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Electrochemistry Communications 8 (2006) 1845–1849

Direct electrochemistry of human and rat NADPH cytochromeP450 reductase

Alka Shukla a, Elizabeth M.J. Gillam a, Paul V. Bernhardt b,*

a Centre for Metals in Biology, School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australiab Centre for Metals in Biology, Department of Chemistry, University of Queensland, Brisbane 4072, Australia

Received 19 July 2006; received in revised form 10 August 2006; accepted 10 August 2006Available online 15 September 2006

Abstract

The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cyto-chrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefullyorchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function,no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purifiedhuman CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltam-metry. For human CPR redox responses at �2 and �278 mV (with a ratio of 1e�:3e�) vs NHE were seen at pH 7.9 while the potentialsfor rat CPR at pH 8.0 were �20 and �254 mV. All redox responses exhibit a pH dependence of approximately �59 mV/pH unitconsistent with proton coupled electron transfer reactions of equal stoichiometry.� 2006 Elsevier B.V. All rights reserved.

Keywords: Cytochrome P450 reductase; Protein; Voltammetry

1. Introduction

Cytochrome P450s (P450s) are heme containingenzymes that play a major role in the metabolism of xeno-biotics [1]. Most P450s catalyze a 2-electron oxidation reac-tion of an organic substrate coupled to O-transfer (Eq. (1)).Dioxygen and the substrate (RH) bind at the heme activesite and electrons from NADPH act to cleave the O–Obond leaving a high valent oxo-heme moiety that is capableof O-insertion into an otherwise inert C–H bond [1].

NADPH + RH + O2 + Hþ!NADPþ+ ROH + H2O

ð1ÞDelivery of the two electrons from NADPH to the P450

must be carefully orchestrated in order to avoid so-calleduncoupling i.e., the futile catalytic reduction of dioxygento superoxide, hydrogen peroxide or water without sub-

1388-2481/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.elecom.2006.08.020

* Corresponding author. Tel.: +61 7 3365 4266; fax: +61 7 3365 4299.E-mail address: P.Bernhardt@uq.edu.au (P.V. Bernhardt).

strate turnover. Microsomal P450s require the NADPHcytochrome P450 reductase (CPR) as a relay, which acceptselectrons from NADPH and transfers them sequentially tothe P450.

CPR is a rare example of a mammalian protein contain-ing both flavin adenine dinucleotide (FAD) and flavinmononucleotide (FMN) cofactors within a single polypep-tide chain [2]. The FMN domain has strong resemblance tobacterial flavodoxins whereas the FAD domain is relatedto ferredoxin NADP+-reductase. It has been proposed thatCPR and other FMN/FAD containing proteins haveevolved as a fusion of two ancestral genes [3]. CPRs froma number of eukaryotic organisms (including humans)are highly homologous [4]. Crystal structures of wild typeand mutant forms of CPR from rat liver have been solved[5,6]. These structures reveal that NADP+ binds at theFAD site. Electrons are passed sequentially to the adjacentFMN cofactor (�5 A away) and then on to any of a num-ber of P450s; a single form of CPR serves all microsomalP450s [1,2].

1846 A. Shukla et al. / Electrochemistry Communications 8 (2006) 1845–1849

To facilitate isolation and solution studies, the hydro-phobic amino terminal domain of CPR may be cleavedby treating the enzyme with pancreatic steapsin or trypsin,releasing the soluble C-terminal 72-kDa hydrophilicdomain. This soluble CPR contains both the FAD andFMN cofactors and is catalytically active with many elec-tron acceptors (such as cytochrome c), but it does notreduce cytochromes P450 in its modified form [7].

Potentiometric studies on human [8] and rabbit liver [9]CPR have been reported, where the redox potentials of theFAD and FMN cofactors were determined. Redox proper-ties of the closely related methionine synthase reductase(MSR) [10], nitric oxide synthase (NOS) [11] and humannovel reductase 1 (NR1) [12] have also been determinedrecently by potentiometric titrations. A feature of CPR,and its related di-flavoproteins, is a capacity to stabilizesingle-electron reduced (‘semiquinone’) forms of both itsFAD and FMN cofactors. This is in contrast to the freeFMN and FAD cofactors which undergo obligate cooper-ative 2-electron transfer reactions.

In the case of CPR, four redox couples (FADox/FADsq,FADsq/FADred, FMNox/FMNsq and FMNsq/FMNred)may be resolved potentiometrically. In human and rabbitCPR the FMNox/FMNsq couple is found at a potentialalmost 200 mV positive of the other three redox couples.Indeed the singly reduced form of CPR (FMNsq:FADox)is the resting state of the enzyme.

Given its importance in P450 catalysis, it is perhaps sur-prising that there has been very little work on the electro-chemistry of any CPR. Recently a report of the cyclicvoltammetry of insect cell microsomes containing recombi-nant human P450s 1A2 and 3A4 (comprising a mixture ofCPR, lipid, cytochromes P450 and other membrane pro-teins) has appeared [13]. A single low potential voltammet-ric response (�250 mV vs NHE, pH 7.4) was reported,which was assigned to that of CPR and not due to anyP450s present or other proteins in the mixture. A similarresponse was seen in the voltammetry of isolated rabbitCPR. No mention was made of the absent high potentialFMNox/FMNsq response of CPR, which apparently wasnot observed [13]. Herein we report the first direct electro-chemical study of isolated recombinant human CPR and aparallel study with recombinant rat CPR. We have identi-fied both low and high potential responses that for the firsttime unequivocally show that CPR may undergo electro-chemically driven redox reactions. This is a first importantstep towards the construction of direct electrochemicallydriven, reconstituted CPR/P450 systems that may leadtoward a protocol for rapid screening of P450 metaboliteson the basis of electrochemical detection.

2. Materials and methods

2.1. Materials

2 0AMP (2 0 adenosine monophosphate), FMN and aden-osine were obtained from Sigma Chemical Company. Cel-

lulose ester dialysis membrane (MWCO: 8000) wasobtained from Spectrum Laboratories (Los Angeles, CA,USA). Aprotonin was obtained from Boehringer Mann-heim Australia Pty Ltd. (Castle Hill, NSW, Australia).Bactotryptone, yeast extract and bactopeptone were pur-chased from Difco (Detroit, MI, USA), Ampicillin, chlor-amphenicol and isopropyl b-D-thiogalatopyranoside(IPTG) were obtained from Progen Industries Pty Ltd.(Darra, Qld, Australia). All other reagents were obtainedfrom local suppliers at the highest quality commerciallyavailable.

2.2. Protein expression and purification

Human and rat CPR expression was performed usingthe expression construct pCW/hNPR and pOR263 inDH5aF’IQe strain of E. coli (Invitrogen, Life Technolo-gies, Carlsbad, USA) cotransformed with the chaperoneexpression plasmid pGro7 [14] using the method of Inoueet al. [15]. The same procedure was used for the expressionand purification of human and rat CPR. The rat CPR waspurified by Rebecca Wunsch. Expression was done accord-ing to established procedures [16].

Spheroplasts were resuspended in 50 mM Tris/HCl buf-fer pH 8.0, containing 5 mM EDTA, 20% glycerol and pro-tease inhibitors (0.1 mM PMSF, 10 lg/mL aprotinin and2 lM leupeptin), then sonicated in salt/ice bath, and centri-fuged at 10,000g for 40 min at 4 �C. The resultant superna-tant was centrifuged at 105,000g for 65 min at 4 �C tocollect membranes which were then solubilized in�120 mL 100 mM Tris/HCl pH 7.7, containing 1 mMEDTA, 20% glycerol, 0.1% Tergitol, 0.6% CHAPS, 1 lMFMN, 1 mM DTT, 0.1 mM PMSF, and 10 lg/ml aprotininwith gentle stirring at 4 �C for 2–3 h.

The solubilized membrane fraction was centrifuged at4 �C for 65 min at 105,000g. The supernatant was adjustedto 0.3 M KCl and then loaded onto an ADP Sepharose col-umn pre-equilibrated with 50 mM potassium phosphatebuffer pH 7.7, containing 300 mM KCl, 20% w/v glycerol,0.1 mM EDTA, 0.35% w/v cholate, 1 lM FMN, 0.2 mMDTT, and 0.4 mM PMSF. The column was washed with�100 mL 50 mM potassium phosphate buffer pH 7.7, con-taining 20% w/v glycerol, 0.35% cholate, 5 mM adenosine,1 lM FMN, 0.2 mM DTT, 0.4 mM PMSF. The columnwas eluted with 50 mM potassium phosphate buffer pH7.7, containing 20% glycerol, 0.1 mM EDTA, 0.35% cho-late, 1 lM FMN, 5 mM 2 0AMP, and 0.4 mM PMSF and�2–5 mL colored fractions were collected. The reductasewas dialyzed against two changes (1 L and �12 h each)of 10 mM potassium phosphate buffer pH 7.7, containing20% w/v glycerol, 0.35% w/v cholate, 1 lM FMN, and0.4 mM PMSF.

The post-dialysis sample was loaded onto hydroxyapa-tite column equilibrated with 10 mM potassium phosphatebuffer pH 7.7, containing 20% w/v glycerol, 0.35% w/vcholate, 0.1 mM DTT, 0.1 mM PMSF and washed with�250 mL 10 mM potassium phosphate buffer pH 7.7,

E (mV vs NHE)

-400 -300 -200 -100 0 100

CV (background subtracted)

400 nA

SWV (offset)

200 nA

Fig. 1. Background subtracted cyclic voltammogram of human CPR (redcurve, bottom). Sweep rate 20 mV s�1, pH 7.9. Square wave voltammo-gram also shown (blue curve, top) square wave amplitude 5 mV, frequency8 Hz. Note different current scales.

A. Shukla et al. / Electrochemistry Communications 8 (2006) 1845–1849 1847

containing 20% w/v glycerol, 0.1 mM DTT, and 0.1 mMPMSF. Elution was performed with 300 mM potassiumphosphate buffer pH 7.7, 20% w/v glycerol, 0.1 mMEDTA, and 0.1 mM PMSF, and �1 mL fractions contain-ing reductase were pooled and dialyzed against 2–4changes of 1 L each of 10 mM Tris acetate pH 7.4, con-taining 1 mM EDTA, and 20% w/v glycerol. The concen-tration of human (6.9 lM) and rat CPR (51 lM) wasdetermined from its activity towards cytochrome c usinga specific activity of 3200 nmol cytochrome c reduced/min/nmol [17].

2.3. Electrochemistry

Cyclic and square wave voltammetry were performed ona BAS 100B/W analyzer employing an edge-plane pyrolyticgraphite (EPG) electrode (modified as described below), aPt wire counter and Ag/AgCl (3 M KCl) reference elec-trode (Eo +196 mV vs NHE). All measurements were car-ried out in a glove box (Belle Technology) with oxygenlevel below 10 ppm and at 25 �C. The electrochemical cellcontained 0.4 mL of 50 mM of bis-tris propane and 2-amino-2-methylpropane hydrochloride buffer (adjusted tothe desired pH with AcOH or NaOH) in addition to10 mM NaCl as electrolyte. The EPG electrode wascleaned by cleaving several 2 lm layers from the surfaceusing a microtome then the electrode was thoroughlyrinsed with water, sonicated twice and allowed to dry atroom temperature. Purified human or rat CPR (5 lL)was mixed with 5 lL of an aqueous didodecyldimethylam-monium bromide (DDAB) solution (5 mM) and then castonto the working electrode surface and the film wasallowed to set overnight at 4 �C. Baseline subtractions wereperformed with either the program UTILS [18], which waskindly provided by the author, or the BAS100W software(vers. 3.2).

3. Results and discussion

Cyclic voltammetry of both purified recombinanthuman and rat CPR confined to an edge-plane pyrolyticgraphite electrode within a didodecyldimethylammoniumbromide (DDAB) surfactant film yielded two well sepa-rated pairs of anodic and cathodic redox responses. Thevoltammogram for human CPR in Fig. 1 at pH 7.9 (redcurve) has the background capacitive current removedfor clarity. The square wave voltammogram at the samepH is also shown (Fig. 1 blue curve) where peaks at �2and �278 mV vs NHE are resolved.

The anodic and cathodic current maxima exhibit a lin-ear dependence on sweep rate which identifies the responsesas due to a surface adsorbed molecule. The higher potentialanodic and cathodic peaks have a width of 110 mV at halfheight and are consistent with a single-electron reaction(theoretically 90/n mV at 25 �C) [19]. Integration of thepeak (after correcting for charging current) gives a chargeof 1.6 nC (840 fmol of electroactive protein). The lower

potential response has a peak width at half height of70 mV and as such is consistent with a convoluted multielectron wave. Integration of this peak yields a charge of4.6 nC; ca. 3 times that of the higher potential response.Similar cyclic voltammetry results were obtained for ratCPR. Integration of the voltammetric peaks gave, oncemore, a ratio of 3:1 for the charge passed in the low andhigh potential peaks and an electroactive coverage of570 fmol rat CPR.

Most importantly, we observed high potential redoxresponses (ca. 150–200 mV higher than the response atca. 0 V in Fig. 1) that were due to surface oxidation pro-cesses on the EPG electrode as described by Evans andKuwana [20]. These responses fall well outside the poten-tial range shown in Fig. 1 are of no consequence to thepresent study.

For both human and rat CPR pH dependences of about�59 mV/pH unit were identified for their high (1 electron)and low potential (3 electron) responses (Fig. 2), which isconsistent with an overall stoichiometry of 1e�/1 H+ (highpotential) and 3e�/3H+ (low potential) transferred duringthe sweep within the range 6 < pH < 9. Redox potentio-metric studies of the FMN-dependent flavodoxins [21–24]have found that the high potential FMNox/FMNsq coupletypically exhibits a dependence of �59 mV/pH unit with nopKa i.e., the FMNsq form is protonated at all accessible pHvalues. The lower potential couple of flavodoxins oftenexhibits a pH dependence below about pH 7, where theFMNred form binds an additional proton. There is greatervariation in the redox potentials of FAD-dependent pro-teins such as the ferredoxin reductases with some beingable to stabilize a semiquinone [25] while others do not [26].

The redox potentials of human CPR as determined byredox potentiometry at pH 7.0 [8] were �66 mV (FMNox/FMNsq), �269 mV (FMNsq/FMNred), �283 mV (FADox/FADsq) and �382 mV (FADsq/FADred). Our voltammetric

wavelength (nm)

400 450 500 550 600 650

abs.

(ar

bitr

ary

scal

e)

0

Human CPR (DDAB film)Rat CPR (DDAB film)Human CPR (in solution)DDAB film (no protein)

(a)

(b)

(c)

(d)

Fig. 3. Visible spectra of (a) human CPR in a DDAB film (cast on a glassslide); (b) rat CPR in a DDAB film (cast on a glass slide); (c) human CPRin solution and (d) a DDAB film cast on a glass slide with no protein. Theabsorption scale of each spectrum has been normalised to correct forvariations in protein concentrations and film thicknesses and also offsetfor clarity.

pH6 7 8 9

E (

mV

vs

NH

E)

-400

-300

-200

-100

0

100

200E1 human CPRE2 human CPRE1 rat CPRE2 rat CPR

m

Fig. 2. pH dependence of the redox potentials of human (circles andsquares) and rat (triangles) CPR as determined by square wave voltam-metry (square wave amplitude 5 mV, frequency 8 Hz). The high potential(E1) and low potential (E2) responses are indicated.

1848 A. Shukla et al. / Electrochemistry Communications 8 (2006) 1845–1849

data at the same pH are +58 mV (FMNox/FMNsq) and�228 mV for the three remaining couples. The pH depen-dences of the redox potentials of human or rat CPR havenot been reported before. Regardless of pH, we were unableto resolve the lower potential 3-electron response into morethan one component.

The anodic shift in redox potentials seen here relative tothose reported from solution potentiometric redox titra-tions may be a consequence of the surfactant used in immo-bilizing the protein to the electrode surface. We [27] andothers [28] have noted similar anodic shifts in the voltam-metry of P450s relative to their potentials determined insolution using redox potentiometry.

Voltammetry of the individual protein-free FAD andFMN cofactors was conducted under identical experimen-tal conditions as those employed for the CPR holoprotein.Single, reversible, 2-electron voltammetric waves were seenat �280 mV (pH 8) for both FAD and FMN. This is con-sistent with previous studies of these compounds [29–33].Coincidentally these waves fall in a similar region as thelow potential couple of both human and rat CPR. How-ever, the free flavins lack the high potential, single electronwave of both CPR proteins as they cannot benefit fromspecific hydrogen bonding interactions within the proteinthat stabilize the semiquinone form of the FMN cofactorin particular at close to neutral pH [34].

In order to examine whether any major structuralchanges might have occurred upon immobilizing the pro-tein in a DDAB surfactant film, we measured the electronicspectra of both human and rat CPR within a DDAB filmcast onto a glass slide. The results are shown in Fig. 3 incomparison with the solution spectrum of human CPR(Fig. 3c), which is essentially indistinguishable from thatof rat CPR in solution (not shown). The spectrum of theblank DDAB film is also given for comparison (Fig. 3d).

The most prominent electronic maxima apparent in thesolution spectrum of human CPR emerge at 380 nm,450 nm and a very broad peak around 600 nm. The twohigher energy maxima are associated with the fully oxi-dized flavin chromophore (FMN or FAD) while the lowenergy maximum is from the flavin semiquinone [8]. Thehuman CPR sample used here is evidently already in a par-tially reduced form (Fig. 3c). The DDAB film spectra ofhuman (Fig. 3a) and rat (Fig. 3b) CPR exhibit the samefeatures as the solution spectrum although the quality ofthe spectra are affected by a sloping baseline (opaque films)and inherently small optical density changes due to the thinfilms (short path lengths). Nevertheless, there is no evi-dence to suggest that the protein has been altered by immo-bilization in the DDAB film.

4. Conclusions

Having established that direct electron transfer is possi-ble between either human or rat CPR and a working elec-trode, future studies will focus on using these CPRs as anelectron bridge between the electrode and human P450sunder turnover conditions i.e., in air and in the presenceof substrate. It is our hope that the use of a natural electronpartner will lead to tighter coupling of the electrochemi-cally driven catalytic reaction and greater overall efficiencythan seen in previous work using artificial mediators or noelectron relay to the heme active site.

Acknowledgments

We wish to acknowledge the help of Dr. Pavel Soucek inthe purification of human reductase and of Rebecca

A. Shukla et al. / Electrochemistry Communications 8 (2006) 1845–1849 1849

Wunsch in purifying the rat reductase. Financial support ofthe Australian Research Council and an Australia AsiaAward for India (to AS) is gratefully acknowledged.

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