
Stopped-flow, multiple-wavelength absorption studies
were carried out using a photodiode array detector and
X
-
SCAN
software (Applied Photophysics Ltd). Spectral
deconvolution was performed by global analysis and
numerical integration methods using
PROKIN
software
(Applied Photophysics Ltd). In single wavelength studies,

flavin reduction by NADPH was observed at 454 nm or
600 nm. Transients at 454 nm were found to be mono-
phasic and were analysed by fitting to a standard single
exponential expression. For studies of electron transfer
from 2 electron-reduced FAD/NADPH domain to
NADP
+
, the FAD/NADPH domain was titrated to the
2-electron level with sodium dithionite. The reduced FAD/
NADPH domain was then mixed rapidly with NADP
+
.
Reaction transients at 454 nm were monophasic and fitted
using a single exponential expression. In studies of hydride
transfer, the concentration of coenzyme was always at least
10-fold greater than enzyme concentration to ensure
pseudo first order conditions.
Reduction of the FAD/NADPH domain by NADPH
was also analysed using fluorescence detection. Enzyme
concentration was 4 l
M
and oxidation of NADPH was
monitored by fluorescence emission at 450 nm (excitation
340 nm). Emission bands were selected using a bandpass
filter (Coherent Optics; 450 nm #35–3367). Tryptophan
emission was monitored at 340 nm (excitation 295 nm). A
bandpass filter (Coherent Optics; # 35–3003) was used to
select fluorescence emission.
Electron transfer from NR1 FAD/NADPH domain to
the FMN domain was monitored using a sequential mixing

protocol in the stopped-flow instrument. In the first mix the
FAD/NADPH domain (2 l
M
) was reduced with NADPH
(2 l
M
). Following an appropriate delay time to allow
reduction of the FAD the solution was mixed with varying
concentrations of the FMN domain (5–20 l
M
). Reactions
were followed at 454 nm and a double exponential process
best described the absorbance change.
Potentiometry
Redox titrations were performed in a Belle Technology
glove-box under a nitrogen atmosphere. All solutions were
degassed under vacuum with argon. Oxygen levels were
maintained at less than 2 p.p.m. The protein was applied to
a Bio-Rad Econo-Pac 10DG desalting column in the
anaerobic box, pre-equilibrated with degassed 100 m
M
potassium phosphate (pH 7.0) buffer, to ensure removal
of all traces of oxygen. The protein solutions (typically
50–100 l
M
in 5–8 mL buffer, both in the presence and
absence of 10% v/v glycerol) were titrated electrochemically
according to the method of Dutton [27] using sodium
dithionite as reductant and potassium ferricyanide as
oxidant. Dithionite and ferricyanide were delivered in

0.2 lL aliquots from concentrated stock solutions (typically
10–50 m
M
). Mediators were added to facilitate electrical
communication between enzyme and electrode, prior to
titration. Typically, 2 l
M
phenazine methosulfate, 5 l
M
2-hydroxy-1,4-naphthoquinone, 0.5 l
M
methyl viologen,
and 1 l
M
benzyl viologen were included (to mediate in the
range between +100 to )480 mV, as described previously
[23,28]). At least 15 min was allowed to elapse between each
addition to allow stabilization of the electrode. Spectra
(250–750 nm) were recorded using a Cary UV-50 Bio
UV-Visible scanning spectrophotometer. The electroche-
mical potential of the solution was measured using a Hanna
pH 211 meter coupled to a Pt/Calomel electrode (Thermo-
Russell Ltd) at 25 °C. The electrode was calibrated using the
Fe
3+
/Fe
2+
EDTA couple as a standard (+108 mV). A
factor of +244 mV was used to correct relative to the
standard hydrogen electrode.

Data manipulation and analysis were performed using
ORIGIN
(Microcal). For the FMN and FAD/NADPH
domain titrations, absorbance values at wavelengths of
454 nm (close to the absorption maximum for oxidized
flavin) and 585 nm or 600 nm (near the absorption
maximum for the blue semiquinone form of flavin) were
plotted against potential. Data for the titration of the
individual NR1 FAD/NADPH and FMN domains were
fitted to Eqn (1), which represents a 2-electron redox
process derived by extension to the Nernst equation and
the Beer–Lambert law, as described previously [23,28]
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
ð1Þ
In Eqn (1), A is the total absorbance, a, b and c are
component absorbance values contributed by the relevant
flavin in the oxidized, semiquinone and reduced states,
respectively. E is the observed potential, and E
1
¢ and E
2
¢ are
the midpoint potentials for oxidized/semiquinone and
semiquinone/reduced couples, respectively, for the relevant
flavin. In using Eqn (1) to fit the absorbance-potential data
for the single-flavin systems (i.e. the isolated FAD/NADPH
and FMN domains), the variables were unconstrained, and
regression analysis provided values in close agreement to
those of the initial estimates.
1166 R. D. Finn et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Results
Expression and purification of the NR1 domains
We have previously expressed and purified full-length NR1
from insect Sf9 cells [1], but the yields of recombinant
enzyme are insufficient for the detailed biophysical analyses
described in this paper. We have therefore attempted to
develop E. coli-based expression systems to generate suffi-
cient quantities of recombinant enzyme for biophysical
studies. Repeated attempts to express soluble full-length
NR1 in E. coli were unsuccessful, but using a similar

approach to that taken with CPR [23,24,29], we have
dissected NR1 using recombinant DNA methods (see
above) and expressed the individual FMN and FAD/
NADPH domains separately, in soluble form (Fig. 1) that
are suitable for kinetic and thermodynamic studies.
The NR1-FMN domain was constructed from residues
1–174, and encodes a polypeptide with a calculated
molecularmassof% 20 kDa, while the NR1-FAD/
NADPH domain encodes a % 48 kDa peptide spanning
residues 194–597. The peptide sequence between residues
175–193 that forms the interdomain linker region was
absent from the constructs as it led to insolubility of both
domains. The presence of this linker region might explain
the problems we experienced with the expression of soluble
full-length NR1 in E. coli. The recombinant domains were
His-tagged to facilitate affinity purification by nickel
agarose chromatography, and purified to homogeneity
(Fig. 2A). In the case of the NR1-FAD/NADPH domain, a
second 2¢,5¢-ADP-Sepharose affinity step was also incor-
porated in the purification scheme, taking advantage of its
nucleotide binding capacity of the resin. Following purifi-
cation, the His-tag was removed from the NR1-FMN
domain by protease cleavage, but routinely not from the
NR1-FAD/NADPH domain as the His-tag was ineffi-
ciently cleaved from this domain. The presence of the His-
tag on the FAD/NADPH domain had no apparent effect
on catalytic activity.
The purified domains were yellow, indicating the presence
of bound cofactor, and they displayed UV-visible absorp-
tion spectra characteristic of flavin containing enzymes

(Fig. 2B). The fully oxidized FMN domain had absorbance
maxima at 376 and 454 nm, and the FAD/NADPH
domain absorbed maximally at 376 and 453 nm. The
FMN domain was stable for several weeks upon storage at
)20 °C at high concentration (>100 l
M
). However, the
FAD/NADPH domain appeared less stable and had a
tendency to aggregate over time, particularly if subjected to
Fig. 1. Schematic overview of the domains generated for this study in the
context of full-length NR1.
Fig. 2. Expression of NR1 domains in E. coli. (A) SDS/12% PAGE
analysis of purified NR1-FMN (FMN) and NR1-FAD/NADPH
(FAD) domains (2 lgÆlane
)1
). (B) Absorption spectra of purified NR1-
FMN and NR1-FAD/NADPH domains (approx. 7 and 8 l
M
protein,
respectively).
Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1167
cycles of freezing and thawing. In addition, potentiometric
studies (see below) revealed a strong tendency for the FAD/
NADPH domain to aggregate at low potentials.
Steady-state enzyme activities
The catalytic activity of the NR1-FAD/NADPH domain
was examined and compared with the CPR FAD/NADPH
domain. The CPR FAD/NADPH domain retains trans-
hydrogenase activity, and is capable of reducing a range of
electron acceptors [29]. To assess the functional activity of

the NR1-FAD/NADPH domain, we measured the specific
activities for ferricyanide and DCPIP (Table 1). The
ferricyanide and DCPIP reductase activities of the NR1-
FAD/NADPH domain were approximately 29-fold and
5-fold lower than CPR-FAD/NADPH, respectively, con-
sistent with differences in activity levels found between the
full-length enzymes [1].
We hypothesized that differences in catalytic activity
between CPR and NR1 might be explained by differences in
the apparent affinity for NADPH. Thus, the apparent K
m
values for NADPH were determined by further steady-state
kinetic assays of potassium ferricyanide reduction. The
ferricyanide was maintained at saturating concentration
(250 l
M
), and NADPH concentration was varied between
0.5 and 100 l
M
. In this system, the apparent K
m
for
NADPH was lower for the FAD/NADPH domain of NR1
(1.08 ± 0.12 l
M
) than for the FAD/NADPH domain of
CPR (2.62 ± 0.40 l
M
). However, both domains clearly
bind NADPH tightly, and differences in catalytic activity

are evidently not associated with NADPH binding.
Mixing the FAD/NADPH and FMN domains of NR1
under the conditions described in Experimental procedures
did not stimulate to any considerable extent (<10%) the
cytochrome c reductase activity of the system over that
observed for the isolated FAD/NADPH domain, which
had a k
cat
of 2.2 ± 0.25 min
)1
(0.037 ± 0.004 s
)1
). Intact
CPR-like diflavin reductase enzymes typically have high
levels of cytochrome c reductase activity, with electron
transfer to the cytochrome mediated by the FMN cofactor.
A more substantial increase in specific activity for cyto-
chrome c reduction was observed through mixing the
human CPR FMN domain with its FAD/NADPH domain
(% 16-fold) (29). However, the domain mixture still recon-
stituted only % 2% of the activity of the full length CPR. In
thecaseofNR1,thek
cat
for the full length enzyme is low
(1.3 s
)1
) and is clearly gated largely by the hydride transfer
event [1]. While domain reconstitution in NR1 only has a
marginal effect on cytochrome c reduction rate, the
reconstituted activity is % 3% of that in full length NR1,

similartotheratioachievedwithCPRdomains.
To explore further the influence of the hydride transfer
step on catalytic activity of the FAD/NADPH domains of
NR1 and CPR, we compared ferricyanide reduction rates
using both NADPH and A-side deuterated coenzyme
(NADPD) under saturating substrate conditions (100 l
M
NADPH/D and 250 l
M
ferricyanide) under standard
conditions described in Experimental procedures. In the
steady-state, ferricyanide reduction was slower for both
enzymes using NADPD as reductant. A deuterium isotope
effect of 2.5 was observed on CPR FAD/NADPH domain-
catalysed ferricyanide reduction, and of 3.5 for the NR1
FAD/NADPH-catalysed process. The value of the KIE on
steady-state activity for NR1 FAD/NADPH domain is
consistent with that observed in stopped-flow studies of
hydride transfer to the flavin (see below) and with this event
being rate-limiting in reductive catalysis using artificial
electron acceptors.
Potentiometric analysis of the component domains
Anaerobic spectroelectrochemical titrations of the isolated
FAD/NADPH and FMN domains of NR1 enabled the
determination of the midpoint reduction potentials for the
oxidized/semiquinone (E
1
) and semiquinone/hydroquinone
(E
2

) flavin couples. Data for both domains were fitted to the
2-electron Nernst function described in Experimental pro-
cedures [23,28]. The FMN domain did not aggregate to any
extent over the course of % 5–8 h required to complete the
titrations. However, for the FAD/NADPH domain of
NR1, considerable aggregation and precipitation of the
protein occurred within 1 h of initiating the titrations, and
this precipitation was accelerated at more negative poten-
tials (<350 mV). To correct for baseline shifts in the
titrations for both NR1 FAD/NADPH and FMN
domains, spectra were manipulated by subtracting absorp-
tion at 800 nm in each sample (where there is negligible
absorption contribution from flavins in any redox state)
across the entire spectrum. In the case of the FAD/NADPH
domain, attempts were made to account for turbidity
caused by protein aggregation by multiplying individual
spectra by a correction factor (1–1/k;wherek is the
absorption wavelength). However, correction did not
improve to any large extent spectra for which the A
800
value had increased above approximately 0.05 units. To
enable accurate determination of the FAD potentials,
titrations of the NR1 FAD/NADPH domain were per-
formed on samples of identical concentration over small
ranges (100–150 mV) of the potential range, moving on to a
fresh sample when turbidity proved excessive. In this way,
data across the entire range were collected and were of
suitable quality for determination of both redox couples for
the FAD/NADPH domain flavin.
Table 1. Apparent turnover numbers for DCPIP and ferricyanide

reduction by the FAD/NADPH binding domains of CPR and NR1.
Apparent turnover numbers were determined at 25 °Cin50m
M
potassium phosphate buffer, pH 7.0, as described in Experimental
procedures, by monitoring the reduction of substrate (DCPIP or
ferricyanide) at appropriate wavelengths (600 nm and 420 nm,
respectively). Results are the mean and standard deviation of triplicate
assays. For the determination of apparent k
cat
values, experiments
were performed at a saturating concentration of NADPH (200 l
M
),
over a wide range of concentrations of the substrate (0–100 l
M
for
DCPIP and 0–700 l
M
for ferricyanide). Values were determined by
curve fitting to the Michaelis–Menten equation and are the mean and
standard deviation of three separate experiments.
Domain
Substrate
DCPIP (k
cat
, s
)1
) Ferricyanide (k
cat
, s

)1
)
CPR FAD/NADPH 4.57 ± 0.18 65.33 ± 2.20
NR1 FAD/NADPH 0.86 ± 0.02 2.27 ± 0.13
1168 R. D. Finn et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Visible absorption spectra collected during the redox
titration of the NR1 FMN domain are shown in Fig. 3. The
oxidized domain has typical flavin absorption maxima at
454 nm and 376 nm, and forms a neutral blue semiquinone
during dithionite titration, indicating that the potential for
the ox/sq couple is more positive that that for the sq/red
couple. The semiquinone has absorption maximum at
585 nm, with a shorter wavelength maximum at 352 nm.
Data from the absorption maximum of the oxidized flavin
(A
454
) and near the semiquinone maximum (A
600
)were
fitted to the 2-electron Nernst function (Fig. 4), and yielded
essentially identical data for the ox/sq ()152±4mV;
)146 ± 5 mV, respectively) and sq/hq couples ()304 ±
8mV;)305 ± 5 mV) (Fig. 4).
Absorption spectra collected during the redox titrations
of the NR1 FAD/NADPH domain are shown in Fig. 5. As
with the FMN domain, a neutral blue semiquinone is
stabilized. However, the maximal intensity of the semiqui-
none is much less than that observed for the NR1 FMN
domain, suggesting that the midpoint reduction potentials
for the FAD ox/sq and sq/hq couples are much closer

together than those for the FMN. Typically for this class of
diflavin enzymes, the potentials for the FAD/NADPH
domains are rather more negative than those for the FMN
domain, reflecting the direction of electron transfer from
NADPH through FAD, then FMN and on to the final
electron acceptor(s) [23,30]. The NR1 FAD/NADPH
domain follows this trend, with midpoint reduction poten-
tials of )315 ± 5 mV (ox/sq) and )365 ± 15 mV (sq/hq)
derived from fitting titration data at the semiquinone
absorption maximum (585 nm) to the 2-electron Nernst
function (Fig. 6). The tendency of the NR1 FAD/NADPH
domain to aggregate and precipitate during the redox
titrations (and to do so particularly rapidly at more negative
potentials) explains the larger error for the midpoint
potential for the sq/hq couple. This aspect of FAD/
NADPH domain behaviour is shared also by the homo-
logous FAD/NADPH domains of human CPR [23] and
flavocytochrome P450 BM3 [28]. Aggregation of the NR1
FAD/NADPH domain was much less extensive in the
absence of cofactor reduction.
Stopped-flow kinetic studies
Reduction of the FAD/NADPH domain of NR1 was
investigated by stopped-flow methods using a photodiode
array detector. Aggregation of the domain was not observed
over the short time periods used in stopped-flow experi-
ments. The spectral changes accompanying flavin reduction
(Fig. 7) revealed the absence of major spectral change in the
long wavelength region (550 nm to 650 nm). This contrasts
with similar studies with the FAD/NADPH domain of
human CPR where rapid absorption increases in this region,

attributable to the formation of an oxidized enzyme-
NADPH charge-transfer species, that accumulates prior
to flavin reduction. Global fitting of the spectral changes for
the NR1 FAD/NADPH domain indicated the presence of
only one detectable kinetic phase corresponding to FAD
reduction; FAD reduction proceeds with an observed rate
constant of 1.07 ± 0.02 s
)1
. In single wavelength studies at
454 nm the absorption changes reporting on FAD reduc-
tion were monophasic, consistent with a single step kinetic
model, and the observed rate of FAD reduction was found
to be independent of coenzyme concentration in the pseudo
first order regime (Fig. 8A; Table 2). Studies at 600 nm
indicated that a spectroscopically distinct NADPH-E
ox
Fig. 3. Spectral changes during redox titration of the FMN domain of human NR1. Anaerobic spectroelectrochemical titration was performed as
described in Experimental procedures. The oxidized FMN domain is shown as a thick solid black line, and has the highest absorption at 454 nm,
with the second major band at 376 nm. The other spectrum shown by a thick solid line is that at which the blue FMN semiquinone is maximally
populated. The spectral maxima for this species are located at approximately 585 nm and 352 nm. Spectra collected during addition of the first
electron (oxidized-to-semiquinone transition) are indicated by thin, solid black lines. Spectra collected during addition of the second electron to the
flavin (semiquinone-to-hydroquinone transition) are indicated by dotted lines. Isosbestic points for the ox/sq [1] and sq/hq [2] couples are locatedat
approximately 501 nm and 434 nm, respectively. Approximately 100 spectra were collected across the relevant range of potentials. For clarity, only
selected spectra are shown.
Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1169
species did not accumulate prior to flavin reduction, as was
seen for the isolated FAD/NADPH domain of CPR [24]. In
studies with CPR [24], NOS [25] and the adrenodoxin
reductase homologue FprA from Mycobacterium tubercu-
losis (which is related structurally to the FAD/NADPH

domains of the diflavin reductase family [32]; K. McLean,
N. S. Scrutton & A. W. Munro, unpublished results) the
observed rate of hydride transfer accelerates as the coen-
zyme concentration is decreased to levels that are stoichi-
ometric with the enzyme concentration. This unusual kinetic
behaviour has been attributed to the presence of a second,
regulatory coenzyme-binding site the occupation of which
attenuates hydride transfer from the catalytic site at high
NADPH concentrations. This behaviour is not observed
with the FAD/NADPH domain of NR1 and highlights a
major difference in the kinetic properties of NR1 compared
with other diflavin reductase enzymes, despite the overall
inferred structural similarity. The flavin reduction rate for
NR1 FAD/NADPH domain is 0.27 ± 0.1 in studies
performed with A-side deuterated coenzyme (NADPD;
Fig. 8A, inset), yielding a kinetic isotope effect of 3.7. This is
consistent with the absorption change at 454 nm reporting
on the hydride transfer step and with hydride transfer being
fully rate-limiting in steady-state turnover with ferricyanide
as electron acceptor (see above). Reactions performed over
an extended time base under aerobic conditions with
absorption detection at 454 nm gave access to the flavin
re-oxidation rate for stoichiometrically reduced FAD/
NADPH domain. In this case, re-oxidation occurred with
an observed rate constant of 0.025 ± 0.0005 s
)1
.
Given the reduction potentials of the FAD
ox/sq
and

FAD
sq/hq
couples of the isolated FAD/NADPH domain
(midpoint potential for the 2-electron couple, E
12
¼
)340±10mV)inrelationtothatofNADPH()320 mV)
we undertook a study of the reverse hydride transfer
reaction from dihydroquinone FAD/NADPH domain to
NADP
+
. Enzyme was initially titrated to the 2-electron
level with sodium dithionite under anaerobic conditions and
mixed rapidly with NADP
+
. Absorption transients were
monophasic at 454 nm (Fig. 8B), and the observed rate
constants for FAD oxidation were independent of NADP
+
concentration (Table 2). The rate of hydride transfer is
% 2.5-fold faster in the ÔreverseÕ direction and similar
observations have been made with FAD/NADPH domain
of human CPR, where the midpoint potential for the
2-electron flavin couple ()329 ± 7 mV) is also more
negative than that for NADPH [23,24].
Fluorescence detection was also used in stopped-flow
studies of enzyme reduction by NADPH. NADPH fluor-
escence was used in our previous studies with human CPR
and NOS to follow NADPH oxidation. However, reduction
of the FAD/NADPH domain of NR1 by NADPH is not

accompanied by a change in fluorescence emission at
450 nm following excitation at 340 nm for reasons that are
as yet are unclear. Changes in tryptophan fluorescence
emission do, however, accompany reduction of the FAD/
NADPH domain (Fig. 8C). Unlike with CPR FAD/
NADPH domain (which gives rise to a fluorescence
decrease on flavin reduction), fluorescence transients dis-
played an increase in fluorescence emission. The rapid
increase in fluorescence observed with the CPR domain
prior to flavin reduction, which reports on coenzyme
binding, is not observed in the NR1 domain transients.
Observed rate constants for the monophasic fluorescence
increase with the NR1 FAD/NADPH domain are inde-
pendent of coenzyme concentration and are similar in value
to the rate constants determined from absorption measure-
ments at 454 nm for flavin reduction (Table 2).
The ability of the reduced FAD/NADPH domain to
transfer electrons to the oxidized FMN domain was studied
by sequential stopped-flow methods. In the first mix the
FAD/NADPH domain was mixed with stoichiometric
NADPH, and the reduced domain was then mixed with
the oxidized FMN domain in a second mix. Reaction
transients measured at 454 nm were biphasic and the
observed rate constants calculated for both the fast and slow
phases were independent of coenzyme concentration
(Table 2). Technical difficulties owing to aggregation of
the FAD/NADPH domain in dithionite titrations preven-
ted detailed analysis of electron transfer between dithionite
reduced FAD/NADPH domain and the oxidized FMN
domain. The lack of a second order dependence of the

observed rate for interdomain electron transfer as the
Fig. 4. Absorbance vs. potential plots for the FMN domain of human
NR1. (A)PlotofA
600
(near the blue semiquinone maximum) vs.
reduction potential fitted to a 2-electron Nernst function, as described
in Experimental procedures. (B) Plot of A
454
data (at the oxidized
flavin maximum) from the same titration, also fitted to the 2-electron
Nernst function. Midpoint reduction potentials for the ox/sq
()152±4mV and 146±5mV) and sq/hq ()304±8mV;
)305 ± 5 mV) couples of the flavin determined from fits to both data
sets are identical within error.
1170 R. D. Finn et al. (Eur. J. Biochem. 270) Ó FEBS 2003
concentration of the FMN domain is increased indicates
that the reaction rate is controlled by some process other
than collision of the two flavin-binding domains.
Discussion
The ability to dissect CPR into distinct functional domains
has assisted in providing detailed structural and kinetic
information about the redox and structural properties of
CPR [24,33–35]. The results of this study show that NR1,
which is related structurally to CPR, can be dissected into
distinct functional domains. Owing to the difficulties in
expressing the full-length protein, the ability to isolate
individual flavin-binding domains has facilitated the deter-
mination of both thermodynamic and kinetic properties of
NR1, and as we have shown for CPR and P450 BM3
(23,24,28) the properties of the flavin binding domains of

NR1 are likely to mimic the redox properties of the domains
in full-length enzyme.
In steady-state assays, the NR1-FAD/NADPH domain
has significantly lower catalytic activity for prototypical
reductase substrates compared to the CPR-FAD/NADPH
domain, in agreement with previously reported findings for
intact NR1 [1] (Table 1). Stopped-flow studies with this
domain indicate that flavin reduction occurs relatively slowly
(% 1s
)1
) as a monophasic process, and flavin reduction
displays a KIE of 3.7 in reactions with A-side NADPD. The
slow reduction of FADby NADPH is limiting in steady-state
reactions as indicated by the KIE of 3.5 observed for NR1
FAD/NADPH domain-catalysed ferricyanide reduction.
The apparent turnover number with ferricyanide (2.27 s
)1
)
is approximately twice the hydride transfer rate (% 1s
)1
)
measured in stopped-flow studies, consistent with it being a
one-electron acceptor. Comparable studies with CPR indi-
cate more complex behaviour; in this case flavin reduction is
biphasic (observed rate constants % 200 s
)1
and % 3s
)1
)and
the kinetic mechanism for flavin reduction is shown in

Scheme 1 (for further details and experimental data sup-
porting the assignment of observed rate constants to kinetic
phases see [24]). The fast phase (200 s
)1
) represents the rapid
formation of an equilibrium between an oxidized enzyme-
NADPH complex and reduced enzyme-NADP
+
complex
(species CT2). The slow phase (% 3s
)1
) is attributed to the
Fig. 6. Absorbance vs. potential plot for the FAD/NADPH domain of
human NR1. Plot of A
585
(near the blue semiquinone maximum) vs.
reduction potential was fitted to a 2-electron Nernst function, as des-
cribed in Experimental procedures. The fit yields midpoint reduction
potential values of )315 ± 5 mV for the oxidized/semiquinone cou-
ple, and )365 ± 15 mV for the semiquinone/hydroquinone couple.
Fig. 5. Spectral changes during redox titration of the FAD/NADPH domain of human NR1. Anaerobic spectroelectrochemical titration was
performed as described in the Experimental procedures. For clarity, only selected spectra are shown. The highest intensity spectrum is that of
oxidized FAD/NADPH domain, and is shown as a thick solid black line with absorption maxima at 376 and 453 nm. The isosbestic point for the
oxidized-to-semiquinone transition [1] is located at approximately 501 nm. Solid lines indicate spectra recorded during addition of the first electron
(ox/sq transition), whereas dotted lines shows spectra recorded during addition of the second electron (sq/hq transition). The tendency of the protein
to aggregate at negative potentials, along with the low potential for the semiquinone/hydroquinone couple of the FAD ()365 ± 15 mV) prevented
collection of useful spectral data at potentials below %)430 mV.
Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1171
release of NADP
+

with concomitant displacement of the
equilibrium distribution of enzyme species towards further
reduction of the FAD (i.e. further transfer of hydride
equivalents from NADPH to the FAD is gated by the release
of NADP
+
). A similar mechanism has also been suggested
Fig. 7. Spectra. (A) Spectral changes accompanying the reduction of
the FAD/NADPH domain of NR1 by NADPH. Conditions: 50 m
M
potassium phosphate buffer, pH 7.0; 25 °C. Protein concentration
13 l
M
; NADPH concentration 130 l
M
. (B) Initial and end spectrum
obtained from fitting to a single step kinetic model. Observed rate
constant for FAD reduction 1.07 ± 0.01 s
)1
.
Fig. 8. Absorbance and fluorescence changes accompanying flavin
reduction in the FAD/NADPH domain of NR1 by NADPH. Condi-
tions: coenzyme concentration, 100 l
M
; enzyme concentration 4 l
M
;
50 m
M
potassium phosphate buffer, pH 7.0, 25 °C. (A) Monophasic

absorption transient at 454 nm for reduction of the FAD/NADPH
domain by NADPH and NADPD (inset); coenzyme concentration
100 l
M
. Observed rate constants calculated at different concentrations
of NADPH are given in Table 2. (B) Monophasic absorption transient
at 454 nm for oxidation of the FAD/NADPH domain by NADP
+
.
Enzyme was initially reduced at the 2-electron level by titration with
sodium dithionite in the presence of methyl viologen. Observed rate
constants calculated at different concentrations of NADP
+
are given
in Table 2. (C) Tryptophan fluorescence emission transient observed
during the reduction of the FAD/NADPH domain with NADPH.
Observed rate constants calculated at different concentrations of
NADPH are given in Table 2. In all panels, the solid black line is the fit
to the experimental data (shown in greyscale).
1172 R. D. Finn et al. (Eur. J. Biochem. 270) Ó FEBS 2003
for reactions of the reductase domain of NOS with NADPH
[25]. The steady-state turnover value (65 s
)1
)fortheFAD/
NADPH domain of CPR in reactions with NADPH and
ferricyanide is much faster than the slow NADP
+
release
step observed in stopped-flow studies. With CPR we suggest
therefore that ferricyanide oxidizes the EH

2
NADP
+
form of
the FAD/NADPH domain, and that subsequent release of
NADP
+
from ENADP
+
occurs at a faster rate than from
2-electron reduced enzyme (i.e. EH
2
NADP
+
) (Scheme 1).
That enzyme oxidation occurs from the EH
2
NADP
+
species
of the NADPH/FAD domain of CPR is also consistent with
the KIE value of 2.5 observed in steady-state reactions with
ferricyanide (i.e. hydride transfer and not NADP
+
release is
rate-limiting). Although NR1 is structurally related to CPR
and NOS, the rate of hydride transfer in NR1 (% 1s
)1
)is
substantially less than the rates in CPR (% 200 s

)1
[24]); and
NOS (% 200 s
)1
for the first hydride transfer reaction in the
FAD-FMN reductase domain [25]). In searching for a
structural reason for the substantially reduced rates of
hydride transfer in NR1 we note the absence of a cysteine
residue that corresponds to Cys630 in CPR; the equivalent
residue in NR1 is Ala549 [1]. In CPR, Cys630 forms part of a
catalytic triad with Ser457 and Asp675, and mutagenesis
studies with rat CPR have demonstrated a key role for this
residue in hydride transfer from NADPH to FAD [36,37].
Our own studies with flavocytochrome P450 BM3 also
indicate that mutation of the equivalent cysteine residue in
this enzyme to alanine substantially decreases the rate of
flavin reduction and has an adverse effect on the FAD
reduction potential (O. Roitel, N. S. Scrutton and A. W.
Munro, unpublished work).
Potentiometric studies of the isolated FAD/NADPH and
FMN domains of NR1 have allowed us to establish that
both flavins stabilize neutral blue semiquinones, and to
determine the midpoint reduction potentials for the four
redox couples of NR1 (Fig. 9). These data indicate that the
relative potentials of the flavins are ordered similarly to
Table 2. Summary of observed rate constants from stopped-flow kinetic studies. All reactions were performed in 50 m
M
potassium phosphate buffer,
pH 7.0 at 25 °C. In studies with the isolated FAD/NADPH domain, protein concentration was 4 l
M

.Instudiesofinterdomainelectrontransfer,
the FAD/NADPH domain was reduced with stoichiometric NADPH prior to a second mix with the FMN domain (see text for details). Errors are
those from fitting to the average of at least five kinetic transients.
FAD reduction
(A
454
nm transient)
Trp fluorescence
emission
FAD oxidation
(A
454
nm transient)
Interdomain electron
transfer (A
454
nm transient)
NADPH
(l
M
) k
obs
(s
)1
)
NADPH
(l
M
) k
obs

(s
)1
)
NADP
+
(l
M
) k
obs
(s
)1
)
FMN domain
(l
M
) k
fast
(s
)1
) k
slow
(s
)1
)
5 1.07 ± 0.01 4 1.97 ± 0.02 4 2.60 ± 0.02 5 1.20 ± 0.04 0.20 ± 0.02
15 1.07 ± 0.01 20 1.14 ± 0.01 10 2.74 ± 0.02 7.5 1.70 ± 0.04 0.12 ± 0.01
25 1.09 ± 0.01 40 1.12 ± 0.01 50 2.50 ± 0.01 10 1.29 ± 0.01 0.16 ± 0.01
50 0.99 ± 0.01 100 1.08 ± 0.01 100 2.31 ± 0.02 12.5 1.60 ± 0.02 0.10 ± 0.01
100 0.98 ± 0.01 200 1.02 ± 0.01 200 2.23 ± 0.02 15 1.59 ± 0.05 0.20 ± 0.01
200 1.00 ± 0.01 20 1.45 ± 0.02 0.12 ± 0.01

300 0.96 ± 0.01
Scheme 1. Kinetic mechanism for flavin reduction. A
ox
refers to the electron acceptor ferricyanide.
Fig. 9. Flavin reduction potentials for members of the diflavin reductase
enzyme family. The various midpoint reduction potentials for the
oxidized/semiquinone (grey boxes) and semiquinone/hydroquinone
couples (white boxes) of the FAD and FMN cofactors in the various
diflavin reductases are shown diagrammatically. These are NR1 (this
work), human cytochrome P450 reductase (CPR [23]), neuronal nitric
oxide synthase (NOS [30]), and flavocytochrome P450 BM3 reductase
(BM3 [28]). The midpoint reduction potential for the physiological
reductant NAD(P)H ()320 mV) is shown as a dotted bar.
Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1173
those for human CPR. However, the oxidized/semiquinone
couple for the NR1 FMN is rather more negative than that
determined for CPR ()146 mV cf )66 mV). In addition,
the midpoint potential for the 2-electron reduction of the
NR1 FAD cofactor is also slightly more negative than that
for CPR ()340 mV cf )329 mV) and that for NADPH
()320 mV). The smaller separation between the ox/sq and
sq/hq midpoint potentials of the NR1 FAD domain
(50 mV cf 85 mV for CPR FAD and 159 mV for NR1
FMN) explains the rather low intensity of the blue
semiquinone signature at long wavelength that accumulates
during redox titration. The fact that the FAD potentials
thermodynamically disfavour its reduction by NADPH is
another likely factor in explaining the slow flavin reduction
rate in NR1. As might be predicted on the basis of the
relative reduction potentials, the dithionite-reduced NR1

FAD/NADPH domain catalyses NADP
+
reduction ap-
proximately 2.5-fold faster than the NADPH reduces the
enzyme FAD. A similar phenomenon was observed for
human CPR [24], and we consider that this behaviour
reflects the evolutionary origins of the diflavin reductases,
which have evolved from fusion of genes encoding
ferredoxin NADP
+
-reductase (FDR) and flavodoxin
(FLD) progenitors [9]. The physiological role of FDR
enzymes is to catalyse the reduction of NADP
+
.Thus,it
appears likely that the role of the FLD domain in the
diflavin reductases is to remove one (or both) electrons
from the FAD hydroquinone, thus disfavouring the reverse
reaction.
Our work with the NR1 flavin-binding domains has
highlighted major kinetic differences in the kinetics of
hydride transfer and intermediates populated during
enzyme reduction compared with CPR and NOS, despite
the overall similar thermodynamic properties. In future
work, we intend to establish the physiological role of NR1,
to obtain structural data for the FAD/NADPH and FMN
domains, and to examine in greater detail the reasons
underlying its slow, rate-limiting hydride transfer reaction.
Acknowledgements
This work was funded by grants from the Medical Research Council,

the Lister Institute of Preventive Medicine and the Wellcome Trust. The
authors would like to thank the Biotechnology and Biological Sciences
Research Council, the Medical Research Council and the European
Union for financial support for these studies. We are also grateful for
helpful discussions with Professor Gordon Roberts and Dr Aldo
Gutierrez (University of Leicester). N.S.S. is a Lister Institute Research
Professor. O.R. is a Marie Curie Research Fellow.
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Ó FEBS 2003 Redox properties of human NR1 (Eur. J. Biochem. 270) 1175