Interflavin electron transfer in human cytochrome P450 reductaseis enhanced by coenzyme binding Relaxation kinetic studies with coenzyme analogues Aldo Gutierrez1,2, Andrew W.. Roberts1,
Trang 1Interflavin electron transfer in human cytochrome P450 reductase
is enhanced by coenzyme binding
Relaxation kinetic studies with coenzyme analogues
Aldo Gutierrez1,2, Andrew W Munro1, Alex Grunau1,2, C Roland Wolf3, Nigel S Scrutton1
and Gordon C K Roberts1,2
1 Department of Biochemistry and 2 Biological NMR Centre, University of Leicester, UK; 3 Biomedical Research Centre,
University of Dundee, Ninewells Hospital and Medical School, Dundee, UK
The role of coenzyme binding in regulating interflavin
electron transfer in human cytochrome P450 reductase
(CPR) has been studied using temperature-jump
spectros-copy Previous studies [Gutierrez, A., Paine, M., Wolf,
C.R., Scrutton, N.S., & Roberts, G.C.K Biochemistry
(2002) 41, 4626–4637] have shown that the observed rate,
1/s, of interflavin electron transfer (FADsq) FMNsq fi
FADox) FMNhq) in CPRreduced at the two-electron level
with NADPH is 55 ± 2 s)1, whereas with
dithionite-reduced enzyme the observed rate is 11 ± 0.5 s)1,
sug-gesting that NADPH (or NADP+) binding has an
important role in controlling the rate of internal electron
transfer In relaxation experiments performed with CPR
reduced at the two-electron level with NADH, the observed
rate of internal electron transfer (1/s¼ 18 ± 0.7 s)1) is
intermediate in value between those seen with
dithionite-reduced and NADPH-dithionite-reduced enzyme, indicating that the
presence of the 2¢-phosphate is important for enhancing
internal electron transfer To investigate this further,
tem-perature jump experiments were performed with
dithionite-reduced enzyme in the presence of 2¢,5¢-ADP and 2¢-AMP
These two ligands increase the observed rate of interflavin
electron transfer in two-electron reduced CPRfrom
1/s¼ 11 s)1to 35 ± 0.2 s)1and 32 ± 0.6 s)1, respectively
Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPRare unchanged on binding 2¢,5¢-ADP, supporting the view that enhanced rates
of interdomain electron transfer have their origin in a con-formational change produced by binding NADPH or its fragments Addition of 2¢,5¢-ADP either to the isolated FAD-domain or to full-length CPR(in their oxidized and reduced forms) leads to perturbation of the optical spectra
of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors The binding of 2¢,5¢-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentra-tion (i.e enhanced at low coenzyme concentraconcentra-tion) ob-served in stopped-flow studies The data are discussed in the context of previous kinetic studies and of the crystallo-graphic structure of rat CPR
Keywords: coenzyme binding; cytochrome P450 reductase; electron transfer; flavoprotein; temperature-jump relaxation spectroscopy
Members of the cytochrome P450 mono-oxygenase
super-family catalyse the hydroxylation of a wide range of
physiological and xenobiotic compounds; in eukaryotes the
type II cytochromes P450, located in the endoplasmic reticulum, play a key role in drug metabolism NADPH-cytochrome P450 reductase (CPR; EC 1.6.2.4) catalyses electron transfer to these type II cytochromes P450 [1–5] CPRis a 78-kDa enzyme containing one molecule each of FAD and FMN [6] Sequence analyses [5] and X-ray crystallographic studies of rat CPR[7] have revealed that CPRconsists of an N-terminal membrane anchor, respon-sible for its localization to the endoplasmic reticulum, and three folded domains: an FAD- and NADPH-binding domain related to ferredoxin-NADP+reductase, a flavo-doxin-like FMN-binding domain and a connecting linker domain In addition to the cytochromes P450, CPRcan donate electrons to cytochrome b5[8], haem oxygenase [9], the fatty acid hydroxylation system [10] and a number of artificial redox acceptors [11,12] and drugs [13–17] CPRis related to a number of mammalian diflavin reductases including the isoforms of nitric oxide synthase [18], methionine synthase reductase [19] and protein NR1
Correspondence to N S Scrutton, Department of Biochemistry,
University of Leicester, University Road, Leicester LE1 7RH, UK.
Fax: + 44 116 252 3369, Tel.: + 44 116 223 1337,
E-mail: nss4@le.ac.uk or to G C K R oberts, Biological NMR
Centre, University of Leicester, University Road, Leicester LE1 7RH,
UK Fax: + 44 116 223 1503, Tel.: + 44 116 252 2978,
E-mail: gcr@le.ac.uk
Abbreviations: CPR, NADPH-cytochrome P450 reductase; 2¢,5¢-ADP,
adenosine 2¢,5¢-bisphosphate; 2¢-AMP, adenosine 2¢-monophosphate.
Enzymes: NADPH-cytochrome P450 reductase (CPR; EC 1.6.2.4).
Note: In this paper the term intact CPRrefers to soluble CPR,
containing the FMN-binding, FAD-binding and linker domains and
lacking only the N-terminal membrane-anchoring peptide sequence.
(Received 14 March 2003, revised 17 April 2003,
accepted 24 April 2003)
Trang 2[20] It also shares some mechanistic and structural
similar-ity with prokaryotic diflavin reductases such as cytochrome
P450 BM3 [21] and sulfite reductase [22] The mechanism of
electron transfer in mammalian CPRs has been studied in
detail by stopped-flow kinetic and potentiometric methods
[23–26] The reduction potentials of the flavin couples have
been determined, and key intermediates in the reaction
sequence have been identified Moreover, the roles of
specific residues in flavin reduction by NADPH have been
elucidated by mutagenesis methods [25,27,28] The rate of
interflavin electron transfer in mammalian CPRs has been
determined by flash photolysis [29] and relaxation kinetic
methods [28] We recently demonstrated [28] that this rate is
relatively slow (55 s)1) in human CPR, despite the very close
proximity (3.85 A˚) of the flavin cofactors observed in the
crystal structure of the rat enzyme [7], and is limited by
conformational change and regulated by coenzyme binding
[28] This rate is lower (10 s)1) for dithionite-reduced
enzyme, indicating that nicotinamide coenzyme optimizes
interflavin electron transfer We also showed that mutation
of W676, which is located over the re-face of the FAD in
CPR[7,30], has a major effect on the kinetics of interflavin
electron transfer in both the forward and reverse
direc-tions We have now extended our relaxation kinetic
experiments to include studies of internal electron transfer
in the presence of NADPH, NADH and fragments of
nicotinamide coenzymes We show that binding of the
2¢-phosphate of NADPH, and to a lesser extent other
regions of the reducing coenzyme, specifically enhance the
rate of internal electron transfer Our studies point to a
complex mode of regulation of electron transfer reactions in
human CPR, triggered by coenzyme binding and involving
conformational change over a relatively large distance, and
highlight further the key role of conformational gating in
biological electron transfer reactions
Experimental procedures
Materials
NADPH, NADH, 2¢,5¢-ADP, 2¢-AMP, nicotinamide
1,N6-ethenoadenine dinucleotide phosphate and sodium
dithionite were from Sigma
2¢(3¢)-O-(trinitrophenyl)adeno-sine 5¢-monophosphate was from Molecular Probes All
other chemicals were of analytical grade
Protein purification
Human fibroblast CPR(lacking the N-terminal
membrane-anchoring region) and the functional FAD-binding domain
were expressed in Escherichia coli BL21(DE3)pLysS from
appropriate pET15b plasmid constructs, and purified as
described previously [24] The FAD-binding domain
con-struct includes the so-called linker domain [7]
Temperature-jump and stopped-flow kinetic methods
Temperature-jump experiments, using a TJ-64
temperature-jump instrument (Hi-Tech Scientific), were performed under
anaerobic conditions according to the method recently
described [28], and the heating time was estimated to be 4 ls
using a standard phenolphthalein-glycine buffer test
(T-jump users’ manual, Hi-Tech Scientific, Salisbury, UK) The initial temperature was 20C CPRsamples (140 lM) were prepared in an anaerobic glove box (Belle Technology Ltd) Reduction of the enzyme to the two-electron level by different two-electron donors (dithionite, NADH or NADPH) was monitored optically as described previously [28] Typically, 20 transients were collected and averaged for each reaction condition Optical artefacts caused by the high voltage discharge through the cell were accounted for by control measurements with the oxidized form of the enzyme, as described previously [28] Relaxation transients were fitted to a monophasic process using Hi-Tech software dedicated to the T-jump instrument Stopped-flow experiments with fluorescence detection were performed using an Applied Photophysics SX.18MV stopped-flow instrument Tryptophan fluorescence studies were performed using an excitation wavelength of 295 nm The emission band was selected using a WG320 cut-off emission filter (Coherent Optics) in combination with a UG-11 filter (Coherent Optics) to block stray visible light The photomultiplier voltage was kept constant during the measurements The oxidized form of the CPRenzyme (10 lM) was used in these experiments, which were performed in 50 mMpotassium phosphate buffer (pH 7.0)
at 25C
Inhibition studies The inhibition constant for 2¢,5¢-ADP was determined by using steady-state kinetic assays with cytochrome c as electron acceptor and NADPH as electron donor Reaction mixtures contained 7 nM CPR, 50 mM potassium phos-phate buffer (pH 7.0), 50 lM cytochrome c and variable concentrations of NADPH and of inhibitor 2¢,5¢-ADP; reactions were initiated by making microlitre additions of NADPH to the reaction mix Assays were performed
at 25C and the initial velocity of the reaction was measured by reduction of cytochrome c3+ at 550 nm (De¼ 21.1 mM )1Æcm)1) using a Cary-300 UV/visible spec-trophotometer Data were first analysed graphically using double-reciprocal plots (1/vivs 1/[NADPH]) to determine the type of inhibition Initial velocity values were then fitted
to the equation describing competitive inhibition
vi¼ VmaxS
KM 1þ I
K i
þ S
by nonlinear regression analysis using theGRAFITsoftware package [31]
Potentiometric titrations Spectroelectrochemical titrations were performed within a Belle technology glove box under a nitrogen atmosphere (oxygen maintained at < 5 p.p.m.) in 100 mM potassium phosphate buffer (pH 7.0) containing 10% (v/v) glycerol (titration buffer), at 25 ± 2C, essentially as described previously [26] Anaerobic titration buffer was prepared by flushing freshly prepared buffer with oxygen-free argon CPRprotein samples (typically 50–80 lM) admitted to the glove box were de-oxygenated by passing through a Bio-Rad EconoPack 10 DG desalting column pre-equilibrated
Trang 3in the anaerobic titration buffer The final
CPRconcentra-tion used for the redox titraCPRconcentra-tions was 60 lM Solutions of
benzyl viologen, methyl viologen,
2-hydroxy-1,4-naphtho-quinone and phenazine methosulfate were added to a
final concentration of 0.5 lM as redox mediators for the
titrations Absorption spectra (300–800 nm) were recorded
on a Cary UV50 Bio UV-visible spectrophotometer external
to the glove box, with absorption signals relayed to the
instrument from an absorption probe (Varian Inc.)
immersed in the protein sample, via a fibre optic cable
The electrochemical potential was monitored using a Hanna
instruments pH/voltmeter coupled to a Russell Pt/calomel
electrode immersed in the CPRsolution The electrode
was calibrated using the Fe(II)/Fe(III)-EDTA couple
(+108 mV) as a standard The enzyme solution was titrated
electrochemically using sodium dithionite as reductant and
potassium ferricyanide as oxidant, as described by Dutton
[32] Duplicate titration data sets were collected for CPRin
the presence and in the absence of 2¢,5¢-ADP (60 lM)
Approximately 100 different spectra were recorded during
each redox titration, covering the range between around
)400 mV and +200 mV (vs normal hydrogen electrode)
Reductive and oxidative titrations of the enzyme indicated
that there were no hysteretic effects, and that the enzyme did
not aggregate to any significant extent over the course of the
5–8 h required to complete the titrations
Data analysis was performed using Origin (Microcal),
essentially as described previously [26] Throughout the
titrations, the enzyme remained soluble and corrections for
turbidity were not required The absorption vs spectral data
were fitted by using Eqn (2), which describes a two-electron
redox process derived by extension to the Nernst equation
and the Beer–Lambert Law, or Eqn (3), which represents
the sum of two two-electron redox processes Fitting
procedures are described in detail in our previous studies
[26,33]
A¼a10
ðEE 0
1 Þ=59þ b þ c10ðE 0
2 EÞ=59
1þ 10ðEE 0
1 Þ=59þ 10ðE 0
2 EÞ=59 ð2Þ
A¼a10
ðEE 0
1 Þ=59þ b þ c10ðE 0
2 EÞ=59
1þ 10ðEE 0
1 Þ=59þ 10ðE 0
2 EÞ=59
þd10
ðEE 0
3 Þ=59þ e þ f10ðE 0
4 EÞ=59
1þ 10ðEE 0
3 Þ=59þ 10ðE 0
4 EÞ=59 ð3Þ
In these equations, A is the total absorbance; a, b and c are
component absorbance values contributed by one flavin in
the oxidized, semiquinone and reduced states, respectively,
and d, e and f are the corresponding absorbance
compo-nents associated with the second flavin E is the observed
potential in mV; E1¢ and E2¢ are the midpoint potentials for
oxidized/semiquinone and semiquinone/reduced couples,
respectively, for the first flavin, and E3¢ and E4¢ are the
corresponding midpoint potentials for the second flavin
The complexity of the system (4-electron titration with
probable overlap of midpoint potentials) necessitated the
use of a two-stage fitting process Our previous studies using
intact CPRand the FMN and FAD domain of human
CPRidentified isosbestic points for the
oxidized/semi-quinone and semioxidized/semi-quinone/reduced transitions for both the
FAD and FMN flavins at 500 nm and 430 nm,
respect-ively For intact CPR, data fitting at these wavelengths
yielded, within the uncertainty of the measurements, the same midpoint potentials for the redox couple contributing
to the absorbance change whether Eqn (2) or Eqn (3) was used In using Eqn (2), pairs of variables were set as constant and equal prior to fitting Thus, to determine the oxidized/semiquinone couples, b¼ c and e ¼ f (the absorp-tion values for the semiquinone and reduced forms of the first and second flavins, respectively, at the isosbestic point), and to determine the semiquinone/reduced couples, a¼ b and d¼ e Midpoint potential values obtained from the fits
at isosbestic points were then used as starting points to enable accurate fitting of absorption vs reduction potential data for intact CPRat wavelengths near-maximal for the oxidized flavins (455 nm) and for the neutral blue semi-quinone species observed to accumulate during the redox titration (585 nm) The oxidized flavins were assumed to have equal absorbance coefficients, specifically a¼ d This
is known to be the case from previous studies with the individual domains [26] Reduction potentials for the system were determined using A455 and A585 data and Eqn (2) through an iterative process in which midpoint potentials associated with one isosbestic point were held fixed (e.g FMN and FAD oxidized/semiquinone) while the other pair were varied The process was then repeated with the resultant potentials determined for the second pair fixed and the first pair allowed to vary in the second round of analysis This process was repeated iteratively until there was no further change observed Data derived in this way are similar to those originally estimated from the fits at the isosbestic points [26]
Results
Chemical relaxation reactions with NADH-reduced CPR The optical spectrum of CPRreduced by stoichiometric NADH is identical to that obtained with NADPH (Fig 1A), indicating that binding of the coenzyme 2¢-phosphate does not affect the redox potentials of the flavins Furthermore, the optical spectra obtained with nicotinamide coenzymes are very similar to the spectrum of CPRfollowing reduction with stoichiometric sodium dithio-nite [28], indicating that the potential of the flavin couples is the sole determinant of the redox equilibrium within the enzyme under these conditions, and hence of the nature of the equilibrium perturbation following rapid elevation of temperature in a temperature-jump experiment A detailed discussion of the electron distribution prior and subsequent
to capacitor discharge in the temperature-jump instrument for NADPH- and dithionite-reduced enzyme can be found
in our previous publication [28] Briefly, two different two-electron reduced species of CPRare thought to predominate prior to temperature elevation One species contains the high-potential blue semiquinone form of the FMN (FMNox/sq¼)66 mV) and the semiquinone form of FAD (FADox/sq¼)283 mV) The second species contains oxidized FAD and the hydroquinone form of FMN (FMNsq/hq¼)269 mV) These two species are present in approximately equal concentration (Scheme 1), consistent with our measured values for the relevant couples of the FAD and FMN [26] We have shown that perturbation of this equilibrium by a temperature jump leads to further and
Trang 4transient oxidation of the FAD semiquinone form as it transfers an electron to the FMNsqto produce more of the FAD/FMNH2 species This is observed as a net loss of absorbance at 600 nm (i.e loss of blue semiquinone signature) Transient oxidation of the FAD semiquinone shifts the equilibrium towards the FAD/FMNH2 species (Scheme 1) The relaxation process therefore involves electron transfer between the two flavin cofactors (i.e FADsq–FMNsq (q) FADox–FMNhq) With NADPH-reduced CPR, electron transfer occurs with an observed rate, 1/s, of 55 ± 2 s)1[28]
Although the initial equilibrium distribution is identical, the observed rate of internal electron transfer in CPR reduced with NADH (1/s¼ 18 ± 0.7 s)1)is a factor of three less than the corresponding value for NADPH-reduced CPR(1/s¼ 55 ± 0.5 s)1; Fig 1B), and more similar to that seen for dithionite-reduced CPR (11 ± 0.5 s)1) The midpoint redox potential values for the NADPH/NADP+and NADH/NAD+couples are the same ()320 mV, pH 7.0, 30 C), so that the altered rate of interflavin electron transfer is not related to a change in driving force The presence of a phosphate group esterified
at the ribose 2¢ position in the redox-inactive adenosine moiety of NADPH is the only chemical difference between the two coenzymes The observation of a threefold reduc-tion in rate of interflavin electron transfer in NADH-reduced CPRthus suggests a role for coenzyme binding, and in particular for interactions made by the 2¢-phosphate
of NADPH, in modulating interdomain electron transfer in CPR Temperature-jump experiments performed with frag-ments of NADPH were performed to test this hypothesis and are described in the following sections
Binding of adenosine 2¢,5¢-bisphosphate to CPR and effects on interflavin electron transfer
In the crystal structure of rat CPR[7], NADP+appears to
be bound to the enzyme predominantly through inter-actions involving its adenine end, whose binding site is contained within the FAD-binding domain of the protein (Fig 2) The electron density for the NMN portion of the coenzyme is poorly defined, and the position of the nicotinamide ring is conjectured to be different in each of the two independent molecules present in the crystal [7] To investigate any effect of binding 2¢,5¢-ADP on the flavin environment, difference absorption spectra on binding 2¢,5¢-ADP were measured with both the isolated FAD-binding domain and the intact soluble CPRenzyme (see Note) Binding of a stoichiometric amount of 2¢,5¢-ADP to the isolated FAD-binding domain, in either the oxidized form
or the form reduced to the one-electron level with dithionite, leads to clear changes in the absorption spectrum of the FAD, consistent with a perturbation of the cofactor environment (Fig 3) Similar experiments performed with intact CPR(oxidized and two-electron reduced) show that the optical changes induced on binding 2¢,5¢-ADP (Fig 3C,D) are different from those observed in the isolated FAD domain in both oxidized and reduced states This is especially evident for the reduced forms (Fig 3B,D) in the region 500–700 nm Thus, in intact CPRthe binding of 2¢,5¢-ADP to the FAD domain appears to modify not only the environment of the FAD but also that of the FMN
Fig 1 Relaxation transients observed with CPR reduced at the
two-electron level with nicotinamide coenzymes (A and C) Absorption
spectra obtained after reduction of human CPR(140 l M ) with
NADPH (140 l M ) Conditions: 100 m M potassium phosphate buffer,
pH 7.0, 25 C Identical spectral changes to those shown in (A) were
also observed on adding 140 l M NADH to human CPR (B)
Tem-perature-jump difference absorption transients obtained for CPR
reduced at the two-electron level with NADPH (upper transient) and
NADH (lower transient) Conditions: thermostat temperature, 20 C;
temperature-jump, +7 C; voltage discharge, 12.5 kV; 140 l M CPR,
100 m M potassium phosphate buffer, pH 7.0 For the difference
absorption transient measured at 450 nm for CPRreduced with
NADPH; 1/s ¼ 55 ± 2 s)1 For the difference absorption transient
1/s ¼ 18 ± 0.7 s)1 Difference absorption transients were generated
by subtracting the transient obtained for the oxidized enzyme from
that obtained for reduced CPRas described previously [28].
Trang 5cofactor This structural perturbation transmitted between
the two domains may play a role in the kinetics of internal
electron transfer (see below)
Titration with 2¢,5¢-ADP at protein levels sufficient for
good spectral signals gave linear plots, implying that the Kd
is quite low Thus, it was necessary to measure the inhibition
constant for 2¢,5¢-ADP in steady-state CPR-catalysed
reactions as a guide to its strength of binding to CPR
Steady-state kinetic studies with cytochrome c as electron
acceptor demonstrated that 2¢,5¢-ADP is a competitive
inhibitor with respect to NADPH The Km,appfor NADPH
increases as the 2¢,5¢-ADP concentration is increased,
without effect on Vmax,app (Fig 4) The value of the
inhibition constant for 2¢,5¢-ADP (K ¼ 5.4 ± 1.3 lM) is,
within error, identical to the Km,app for NADPH (6.7 ± 1.3 lM), suggesting that this moiety is the major determinant of coenzyme binding to CPR
The rate of interflavin electron transfer for CPRreduced
at the two-electron level by dithionite (1/s¼ 11 ± 0.5 s)1)
is increased approximately threefold to 35 ± 0.2 s)1in the presence of a stoichiometric amount of 2¢,5¢-ADP, and this
is accompanied by an increase in the amplitude of the absorbance change This is consistent with the notion that the 2¢,5¢-ADP moiety of NADPH makes a major contribu-tion to the enhanced rate of internal electron transfer in NADPH-reduced CPRas compared to the dithionite-reduced enzyme The absence of a comparable rate enhancement in NADH-reduced CPRsuggests that the 2¢-phosphate of NADPH is a major factor in increasing the rate of interdomain electron transfer It should be noted, however, that the binding of 2¢,5¢-ADP to dithionite-reduced enzyme does not lead to as fast an electron transfer rate as that observed in NADPH-reduced samples (35 ± 0.2 s)1vs 55 ± 0.5 s)1) This suggests that other interactions (perhaps with the pyrophosphate bond) can also contribute to the enhancement of the rate of interflavin electron transfer
Similar temperature-jump experiments performed with 2¢-AMP are also consistent with this conclusion The observed rate of interflavin electron transfer obtained with dithionite-reduced CPRin the presence of either 140 lMor
1800 lM 2¢-AMP (Ki 2¢-AMP¼ 180 ± 11 lM) is identical, within experimental uncertainty, to that obtained with 2¢,5¢-ADP (32 ± 0.6 s)1and 35 ± 0.2 s)1, respectively) Spectroelectrochemical titration
Any perturbation in the redox potential of the relevant flavin couples on binding 2¢,5¢-ADP might account for the observed increase in the rate of internal electron transfer, and perhaps for the changes in the flavin absorption spectra
We have therefore determined the redox potentials of CPR
in the absence and presence of 2¢,5¢-ADP Electrochemical titration of CPRwith dithionite has revealed that each of the four flavin couples in CPRcan be distinguished and their reduction potentials determined [26] Spectral changes accompanying a typical titration are shown in Fig 5A and plots of absorbance at 455 nm vs potential in Fig 5B; in these experiments CPRconcentration was 60 lM and 2¢,5¢-ADP was present in equimolar amounts The oxidized enzyme has visible absorption maxima at 455 nm and
381 nm, with the semiquinone species having a maximum at
585 nm Redox titrations performed in the presence of 2¢,5¢-ADP exhibited similar spectral changes to those observed in its absence
Plots of absorption vs potential data for CPRin the presence and absence of 2¢,5¢-ADP were essentially identical (Fig 5B), indicating that there was negligible difference in redox behaviour of the flavins in the presence of this ligand While the midpoint reduction potential values for three of the four couples, estimated as described in Experimental procedures, are slightly more positive than those reported previously, all values are within error of the previously reported data set [26] The redox potentials were not altered
to any significant extent by addition of 2¢,5¢-ADP Thus, the midpoint potentials for both the 2¢,5¢-ADP-bound and
Fig 2 The crystal structure of NADP 9
-bound rat CPR in its oxidized form [7] (A) The overall polypeptide fold The different structural
domains are indicated The FAD-domain contains the binding site for
NADPH and is related to the FNRfamily of flavoproteins For clarity,
the NADPH-binding subdomain is also indicated (B) The
coenzyme-binding site The main residues known to interact with the adenosine
2¢-phosphate group are shown (R298, R597 and K602) W677 (W676
in human CPR), which shields the Re-face of the FAD isoalloxazine
ring position, is also shown The nicotinamide moiety of NADP(H) is
highly disordered in the crystal.
Trang 6ligand-free forms of CPRare)76 ± 5 mV (FMN ox/sq);
)248 ± 10 mV (FMN sq/hq); )260 ± 12 mV (FAD ox/
sq) and)361 ± 7 mV (FAD sq/hq) Our findings contrast
with published work on cytochrome P450 BM3, where
NADP(H) binding to the reductase domain is thought to
induce significant changes in the reduction potentials of the
FAD [34] We conclude that in the case of CPR the observed effects of the binding of coenzyme and coenzyme fragments in increasing the rates of electron transfer result largely from structural perturbations that affect the rate-limiting step(s) for electron transfer) probably inter– domain interactions) without altering the driving force
Effects of binding adenosine 2¢,5¢-bisphosphate
on hydride ion transfer
In previous work with CPRand the isolated FAD-domain we provided kinetic evidence for the existence of
a second kinetically distinct noncatalytic NADPH-bind-ing site in the enzyme, occupation of which led to a
two-to fivefold decrease in the rate of hydride ion transfer [24,25] Similar observations have subsequently been made with the structurally related enzymes neuronal nitric oxide synthase [35] and the adrenodoxin reductase homologue FprA from Mycobacterium tuberculosis [36]
A general model has emerged from these kinetic obser-vations in which occupation of this second site by NADPH hinders the release of NADP+ Because electron transfer is reversible, hydride transfer leads to
an equilibrium distribution of enzyme species comprising oxidized enzyme, oxidized enzyme bound to NADPH, and reduced enzyme bound to NADP+ R elease of NADP+from the latter species will lead to further flavin reduction as the equilibrium distribution is shifted toward reduced enzyme (see [24,35] for a more detailed discus-sion) Thus, if the binding of NADPH to the noncata-lytic site hinders NADP+ release from the catalytic site, the observed rate of FAD reduction will be decreased at
Fig 3 Difference absorption spectra for the binding of 2¢,5¢-ADP to the isolated FAD-domain and CPR (A) FAD-domain Difference absorption spectra (spectrum in the presence of 100 l M 2¢,5¢-ADP minus spectrum in the absence of 2¢,5¢-ADP) of oxidized FAD-domain (100 l M ) (B) As for panel A, but for the FAD-domain (100 l M ) reduced at the one-electron level (blue semiquinone species) with sodium dithionite (C) CPR Difference absorption spectra (spectrum in the presence of 50 l M 2¢,5¢-ADP minus spectrum in the absence of 2¢,5¢-ADP) of oxidized CPR (50 l M ) (D) As for (C) but for CPR(50 l M ) reduced at the two-electron level (blue disemiquinone species) with sodium dithionite.
Fig 4 The inhibition of cytochrome c reductase activity by 2¢,5¢-ADP.
Reaction mixtures contained 7 n M CPR, 50 m M potassium phosphate
buffer, pH 7.0, 50 l M cytochrome c and various concentrations of
NADPH and of the inhibitor 2¢,5¢-ADP The assay temperature was
25 C and the reaction rate was measured by reduction of
cyto-chrome c 3+
Five different concentrations of 2¢,5¢-ADP were used: 5,
10, 20, 40 and 80 l M corresponding to curves a, b, c, d and e,
respectively.
Trang 7high NADPH concentrations, and this is in fact observed
[24,25] By contrast, in stopped-flow experiments
per-formed with NADH a conventional saturable increase in
the rate of flavin reduction with increasing NADH
concentration is seen (Fig 6B) This indicates that
NADH does not bind to the second coenzyme-binding
site and/or that NAD+ dissociates too rapidly to limit
the rate of hydride transfer In light of the major
importance of the 2¢-phosphate in coenzyme binding, the
latter seems a likely explanation
In rapid-mixing stopped-flow experiments, preincubation
of the isolated FAD-domain with stoichiometric 2¢,5¢-ADP
eliminates this inhibitory effect observed at high NADPH
concentration, presumably by preventing the simultaneous
binding of two NADPH molecules (Fig 6A and inset) In
temperature-jump experiments with dithionite-reduced
CPRin the presence of a 10-fold molar excess of
2¢,5¢-ADP, the observed rate of interflavin electron transfer
(1/s¼ 28 ± 0.5 s)1) is only slightly less than that observed
with stoichiometric 2¢,5¢-ADP (1/s ¼ 35 ± 0.2 s)1)
Com-bined, the stopped-flow and temperature-jump data indicate
that increasing the concentration of 2¢,5¢-ADP above
stoichiometric levels does not have any substantial addi-tional effect on the rate of internal electron transfer These results suggest that the observed inhibitory effect of high NADPH concentrations on the reductive half-reaction is associated with the nicotinamide moiety of the coenzyme The role of W676 in interdomain electron transfer Our previous studies have shown that microsecond tem-perature perturbation of CPRreduced at the two-electron level with NADPH yields two different relaxation processes [28] The initial fast relaxation (1/s¼ 2200 ± 300 s)1) is not associated with electron transfer, but is attributed to local conformational changes in the vicinity of the cofactors and induced by NADPH binding [28] It is not observed in the W676H mutant of CPR[28], suggesting that these changes may involve W676, a residue that stacks against the Re-face of the isoalloxazine ring of FAD in CPR(Fig 1B) and which is involved in a series of conformational changes associated with the hydride transfer step [25] This fast relaxation process is not observed in temperature-jump experiments with CPRreduced by NADH, nor in
Fig 5 Redox potentiometry studies (A) Spectral changes during redox titration of human CPR Human CPR (60 l M ) was titrated electrochemically as described in Experimental procedures Progressive reduc-tion of the enzyme leads to bleaching of the oxidized flavin spectrum and accumulation of neutral blue flavin semiquinones with absorption maximum at 585 nm Positions of isosbestic points in the titration are indicated
at 501 nm (oxidized/semiquinone couple for both flavins) and at 435 nm (semiquinone/ reduced couple) (B) Absorption vs potential data for 2¢,5¢-ADP-bound and ligand-free forms of human CPR Plots of absorption data at 455 nm (at the flavin maximum for the oxidized CPR) vs applied potential are shown for both ligand-free (h) and 2¢,5¢-ADP-bound (d) forms of human CPR Enzyme concen-trations in all ticoncen-trations were 60 l M
Trang 8experiments with dithionite-reduced CPRincubated with a
stoichiometric amount of 2¢,5¢-ADP That only NADPH
(and not NADH, or 2¢,5¢-ADP) can elicit this rapid
structural relaxation indicates additional complexity in
the interaction of coenzymes with CPR This suggests that
the nicotinamide ring of NAD+interacts differently with
the enzyme than that of NADPH or that NAD+is bound
weakly to reduced CPR
It is possible that the conformational changes produced
by occupation of the 2¢-phosphate binding site that increase
the rate of electron transfer also affect the nicotinamide ring
binding site and W676 Stopped-flow tryptophan
fluores-cence experiments indicated a rapid but small increase (complete in about 2 ms after the mixing event; instrument dead-time 1 ms) in fluorescence emission on mixing oxidized CPR(10 lM) with a stoichiometric amount of 2¢,5¢-ADP (Fig 7A) However a similar transient is observed in control experiments involving rapid mixing of
Fig 6 Stopped-flow kinetic studies of flavin reduction in the presence of
2¢,5¢-ADP (A) Fluorescence stopped-flow transients (excitation at
340 nm; emission at 450 nm) for NADPH oxidation by the
FAD-domain Conditions: 50 m M potassium phosphate buffer, pH 7.0,
25 C, 10 l M FAD-domain, 200 l M NADPH Transient a:
mono-phasic fluorescence transient (k obs , of 3.2 s)1) obtained for NADPH
oxidation by the FAD-domain Transient b: fluorescence transient
obtained for NADPH oxidation by the FAD-domain preincubated
with a stoichiometric amount of 2¢,5¢-ADP (10 l M ) Data are best
described by a double-exponential expression, yielding values k obs1
and k obs2 of 26 s)1and 2.5 s)1, respectively Inset: Dependence of the
observed rates on NADPH concentration s, n, Estimated values for
k obs1 and k obs2 , respectively, for FAD-domain preincubated with
stoichiometric 2¢,5¢-ADP d, Estimated values for k obs in the
absence of 2¢,5¢-ADP preincubation Note the overlapping of n (k obs2 )
and d (k obs ) (B) NADH concentration dependence of the rate of
flavin reduction in intact CPR Absorption stopped-flow experiments.
Reaction monitored at 450 nm Conditions: 50 m M potassium
phos-phate buffer, pH 7.0, 25 C, 10 l M CPR.
Fig 7 Stopped-flow tryptophan fluorescence studies with oxidized CPR and 2¢,5¢-ADP Conditions: 10 l M CPR, 10 l M 2¢,5¢-ADP, 50 m M
potassium phosphate buffer pH 7.0, 25 C Excitation wavelength
295 nm (A) Transient observed upon rapid-mixing of oxidized CPR and 2¢,5¢-ADP (B) Control experiment Fluorescence change observed when CPRwas mixed against buffer alone The transients observed in (A) and (B) are most likely to originate from fast pressure-pulse pro-pagation after the initial mixing event (apparatus dead time 1 ms) (C) Transient a: same as in (B) Transient b: fluorescence kinetic transient observed during reduction of CPRwith 10-fold excess NADPH (100 l M ) Mutagenesis studies demonstrated that these fluorescence fluctuations are related to changes in the environment of W676 (see Fig 2), accompanying hydride transfer from the nicotind-amide nucleotide to the FAD cofactor [25].
Trang 9CPRagainst buffer alone (Fig 7B), ruling out any specific
effect on the conformation of W676 induced by the binding
of 2¢,5¢-ADP Thus, although coenzyme binding is driven
primarily by interaction of the 2¢,5¢-ADP moiety with CPR,
the larger tryptophan fluorescence changes on mixing
NADPH with CPRin stopped-flow experiments ([24,25];
Fig 7C), attributed to environmental effects on W676,
must derive from additional interactions of the coenzyme
We have previously rationalized the lack of a tryptophan
fluorescence change observed in temperature-jump
experi-ments with NADPH by suggesting that the equilibrium
position of NADP+in the two-electron reduced enzyme is
that observed in the reported crystal structure (i.e the
nicotinamide ring is not tightly associated with the enzyme
and is distant from the isoalloxazine ring) [28]
Discussion
Our previous temperature-jump studies with human CPR
highlighted the importance of conformational change in
limiting the rate of internal electron transfer and pointed to
the role of the coenzyme in enhancing reaction rate [28] We
have now extended our temperature-jump studies to identify
key interactions that are responsible for modulating the rate
of internal electron transfer between the two domains of this
enzyme We have demonstrated that the role of the
2¢-phosphate of NADPH is to optimize electron transfer
between the flavin cofactors Occupation of the
2¢-phos-phate binding site by NADPH, 2¢,5¢-ADP or 2¢-AMP leads
to altered rates of conformational change These
conform-ational changes are transmitted over a relatively large
distance through the protein to optimize electron transfer
between the flavins In the case of 2¢,5¢-ADP binding, optical
spectroscopy provides further evidence for a long-range
perturbation of the environments of the isoalloxazine rings
of the flavins This conformational change appears to
involve significant domain movement, as it is sensitive to the
effects of solution viscosity [28] Reduction of the enzyme
with NADH leads to a slower rate of internal electron
transfer owing to the absence of the phosphate group in the
2¢-phosphate-binding site However, electron transfer in the
NADH reduced enzyme is faster than in dithionite-reduced
CPR, indicating that other interactions made by the
coenzyme play some role in optimizing electron transfer
between the flavins This is further suggested by the fact that
the observed rate of internal electron transfer in the presence
of 2¢,5¢-ADP and 2¢-AMP for dithionite-reduced enzyme is
increased to 60% the value seen for NADPH-reduced
enzyme
In agreement with our kinetic work on human CPR,
crystallographic studies of mutant forms of rat CPRalso
indicate a role for conformational change and domain
re-orientation [30] The multidomain structure of CPRis
highly flexible in solution The crystal structure strongly
suggests that the so-called linker domain plays a key role in
controlling the mutual orientation of the two
flavin-containing domains Our kinetic [24] and NMRstudies
(B Hawkins, I Barsukov, L.-Y Lian, G C K Roberts,
unpublished data) indicate that the two isolated
flavin-binding domains do not form a strong complex in solution,
and electron transfer between the isolated domains is much
slower than in the intact enzyme, with a second-order rate
constant of 9.5· 104
M )1Æs)1[24] By tethering the domains with the linker region the electron transfer rate is optimized, but more importantly new opportunities are presented for regulating the rate of interflavin electron transfer by coenzyme binding to the NADPH/FAD domain
Multiple conformations of CPRin the absence of coenzyme have been suggested from earlier steady-state studies with the rat enzyme [37] It was rationalized that binding of NADPH would then induce conformational change and that a unique conformation would be populated compatible with hydride transfer Our use of equilibrium-perturbation methods with CPRhas now provided evidence that coenzyme binding energy is utilized to optimize reaction steps other than the initial hydride transfer event Given the conserved multidomain structure of other mem-bers of the diflavin reductase family, it seems reasonable to propose that conformational gating of electron transfer may also occur in these enzymes The temperature-jump method may be useful for probing these aspects in related enzymes
is currently in hand
Acknowledgements
We thank C Baxter and Dr Andrew Westlake for assistance in preparing Fig 2 The work was funded by the Medical Research Council, the Wellcome Trust and the Lister Institute of Preventive Medicine N.S.S is a Lister Institute Research Professor.
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