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
Trang 1Determination of the redox potentials and electron transfer
properties of the FAD- and FMN-binding domains of the human
oxidoreductase NR1
Robert D Finn1, Jaswir Basran2, Olivier Roitel2, C Roland Wolf1, Andrew W Munro2, Mark J I Paine1 and Nigel S Scrutton2
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
P450reduc-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)340mV, cf )320mV 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 P450reductase; 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 P450reductase (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 P450BM3 (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 P450are 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 b5[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
cand 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: nss4@le.ac.uk 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: m.j.paine@dundee.ac.uk
Abbreviations: CPR, cytochrome P450reductase; 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)
Trang 2doxorubicin, 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
P450enzymes 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
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 37C in LB
broth containing ampicillin (50 lgÆmL)1) and chloram-phenicol (34 lgÆmL)1) to a D600 of 0.4–0.8 Isopropyl thio-b-D-galactoside was then added (0.5 mM and 1 mM for the FAD-domain and FMN-domain constructs, respectively) to initiate protein expression, and cultures were incubated for a further 12 h at 30C Cells were harvested by centrifugation (5000 g, 20mins) and resus-pended in binding buffer (20mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 5 mM 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 30C for 15 min in the presence of
100 lgÆmL)1lysozyme, followed by 30min at 4C in the presence of 0.1% Triton X-100 The lysates were sonicated (MSE probe, several short bursts at high power) and centrifuged (40 000 g, 30 min, 4C) 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 20mM imidazole The bound protein was eluted with binding buffer containing 350mM imidazole The eluted NR1 FMN domain was exchanged into
thrombin cleavage buffer (20mM Tris/HCl pH 8.4, 150mMNaCl, 2.5 mM CaCl2), and rebound to the nickel resin Cleavage was performed at 4C overnight in the presence of thrombin at a concentration of 0.5 UÆmg)1of 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 20mM Tris/HCl buffer, pH 8.0, and loaded onto a Hi-Trap Mono Q column equilibrated with 20mMTris/HCl buffer,
pH 8.0 The column was washed sequentially with 20 mM Tris/HCl buffer, pH 8.0, containing 50 mMNaCl and then 20mMTris/HCl buffer pH 8.0, containing 100 mMNaCl NR1-FMN domain was eluted from the column with 20mMTris/HCl buffer, pH 8.0containing 200mMNaCl 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 P450reductase substrates dichlorophenol indophenol [DCPIP, 0–100 lM,
e600¼ 22 000M )1Æcm)1] and ferricyanide (0–250 lM,
e420¼ 1020M )1Æcm)1) was carried out using NR1 or CPR FAD/NADPH domain (700pmol and 60pmol enzyme, respectively) in 50mMpotassium phosphate buf-fer, pH 7.0, at 25C The final assay volume was 1 mL Apparent Km values for NADPH for the various FAD/ NADPH domains were determined by measuring the rate
of potassium ferricyanide reduction at 25C in 50 mM potassium phosphate buffer, pH 7.0, essentially as described previously for CPR [26] Ferricyanide concentration was saturating at 250 lM The NADPH concentration range was 0.5 lMto 100 lM
In attempts to reconstitute cytochrome c reductase activity, 100 pmol of either NR1 or CPR FAD/NADPH
Trang 3domain was mixed with various amounts of the NR1 FMN
domain, ranging from 0to 2 nmol, in 50mM potassium
phosphate buffer, pH 7.0 The rate of reduction of
cytochrome c in the presence of 200 lM NADPH and
100 lM horse heart cytochrome c at 25C was then
determined at 550nm (e550¼ 22 640M )1Æcm)1) on a
Varian Cary UV50BioI 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 25C in 50 mM potassium phosphate buffer,
pH 7.0 Protein concentration (NR1 FAD domain) was
13 lM (reaction cell concentration) for photodiode array
experiments and 4 lM (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 lM and oxidation of NADPH was
monitored by fluorescence emission at 450nm (excitation
340nm) Emission bands were selected using a bandpass
filter (Coherent Optics; 450nm #35–3367) Tryptophan
emission was monitored at 340nm (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 lM) was reduced with NADPH
(2 lM) Following an appropriate delay time to allow
reduction of the FAD the solution was mixed with varying
concentrations of the FMN domain (5–20 lM) 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 mM potassium phosphate (pH 7.0) buffer, to ensure removal
of all traces of oxygen The protein solutions (typically 50–100 lM 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 mM) Mediators were added to facilitate electrical communication between enzyme and electrode, prior to titration Typically, 2 lM phenazine methosulfate, 5 lM 2-hydroxy-1,4-naphthoquinone, 0.5 lM methyl viologen, and 1 lMbenzyl viologen were included (to mediate in the range between +100 to)480mV, 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 25C The electrode was calibrated using the
Fe3+/Fe2+ 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]
A¼a10
ðEE 0
1 Þ=59þ b þ c10ðE 0
2 EÞ=59
1þ 10ðEE 0
1 Þ=59þ 10ðE 0
2 EÞ=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 E1¢ and E2¢ 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
Trang 4Expression 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
molecular mass of 20kDa, 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 lM) 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).
Trang 5cycles 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 Km
values for NADPH were determined by further steady-state
kinetic assays of potassium ferricyanide reduction The
ferricyanide was maintained at saturating concentration
(250 lM), and NADPH concentration was varied between
0.5 and 100 lM In this system, the apparent Km for
NADPH was lower for the FAD/NADPH domain of NR1
(1.08 ± 0.12 lM) than for the FAD/NADPH domain of
CPR (2.62 ± 0.40 lM) 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 kcatof 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 the case of NR1, the kcatfor 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, similar to the ratio achieved with CPR domains
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 lM NADPH/D and 250 lM 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 (E1) and semiquinone/hydroquinone (E2) 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 (<350mV) 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; where k is the absorption wavelength) However, correction did not improve to any large extent spectra for which the A800 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 C in 50 m M
potassium phosphate buffer, pH 7.0, as described in Experimental
procedures, by monitoring the reduction of substrate (DCPIP or
ferricyanide) at appropriate wavelengths (600nm and 420nm,
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
Trang 6Visible 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
(A454) and near the semiquinone maximum (A600) were
fitted to the 2-electron Nernst function (Fig 4), and yielded
essentially identical data for the ox/sq ()152 ± 4 mV;
)146 ± 5 mV, respectively) and sq/hq couples ()304 ±
8 mV;)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 P450BM3 [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 (550nm to 650nm) 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
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 located at 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.
Trang 7species 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 FADox/sq and FADsq/hqcouples of the isolated FAD/NADPH domain (midpoint potential for the 2-electron couple, E12¼ )340 ± 10 mV) in relation to that of NADPH ()320mV)
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 reverse 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 450nm following excitation at 340nm 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) Plot of A 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 ± 4 mV and 146 ± 5 mV) and sq/hq ()30 4 ± 8 mV;
)305 ± 5 mV) couples of the flavin determined from fits to both data
sets are identical within error.
Trang 8concentration 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 P450BM3 (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 ( 1 s)1) as a monophasic process, and flavin reduction displays a KIE of 3.7 in reactions with A-side NADPD The slow reduction of FAD by 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 ( 1 s)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)1and 3 s)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 ( 3 s)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 )430mV.
Trang 9release 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: 50m 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 ;
50m 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).
Trang 10for reactions of the reductase domain of NOS with NADPH
[25] The steady-state turnover value (65 s)1) for the FAD/
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 EH2NADP+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 EH2NADP+) (Scheme 1)
That enzyme oxidation occurs from the EH2NADP+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 ( 1 s)1) is
substantially less than the rates in CPR ( 200 s)1[24]); and
NOS ( 200 s)1for 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 Cys630in CPR; the equivalent
residue in NR1 is Ala549 [1] In CPR, Cys630forms 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 P450BM3 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 50m M potassium phosphate buffer,
pH 7.0at 25 C In studies with the isolated FAD/NADPH domain, protein concentration was 4 l M In studies of interdomain electron transfer, 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
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 P450reductase (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 ( )320mV) is shown as a dotted bar.