Cytochrome P450 biosensors—a review

Biosensors and Bioelectronics 20 (2005) 2408–2423

Review

Cytochrome P450 biosensors—a review

Nikitas Bistolasa, Ulla Wollenbergera, Christiane Jungb, Frieder W. Schellera,∗a Department of Analytical Biochemistry, University of Potsdam, Karl-Liebknecht-Street 24-25, 14476 Golm, Germany

b Max-Delbruck-Center for Molecular Medicine, Protein Dynamics Laboratory, Robert-R¨ossle-Strasse 10, 13125 Berlin, Germany

Received 25 August 2004; received in revised form 10 November 2004; accepted 10 November 2004Available online 5 January 2005

Abstract

Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation oftheir structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970sattempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electrontransfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigationof the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of
substrates important in environmental monitoring, pharmaceutical industry and clinical practice.© 2004 Elsevier B.V. All rights reserved.
Keywords:Cytochrome P450; Bioelectrocatalysis; Electrochemistry; Modified electrodes

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2409

2. The reaction cycle of CYP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2414

3. Protein electrochemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2414

4. Direct electron transfer of CYP in electrochemical sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24154.1. Bare electrodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24154.2. Clay modified electrodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24164.3. Phospholipid modified electrodes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24174.4. Electrodes modified with multilayer films. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24184.5. CYP bionsensing at high temperatures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24194.6. Spectroelectrochemistry and surface plasmon resonance of CYP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2419

5. Summary and conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420

6. Future perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2420

∗ Corresponding author. Tel.: +49 331 977 5121; fax: +49 331 977 5150.
E-mail address:[email protected] (F.W. Scheller).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.11.023

N. Bistolas et al. / Biosensors and Bioelectronics 20 (2005) 2408–2423 2409

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2421

1. Introduction

Cytochromes P450 CYP form a large family of heme en-zymes that catalyze a diversity of chemical reactions such asepoxidation, hydroxylation and heteroatom oxidation. Theenzymes are involved in the metabolism of many drugs andxenobiotics and are responsible for bioactivation. Many ofthese compounds are even inducers for CYP expression indifferent organs. (Poulos, 1995; Ortiz de Montellano, 2004;Lewis, 1996).

The catalytic abilities of the CYP family have attracted theinterest of enzyme engineers already in the 1970s (Brunnerand Loesgen, 1977). Derived from their physiological func-tion, intact hepatocytes or microsomes were selected as bio-catalytic components in extracorporal detoxification reac-tions. Later on, the CYP mediated specific hydroxylation of abroad spectrum of substrates, including highly inert alkanesor steroids in a preparative scale, were the target. However,these studies had to face complications both due to the lim-ited stability of the labile multi-enzyme system and the needof the regeneration of the cofactor NADPH or NADH.

Considerable improvement has been achieved by the de-velopment of appropriate methods of immobilization of CYP.To simplify the technology, the multi-component system hasbeen restricted to the substrate converting part, i.e., the termi-nal oxidase. In this way the need of the electron transferringp sters

d bye 6-p al conc rna-t inalo type.T theticg ver,

TT n, thet s ared

P

CCCCC dC pedC

dioxygen – co-substrate for the substrate conversion – reactswith these potent reductants thus consuming in a parasiticreaction the reduced mediator to form hydrogen peroxide.Some progress has been achieved by using Co2+ sepulchrateas mediator, which reacts only slowly with the ambient oxy-gen (Estabrook et al., 1996). Attempts have also been made toattach CYP-enzymes to electrodes by introducing electroac-tive bridges covalently coupled to the protein (Lo et al., 1999;Shumyantseva et al., 2000). Such redox relays have been in-troduced at specifically selected sites generated by proteinengineering or randomly, e.g., CYP2B4 with covalently at-tached riboflavin (Shumyantseva et al., 2000). A list of CYPbionsensors based on mediated electron transfer is presentedin Table 2.

The ultimate approach is the direct (mediatorless) elec-tron supply from a redox electrode to the redox active groupof the CYP (Table 2). This concept was established by us(Scheller et al., 1977) in parallel with the development ofthe ‘promoted’ direct electron exchange of modified elec-trode forc-type cytochromes (Eddowes and Hill, 1977; Yehand Kuwana, 1977). In addition to the potential applica-tion of CYP for preparative purposes, its ability to metab-olize a broad spectrum of endogenous substances, e.g., fattyacids, steroid hormones, prostaglandins like mediators andforeign compounds, e.g., drugs and environmental toxinshas made this enzyme family interesting as recognition el-e en-d strated tal-y re-t ac-t reat-m fromo oft fore,f nt oft ctionw esb nce.T lest ive ab

type

( ring

a trate

roteins containing the redox active flavine or FeS cluhould be avoided (Table 1).

Regeneration of the reduced cofactor can be realizenzymatic reduction of NADP+, e.g., by using glucose-hosphate dehydrogenase. This represents the classicept of preparative application of dehydrogenases. Alteively, the electrons can be supplied directly to the termxidase by reduced mediators, e.g., of the viologenehese substances are well suited to reduce the prosroup of CYP due to their negative redox potential. Howe

able 1he isoforms of CYP enzyme that are used for sensor constructio

ype of electrodes together with the different electrode modificationisplayed

450 enzymes studied Electrodes Electrodemodifications

YP 1A1 CYP 17A Au BareYP 1A2 CYP 102 Platinum ClayYP 2B4 CYP 101 Tin oxide PhospholipidsYP 2C19 CYP 176A1 Glassy carbon MultilayersYP 2D6 CYP 11A1 Pyrolytic graphite Screen printeYP 2E1 CYP 4A1 Edge-plane graphite Antimony doYP 3A4 CYP 4A1 Carbon cloth Thiol

-

ment for biosensing. The highly specific conversion ofogenous substances like steroid hormones makes subetermination in complex media feasible. The CYP casis leads usually to detoxification with following excion of drugs but may also form reactive products andivate procancerogens. Adverse effects in multi-drug tents have been seen in many patients originating

verlapping substrate specificities or inhitibory effectshe different isozymes or polymorphic enzymes. Thereor drug or xenobiotics risk assessment the measuremehe substrates specificity and concentration in conjunith distribution of CYP isozyme and polymorphic enzymased on biosensors would be of high clinical relevahe reaction stoichiometry offers two traditional princip

o couple the substrate conversion to a transducer to giosensor:

(i) measurement of the oxygen consumption by a Clark-electrode;

ii) consumption of the reduced co-substrate by measuthe optical absorbance of NAD(P)H.

However, both reaction partners (O2 and NAD(P)H)re not only consumed in the CYP catalyzed subs

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Table 2Survey of CYP biosensors using electrochemical and optical biosensors

Sensors based on direct electron transferReference CYP species Technique Electrode/modification E◦′ (formal potential) Substrates tested – catalysis Comments

1. Bare electrodesKazlauskaite et al. (1996) Recombinant CYP101 CV EPG −526 mV (ls),−390 mV

(hs)Binding of camphor seen.Catalysis not described here

Arg 72,112, 364 and Lys344that interact with Pdx interactalso with bare EPG.

Lo et al. (1999) CYP101—WT,mutants

CV EPG, Au −428 to−449 mV No catalysis Surface cys replaced withAla. Electrochem. due to O2reduction to a certain extent.

Fantuzzi et al. (2004) CYP2E1 CV, chronoamperometry GC, Au −334 mV p-Nitrophenol 1 electron transfer, catalysiswith different modifications(see below)

2. Electrode modifier: phospholipidsIwuoha et al. (1998) CYP101 CV, amperometry DAB-BSA-

glutaraldehyde -GCE

−260 mV (ls) Catalysis with camphor,adamantanoneand fenchone(direct ET)

Calibration curve done withamperometry.Km′ = 1,41–3,9 mM. Also useof Co(Sep)3+

Zhang et al. (1997) CYP101 CV, SWV DMPC—PG, DDAB - PG −250 mV (DDAB),−357 mV (DMPC)

Catalysis seen with O2 andTCA

� = 7,2 mol cm−2 (DMPC),� = 4,9 mol cm−2 (DDAB),ks = 25(DMPC) s−1, ks = 26(DDAB) s−1

Fantuzzi et al. (2004) CYP2E1 CV, chronoamperometry Bare, thiol, DDAB – GC,Au

−334 mV Catalysis withp-nitrophenol

CYP2E1 electrochemistryand catalysis with differentelectrodes and modifications

Oku et al. (2004) CYP119 CV DDAB-plastic formedcarbon electrode

−250mV vs. SSE (20◦C),−50 mV vs. SSE (80◦C)

No catalysis Electrochemistry alsoobserved at 80◦C.,�E (at20◦C) = 90mV,�E (at80◦C) = 30mV

Agu sis Fe2+/Fe3+ redox potentialunaffected by subsratebinding. pH dependence−59 mV/pH unit

Flem with O2 and H2O2 ks: 221 s−1 no shift ofE◦′with substrate. Slope ofpH change (3–8):−33 mV/pH unit, (8–10):−126 mv/pH unit

3. MEsta with styrene Electrolysis done at

−600 mV at 4◦C. Catalaseinhibited catalysis.

Lvo with styrene Cationic PEI and PDDA andanionic PSS films were used.Au-MPS-(PEI/PSS-CYP101)- CYP101 pH5,2

ey-Zinsou et al., 2003 CYP176A1 CV, potentiometry DDAB-EPG electrode −360 mv No cataly

ing et al., 2003 CYP102 CV DDAB-EPG electrode −250 mV Catalysis

ultilayers on electrodesvillo et al. (2003) CYP1A2 CV (direct electrochem.),

electrolysis (reductasemediated)

Multilayers with PSS -Carbon cloth

−310 mV Catalysis

v et al. (1998) CYP101 CV, QCM, productanalysis

Multilayers with PEI,PDDA, PSS—Au

−250 mV Catalysis

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Munge et al. (2003) CYP101 QCM, CV, electrolysis,product analysis

Multilayers with PEI - PG −250 mV Catalysis with styrene Optimization of multilayersconditions. Influence of pHstudied.� = 0,1 nmol cm−2,Turnover rate = 6,3 h−1

Rusling et al. (2000) CYP101 CV, SWV PEI multilayers – roughPG electrode

−270 mV Catalysis with O2 and H2O2 � = 0,15 nmol cm−2

Lei et al. (2000) CYP101 CV Clay—GC −368 mV No catalysis � = 3,54 pmol cm−2,ks = 5–152 s−1.

Zu et al. (1999) CYP101 CV, electrolysis, productanalysis

Au-MPS—PDDA,DDAB, multilayerscarbon cloth

−250 mV Catalysis with styrene andcis-β-methylstyrene

CYP101 immobilised and insolution, Product turnoverrates higher in multilayersthat in solution

Shumyantseva et al. (2004) CYP2B4 CV, chronoamperometry Clay/detergent-GC −292 to−305 mV Catalysis with aminopyrine,benzphetamine

Product analysis,� = 40.5 pmol cm−2,kcat= 1.54 min−1

Joseph et al. (2003) CYP3A4 QCM, CV, SWV,amperometry electrolysis,product analysis

Au – MPS – PDDAmultilayers

342 mV CV), 335 mV(SWV)

Catalysis with verapamiland medazolam

Amperometry done at−500 mV vs. Ag/AgCl.Response time = 15–25 s,Km′ = 271–1082�M.

Nicolini et al. (2001) CYP11A1 CV Langmuir Blodgett films(mono- and multilayers),ITO glass plate

−295 to−318 mV No catalysis ks = 0,45 s−1, E0′ =−470 mV

vs. Ag/AgCl, Binding ofcholesterol seen. Binding alsocharacterized with X-Ray,QCM,CD, Ellipsometry,Brewster angle microscopy

4. Various modifiersIwuoha et al. (2004) CYP2D6 CV, amperometry Polyaniline doped GCE −120 mV Catalysis with fluoxetine Km′ = 3,7�mol/L., E◦′

shifted anodically in thepresence of substrate.

Bisto o catalysis Native state of enzyme duringelectrolysis.

Dav No catalysis STM, QCM, K334C mutantgreater affinity for Au, andenhanced electrochemistrycompared to the wild type.Wild type more mobile thanmutant (from QCM study).

P451. BEsta Catalysis with

progesterone andpregnenolone

Set potential−650 mV andthe decrease in oxygencontent was monitored. Useof Co(Sep)3+.

las et al. (2004) CYP101 Spectroelectrochemistry Dithionite and aldrithiol -Au

−380 mV N

is et al. (2000) CYP101 (K334Cmutant)

CV, SWV Polycrystalline Auelectrode

None mentioned

0-biosensors based on mediated electron transferare electrodesbrook et al. (1996) CYP17A, CYP4A1,

CYP3A4, CYP1A2,CYP102

Fusionprotein—electrolysis (O2monitorin)

Strip of mesh of Pt gauzeattached to Pt wire

None

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Table 2 (Continued)

Sensors based on direct electron transferReference CYP species Technique Electrode/modification E◦′ (formal potential) Substrates tested – catalysis Comments

Gilardi et al. (2002) CYP102, CYP2E1 Staircase CV GC disc −500 mV Neomycin promoter, Fusionprotein: Use of heme (fromCYP102 andCYP2E1)-flavodoxin (fromDesulfovibrio vulgaris)

2. Screen printed electrodesShumyantseva et al. (2001) PfCYP1A2,

RfCYP2B4,RfCYPscc

CV,amperometryspectropho-tometry

Screen printed -thick filmRh-graphite electrode

−555 mV (cholesterolfree),−357 mV(cholesterol bound)

Aminopyrine, aniline.7-ethoxyresorufin,7-pentosyresorufin

Use of 11A1 P452B4,CYP1A2 and CYPscc withcovalently attached riboflavin(Rf) to improve catalysis,Catalytic substrate reductionmeasured at−500 mV

Shumyantseva et al. (1999) CYP2B4 Electrolysis Screen printed -thick filmgraphite electrode

Reduction at−507 mV No catalysis but direct ETwas observed

Truncated flavo-CYP2B4,complex with CO formedunder reduction conditions.Use of riboflavin.

Shumyantseva et al. (2000) CYP1A2, CYP2B4 CV, amperometryspectrophotometry

Screen printed -thick filmRh-graphite electrode

−547 mV (RfCYP2B4),−557 mV (RfCYP2B4)

Catalysis with aminopyrine,aniline, 7-ethoxyresorufin,7-pentosyresorufin at−500 mV

P452B4 with covalentlyattached riboflavin forcatalysis, increased catalyticrates of substrates.

3. Various modifiersIwuoha et al. (1998) CYP101 CV, amperometry DAB-BSA-

glutaraldehyde -GCE

−260 mV (ls) Catalysis with camphor,adamantanoneand fenchone(direct ET)

Calibration curve done withamperometry.Km′ = 1,41–3,9 mM. Use ofCo(Sep)3+

Reip sis seen withor.

Use Pdx as mediator. Noproduct in absence of Pdx.Turnove rate 0,5 s−1

Reip sis with camphor ande

NADH turnover rates: WT:852 nmol−1 s−1 (cam bound),56 nmol−1 s−1 (styrenebound), Y96F: 29 nmol−1 s−1

(cam bound), 130 nmol−1 s−1

(styrene bound),Phenosafranine used asmediator

Mayh ysis with styrene. Use of Pdx as mediator inelectrolysis. Productquantification usingspectrophotometry: Turnoverof product 8 min−1 (WT),70 min−1 (Y96GF) usingNADH, PdR and Pdx

a et al. (1997) CYP101 CV, AC voltammetry,spectroele-ctrochemistry

Antimony-doped - Tinoxide

−437 mV (Pdx-normal−427 mV)

Catalycamph

a et al. (2002) CYP101 (Y96Fmutant)

Spectroelectrochemistry Nano-crystallineSb-dobed tin oxideelectrode

WT: −414 mV to−550 mV, Y96F:−448 mV to−492 mV

Catalystyren

ew et al. (2000) CYP101 (Y96Fmutant)

Spectroelectrochemistry Antimony-doped tinoxide electrode

None Catal

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Faulkner et al. (1995) CYP4A1 Mediatedelectrochemistry,Electrolysis

Thiol—Au None Catalysis with lauric acid Fusion protein, No direct ETbetween CYP and Auelectrode, Co(Sep)3+,Electrolysis at−450 mV vs.NHE

Optical biosensors based on P450 and ISFET-biosensorIvanov et al. (1999) CYP2B4 SPR, Spectrophotometry EDC/NHS Dextran

coated cuvettekon = 0.5–4× 106 M−1 s−1,koff = 0.5 s−1

No catalyis Interprotein ET occursbetween CYP2B4, cytb5 andNADH-CYP reductasethrough complex formationand random collision.

Makings and Zlokarnik (2000) CYP3A4, CYP2C19 Fluorescence Optical molecular sensor None Catalysis with varioussubstrates

Use of an optical probe whichattaches to the substrate andgive rise to fluorescence

Ivanov et al. (2000) CYP2B4 Optical EDC/NHS Dextrancoated cuvette

None No catalysis Formation of ternary complexbetween CYP2B4,NADPH-cyt CYP reductaseand cytb5

Ivanov et al. (2001) CYP2B4, CYP11A1,CYP101

Optical EDC/NHS Dextrancoated cuvette

None No catalysis Formation of binary-ternarycomplex between CYP2B4,CYP11A, CYP101 and theirrespective redox partners.

Ivanov et al. (2001) CYP2B4 Opical biosensorspectropho-tometry

DLPE, DSPEphospholipids -

No catalysis kon and koff CYP2B4, cytb5and NADH-CYP reductaseincorporated readily into thephospholipid layer.

Hara et al. (20 None Catalysis withdichlorophenols

Fusion enzyme

Potentials are

aminosilane cuvette

02) CYP1A1 Voltage output,fluorescence

IFSET

referred to SCE if not otherwise stated.

2414 N. Bistolas et al. / Biosensors and Bioelectronics 20 (2005) 2408–2423

Fig. 1. Catalytic cycle of CYP. The first step is the binding of RH substratefollowed by one electron reduction. The binding of substrate involves a shiftof the spin state of heme from low to high spin. After activation of the hemewith O2 and a second one-electron reduction, an oxygen atom is transferredto the substrate leading to the formation of ROH product.

conversion but also in a parasitic “uncoupling” hydrogenperoxide release via NAD(P)H without product formation.Therefore, the indication of the hydroxylated product givesthe only real measure of the enzyme activity. Only for a fewsubstances, e.g., aniline (Renneberg et al., 1978) this can beperformed by direct electrochemical quantification.

However, coupling of direct cathodic reduction of theterminal oxidase with substrate turnover can overcome theNAD(P)H dependent uncoupling. The generation of “cat-alytic currents” is therefore the direct indicator of CYP depen-dent electrocatalysis. The different approaches of this conceptwill be presented in this review.

2. The reaction cycle of CYP

Since the first three-dimensional structure of the bacte-rial CYP101 was elucidated by Poulos (Poulos et al., 1985)several other structures have been resolved including micro-somal ones, CYP2B4 (Scott et al., 2003; Werck-Reichhartand Feyereisen, 2000).

The active center is the iron-protoporphyrin IX with anaxial thiolate of a cysteine residue as fifth iron ligand. In theabsence of a substrate at the beginning of the cycle (Fig. 1)CYP is in the hexa-coordinated low-spin ferric form withwater being the sixth ligand.

YPm f theo m ofo duc-i

R

xin-l sferc strateh gh

several details remain still unsolved (Auclair et al., 2002).Substrate binding to the hexa-coordinated low-spin ferric en-zyme excludes water from the active site, which is causinga change to the 5-coordinate high-spin state. The decreaseof polarity is accompanied with a positive shift of the re-dox potential by about 130 mV that makes the first electrontransfer step thermodynamically favourable. The transfer ofone electron from a redox partner reduces the ferric iron tothe ferrous enzyme. This can now bind molecular oxygenforming a ferrous-dioxygen (FeII -O2) complex. The secondelectron is transferred along with a proton gaining an iron-hydroperoxo (FeIII –OOH) intermediate. The OO bound iscleaved to release a water molecule and a highly active iron-oxo ferryl intermediate. This intermediate abstracts one hy-drogen atom from the substrate to yield a one-electron re-duced ferryl species (FeIV OH) and a substrate radical orreacts in a concerted reaction with the substrate CH bondwithout intermediate radical formation. Then, it follows im-mediately the enzyme-product complex formation and re-lease of the product ROH to regenerate the initial low-spinstate. The iron-oxo intermediate may however also inducethe formation of protein radicals (Schunemann et al., 2002).

3. Protein electrochemistry

is ane dingb atal-y lvesp heree se top opri-a dingo rge tot igat-i theirm realt tronta ndd agni-t d thea ndon( po-t

eb

k

T d byt ed dox

The overall reaction of substrate hydroxylation of the Conooxygenase function is the insertion of one atom oxygen molecule into an substrate RH, the second atoxygen being reduced to water while consuming two re

ng equivalents under formation of ROH (Eq.(I)).

H + O2 + 2e− + 2H+ → ROH + H2O (I)

The electrons are delivered by flavoproteins or ferredoike proteins and NAD(P)H in a complex electron tranhain. The most generally accepted mechanism for subydoxylation by CYP includes the following steps althou

The transfer of electrons between and within proteinsssential feature for many physiological processes, incluiological energy transfer, metabolism and enzymatic csis. The mechanism of electron transfer usually invorotein–protein interactions as it is the case of CYP, wlectrons are transferred, i.e., from putidaredoxin reductautidaredoxin and then to CYP101. In enzymes an apprte conformational arrangement is important for the binf the substrate to the active site and the transfer of cha

he enzyme. The use of electrochemistry allows investng the electrochemical properties of redox enzymes and

echanism by observing the direct electron transfer inime. Much of the knowledge of the mechanism of elecransfer in proteins is based on the Marcus theory (Marcusnd Sutin, 1985). The factors that govern a highly specific airectional protein-mediated electron transfer are the m

ude of the electronic interaction between the donor ancceptor centres, and the contribution of the Franck–CoFC) factor involving the differences between the redoxentials of electron donator and acceptor.

The rate constant of electron transfer (kET) can therefore expressed using the following equation:

ET = 2π

h|HDA |2FC (1)

he magnitude of the electronic interaction as describehe electronic coupling matrix element (HDA) depends on thistance (r) and the nature of the donor and acceptor re


centres and can be described by

|HDA |2 =∣∣∣H0

DA

∣∣∣2 exp[−2β(d − 3)] =∣∣∣H0

DA

∣∣∣2 exp[−β′d

](2)

where H◦DA is the value of the matrix element at a predefined

distance (3A) andβ is the coefficient that describes the de-cay of electronic coupling withd. The Franck–Condon (FC)factor depends on the thermodynamic driving force for thereaction (�G◦) and the reorganisation energy (λ):

FC = 1√4πλkBT

exp

[− (�G0 + λ)

2

4λkBT

](3)

wherekB is the Boltzmann’s constant.From Eq.(3) can be seen that lnKET increases as the

driving force�G◦ increases until a maximum is reached at−�G◦ =λ. Beyond this point by increasing the�G◦ furthercauses a decrease in the lnKET providingλ remains constant.

The driving force is derived from the difference in mid-point reduction potentials of the electron donor and acceptorcentres, which is modulated by the protein environment sur-rounding the redox active sites. The reorganization energyis associated with the rearrangement of the atomic nuclei ofthe reactants into the configuration that they occupy in theproducts.

oryr alsoa n thep e isa ic in-t elf-a d theo , in-c andt lec-t

rans-f larlyu ithe ands me-t einsl ntrei y ana entf

4s

andt e in-v art-n ctri-

Fig. 2. Schematic representation of CYP sensor. The enzyme could eitherbe adsorbed or immobilized on a variety of metal and non-metal electrodes(see text). Addition of substrate leads to the formation of product, in thiscase hydroxylation, in the presence of O2.

cal contact to CYP-enzymes should be possible at suitablesurface modifications of electrodes.

The electrochemistry of CYP has been investigated usinga variety of metal electrodes (Table 1) such as Au, Pt and Tinoxide, as well as non-metal electrodes such as glassy carbon(GC), pyrolytic graphite (PG), edge-plane graphite (EPG),and carbon cloth (CC). Although direct electron transfer hasbeen observed on bare electrodes, modifying the electrodewith an appropriate medium like a polymer or a polyelec-trolyte, in order to attain native structure and appropriateorientation so increasing electron transfer between the en-zyme and the electrode has been very popular in recent years(Fig. 2). The bioelectrocatalysis by proteins and enzymessuch as cytochromec, CYP, glutathione peroxidase and cel-lobiose dehydrogenase (a heme-flavo enzyme) at modifiedelectrodes has been recently reviewed (Scheller et al., 2002).In the first biosensor based on the direct electron transferbetween the electrode and CYP (Scheller et al., 1977), solu-bilized CYP from rabbit liver showed a polarographic reduc-tion step at a mercury electrode of−580 mV versus SCE andwas partially reduced in constrast to the microsomal CYPwhich was not detectable. Catalytic currents were obtainedfollowing demethylation and hydroxylation of 1.8 mM ben-zphetamine, 2 mMp-nitroanisole and 2 mM aminopyrine.Furthermore, aniline (Renneberg et al., 1978) and steroidssuch as deoxycorticosterone (Scheller et al., 1979) could alsob mi-cf alsob directe .,1

4

erizet 101( c( nd int -b ingo ns-f tion.

Although the above description of the Marcus theefers for the homogenous electron transfer, it can bepplied to the heterogeneous electron transfer betweerotein and the electrode. In this case a redox enzymdsorbed on the surface of an electrode. The electron

eraction (coupling) can be varied by using different sssembled monolayers thus changing the distance anrientation of the protein from the electrode. In additionreasing the applied potential increases the driving forceherefore the number of protein molecules that allows eron exchange.

Several ways have been used to optimize electron ter between the redox protein and the electrode particusing chemically modified electrodes in combination wlectrochemical techniques like cyclic voltammetry (CV)quare wave voltammetry (SWV) in addition to amperory. This is particularly important in the case of heme protike CYP in which the electrochemically active heme ces buried in the protein structure and it is surrounded bmino acid chain in order to gain a hydrophobic environm

or catalysis.

. Direct electron transfer of CYP in electrochemicalensors

On unmodified electrodes enzymes tend to denatureo passivate the electrode. However, CYPs naturally arolved in electron transport pathways of protein redox pers, which require specific docking sites. Therefore, ele

e electrocatalytically determined using CYP from liverrosomes at a mercury electrode with an apparentKm (K′

m)or aniline of 18 mM. In the last decade attempts haveeen made to construct CYP biosensors based on thelectrode transfer using bare electrodes (Kazlauskaite et al996; Lo et al., 1999).

.1. Bare electrodes

Hill and co-workers have used bare EPG to characthe unpromoted electrochemistry of recombinant CYPKazlauskaite et al., 1996). In this work, cyclic voltammetriCV) measurements were carried out in the presence ahe absence of the substrate camphor at 6◦C. Strictly anaeroic conditions were used to prevent formation of the bindf oxygen to Fe2+ and the possibility of second electron tra

er. The results indicated reversible oxidation and reduc


The interaction of the CYP101 with the bare EPG has beenproposed by the authors to be possible via the positivelycharged Arg-72, Arg-112, Arg-364 and Lys-344 residues onthe surface of CYP101.

The formal potential (E◦′) was−526 mV versus SCE withthe camphor-free CYP101 and anE◦′ of −390 mV versusSCE for the camphor-bound form. These values are in agree-ment with the redox potential of CYP101 in solution forthe substrate-free form with anE◦′ of −547 versus SCEand substrate-bound form, with anE◦′ of −394 versus SCE(Sligar and Gunsalus, 1976). The binding of camphor to theactive site of the enzyme shifts the spin state of the heme pros-thetic group from low to high. The authors also claim that acatalytic response upon camphor addition was observed, al-though details are not given.

In order to investigate the role played by amino acids onthe immobilisation of CYP101 on the electrode surface, Loand co-workers have carried out site-directed mutagenesis tomodify the surface of the enzyme (Lo et al., 1999). In par-ticular, the wild type (WT) CYP101 contains eight cysteineresidues and cysteines 58, 85, 136, 148, and 334 are at ornear the protein surface, with Cys-334 being the most ex-posed one (Poulos et al., 1985). In this study these cysteineresidues were replaced by chemically inert alanines. Electro-chemistry of WT CYP101 and the mutants could be observedwith bare EPG electrodes with formal potentials ranging from− /s).H istryw istryo ablew me,w heser ouldb ial of− rbedo -ni

4

evedb fort tiono ans-f riateo Fasth en GCe C)( heg o in-c rche.C s ing , andm col-l ace

forces. Their colloidal and rheological properties have re-viewed previously by Luckham (Luckham and Rossi, 1999).Direct evidence of clay-mediated charge transfer has pre-viously been shown by Teng (Teng et al., 1997) for mont-morillonite K10 (iron-containing clay). Although SMC con-tains no iron atoms in its structure (the general formula isNa0.67(Si)8(Al3.3Mg0.67)-O20(OH)4), charge transfer may bemediated by either Si or Mg.

In addition to SMC, other clays such as kaolinite, talc,goethite and orche have also been used to modify electrodesand investigate the electrochemistry of heme proteins. In par-ticular voltammetric studies were carried out forc-type cy-tochromes, such as cytochromec, c3 andc553, where theirelectrochemical response were investigated using kaolinite,talc, goethite and orche modified PG electrodes (Sallez et al.,2000).

Investigation of the electrochemistry of CYP101 (Lei etal., 2000) was carried out in our group using SMC-Pt modi-fied electrodes. The approach was also used for CYP106A2(unpublished). In particular GC electrodes were modifiedwith SMC that was mixed with colloidal Pt nanoparticles.CYP101 was then adsorbed on the electrode and its redoxactivity was examined. Direct electron transfer between theelectrode and the heme group of the substrate-free enzymewas observed, with anE◦′ of −383 mV versus SCE. Sim-ilar results were also obtained when the GC electrode wasm heE ates ymei psh ial oft ilay-e ,1 rt y orw emeem bei eena2 allyo et asi eta pro-c ught dif-f 0, thed ctrontC r-i oft im-i ,1 doxp tiveo rbed

428 to−449 mV versus SCE at high scan rate (10 Vowever, a quasi-reversible or irreversible electrochemas obtained with bare Au electrodes. The electrochemf cysteine free CYP101 was in both cases indistinguishith that of the WT and the single cysteine mutant enzyhich indicate that electron transfer, is not affected by t

esidues. Furthermore, electrochemistry of CYP2E1 ce seen with bare GC electrodes with a midpoint potent334 mV versus SCE, indicating that CYP2E1 was adson the electrode surface (Fantuzzi et al., 2004). The heterogeeous electron transfer rate (ks) was found to be 5 s−1, which

s rather low.

.2. Clay modified electrodes

At bare electrodes the rather low electron transfer achietween the protein and the electrode limits their use

he construction of efficient CYP biosensors. Modificaf electrodes with compounds that facilitate electron tr

er, prevent denaturation of protein and cause approprientation of the protein have thus been widely used.eterogeneous electron transfer has been observed whlectrodes are modified with sodium montmorillonite (SMLei et al., 2000). Sodium montmorillonite is a member of teneral mineral group of clays, which among others alsludes smectite, laponite, kaolinite, talc, goethite and olay minerals are layer type aluminosilicates, ubiquitoueologic deposits, terrestrial weathering environmentsarine sediments. Clay particles are also very small,

oidal in size, so their behaviour is controlled by surf

odified with SMC and Pt colloid mixed with CYP101. T◦′ lies in this case at−399 mV SCE. These values devitrongly from the redox potential of the substrate-free enzn solution, which lies at−547 mV versus SCE. Other grouave also obtained large deviations on the formal potent

he substrate free enzyme by using polyion-protein multrs ranging from−238 to−357 mV versus SCE (Zhang et al.997; Lvov et al., 1998; Munge et al., 2003). The reason fo

hat may be that the interaction of CYP either with the claith surface of the electrode cause slight alteration to hnvironment as to make it less stable, thus shifting theE◦′ore positive. A positive shift of the redox potential may

ndicative for low to high spin-state conversion that has bscribed to strong interaction of CYP with surfaces (Niki,002). The positive shifts of the redox potential are generbserved when water is excluded from the heme pock

n the case of camphor binding (Poulos et al., 1986; Jungl., 2003) and therefore we suggested that the adsorptioness leads to a dehydration of the CYP structure. Althohe spectrum of CYP101 is not affected by the clay, COerence spectrum has shown a small increase in P42enatured form of CYP, by 4%. The heterogeneous ele

ransfer rate constants reached values as high as 152 s−1 forYP101 and 300 s−1 for CYP101 mixed with clay compa

ng to rates between 27 and 84 s−1 reported for the transferhe first electron from putidaredoxin to CYP101. This slarity suggests that the negatively charged clay (Ege et al.985) obviously mimics the electrostatics of the natural reartner putidaredoxin and may hold the CYP in a producrientation. In this orientation the active site of the adso


CYP101 is still accessible for small iron ligands like CO anddioxygen (Lei et al., 2000).

Moreover, liver microsomal phenobarbital inducedCYP2B4 has been incorporated in montmorillonite on glassycarbon electrodes (Shumyantseva et al., 2004). In contrast toCYP101, CYP2B4 has a flavoenzyme as redox partner anddoes not need an iron-sulphur helping protein for delivery ofelectrons. Using cyclic voltammetry at low scan rates a reduc-tion peak is observed at around−430 mV versus (Ag/AgCl).The electron transfer reaction is obviously very slow. Thisprocess is enhanced in the presence of a non-ionic detergentsuch as Tween 80. CYP2B4 is a membrane bound enzymeand detergent is needed to monomerize CYP2B4 (Kiselyovaet al., 1999) as was confirmed also by AFM-studies. From thecyclic voltammograms the amount of electroactive protein of40.5 pmol cm−2 was calculated. Cyclic voltammetry demon-strates also a reversible one-electron surface redox reactionwith a formal potential of about−302 versus SCE and a het-erogeneous electron transfer rate constant of 80 s−1. As inmany of the published cases, the formal potential of CYP atthe surface determined by the heterogeneous redox reactionis more positive than the redox potential in solution. There-fore, an influence of the detergent on the heme environmentas above can also be discussed.

The studies of direct heterogeneous electron transfer havebeen carried out in most cases by cyclic and square wavev ronsr spitet upons ta allc arelyc

reo-s xo-h ch asc alsob lysise ughp . Then idgew ndA eenP ni cinge 101c oxin( iths oterp a-t tetra-c withm 0W

onm er-

Fig. 3. Relationship of catalytic current, obtained with CYP2B4 bionsensorafter addition of aminopyrine, with increasing concentrations of aminopy-rine.

ized CYP2B4 in montmorillonite (Shumyantseva et al.,2004). When substrates were added to air saturated buffersolution, there was an increase of the reduction current.A typical concentration depedence measured in chronam-perometry is shown for aminopyrine and benzphetamine(Fig. 3). The reaction was inhibited by metyrapone. This in-dicates that CYP2B4 possess catalytic activity in the pres-ence of substrate. A further evidence was delivered by prod-uct analysis. After 1 h of controlled potential electrolysis at−500 mV versus SCE formaldehyde was measured. The ap-parent catalytic rate related to the amount of electroactiveprotein iskcat= 1.54 min−1 which is comparable to the valuekcat= 3.5 min−1 of the microsomal system (Shumyantseva etal., 2001).

4.3. Phospholipid modified electrodes

The majority of CYP enzymes are located in a hy-drophobic environment in the endoplasmic reticulumof cells, although cytosolic enzymes also exist, such asCYP101 (Lewis, 2001). In order to mimic the physiologicalenvironment of CYP enzymes, a number of groups haveused phospholipids, such as didodecyldimethylammoniumbromide (DDAB), dimeristoyl-l-�-phosphatidylcholine(DMPC), dilauroylphosphatidylethanolamine (DLPE)a thec tablev witht antm nt isc ymesr

atedfb 101c thep nkinga -freeb emegb sa olic

oltammetry. In these studies the first of the two electequired for the catalytic reaction has been transferred dehe authors do not see the shift of the reduction potentialubstrate addition as has been reported by (Kazlauskaite el., 1996) and is known for the reaction in solution. Inases catalytic oxygen reduction is observed but only ratalytic substrate conversion could be achieved.

CYP101 predominantly catalyzes the regio- and stepecific hydroxylation of (1R)-camphor to exclusively 5-eydroxycamphor. Other compounds than camphor, suompounds of environmental and industrial interest haveeen identified as substrates for CYP101. During catalectrons are transferred from NADH to CYP101 throutidaredoxin reductase (PdR) and putidaredoxin (Pdx)egatively charged group of Asp38 in Pdx forms a salt brith Arg112 in the positively charged patch (Arg112 arg109, Arg79) in CYP101 to shuttle electrons betwdR and CYP101 (Roitberg et al., 1998). Thus, a ferredoxi

n DET contact to an electrode may deliver the reduquivalents to CYP. On indium–tin oxide electrodes CYPonducts camphor hydroxylation mediated by putidaredReipa et al., 1997) and dehalogenation of haloalkanes wpinach ferredoxin in the presence of polylysine as promroceed (Witz et al., 2000). Acceleration of styrene epoxid

ion and dehalogenation of hexachloroethane, carbonhloride and other polyhalomethanes was successfulutated CYP101 (Reipa et al., 2002; Mayhew et al., 200;alsh et al., 2000).We succeeded in developing biosensors based

ediator-free CYP2B4 catalysis by immobilizing monom

nd distearoylphosphatidylethanolamine (DSPE), foronstruction of biosensors. Phospholipid layers form sesicular dispersions that bear structural relationshiphe phospholipid components of biologically importembranes. By this way a membranous environme

reated that facilitates electron transfer between the enzedox centre and the electrode.

Using this approach a CYP101 biosensor was creor monitoring drug conversion (Iwuoha et al., 1998). Theiosensor comprised a GC electrode modified with CYPontained in DDAB vesicle dispersion. Glutaraldehyde inresence of bovine serum albumin was used as a cross-ligent. CV measurements of the CYP electrode in airuffer indicated direct electron exchange between the hroup of CYP101 and the electrode. TheE◦′ was found toe−260± 10 mV versus SCE at a scan rate of 500 mV−1

nd a peak separation of 36 mV. When 3 mM of ethan


solution of camphor was added a catalytic response was ob-served in an aerobic measuring buffer. The peak cathodicpotential and current were−375 mV versus SCE and 44�Ain the absence of camphor and−350 mV versus SCE and50�A in presence of camphor, respectively. Under anaerobicconditions addition of 3 mM ethanolic solution of camphorproduced a catalytic response with a peak cathodic poten-tial of −430 mV versus SCE and a peak cathodic current of14�A. This indicated a typical fast reversible electrochem-istry of heme Fe3+/2+ redox species coupled to subsequentprocess, such as hydroxylation of camphor or H2O2 produc-tion by uncoupled turn-over. The authors therefore claim thateven in degassed buffer the oxygen in the aerobic ethanoliccamphor solution was sufficient for the reaction to take place.

In a similar study Zhang and co-workers in addition toDDAB, used DMPC to incorporate CYP101 and then mod-ify PG disc electrodes (Zhang et al., 1997). CV responseswith DMPC showed reversible electrochemistry with anE◦′of −357± 4 mV versus SCE, whereasE◦′ with DDAB was−238± 10 mV versus SCE. These values are more posi-tive than the redox potential of CYP101 in solution, that is−547 mV versus SCE, as a result of protein–lipid interactionsand/or possible lipid-dependent electrical double layer effectson electrode potential (Bard and Faulkner, 1980). CV mea-surements in the presence of CO showed a positive shift of theE◦′ from −357 to−297 mV for DMPC and from−238 mVv fortt dG r in-v ge-n tob nsew reem

4

eenA andc ry ofC ns( ua se-q d/orb cre-a nce( ciblel 101f -s nicP andn tein.D 101a oughv The

E◦′ was−250 mV versus SCE at neutral pH. The positive shiftin comparison to the redox potential of CYP101 in solution(−547 mV versus SCE) may be due to the surfactant headgroup charge and its association with the protein’s surface.Furthermore, oxidation of styrene was successfully catalysedby polyion films containing cytochrome CYP101. GC–MSproduct analysis (styrene oxide) showed a turnover numberof 9.3 h−1, which is larger than the turnover number whenCYP101 was in solution, that is 0.35 h−1.

Catalysis of styrene as well as benzaldehyde oxidationhas also been observed by the same laboratory using protein-polyion modified carbon cloth (CC) electrodes (Zu et al.,1999). The formal potential was the same as that reportedwith CYP101-multilayer modified Au electrode (Lvov et al.,1998), but the turnover number for the catalysis of styrenewas slightly lower 7.2 h−1. However, using CYP101 in solu-tion the turnover was 7.0 h−1, which approached that of im-mobilised CYP101. It was also significantly larger than theturnover number observed with Au electrode, i.e., 0.35 h−1

(Zhang et al., 1998). All turnover numbers were calculatedafter product analysis was carried out with GC–MS. A rea-son for the larger turnover number with CC electrodes couldbe the 20-fold larger active surface area of the CC electrodeas compared to the Au electrode. Addition of 3000 units ofcatalase destroyed the H2O2 and thus decreased the turnovernumber significantly to 0.2 h−1. This reflects the fact thatcatalysis of styrene or benzaldehyde with CYP101 is carriedout through H2O2.

Conversion of styrene-to-styrene oxide has also been stud-ied with a biosensor based on CYP1A2 (Estavillo et al.,2003). Cytochrome CYP1A2 is the main enzyme that me-tabolizes caffeine but it is also relatively active in convertingstyrene, although not as active as CYP2E1 and CYP2B6. Inthis study, however, PSS-CYP1A2 multilayers were grownon CC electrodes until films denoted PSS/(cyt CYP/PSS)2were obtained. TheE◦′ obtained was−310 mV versus SCE,which is similar to values obtained in the other works withmultilayers described above. Oxidation of 10 mM styrenewas monitored using a PSS-CYP1A2 modified CC electrodepoised at−600 mV versus SCE. Results obtained indicated aturnover rate of 39 h−1, which is significantly higher than 9.3and 7 h−1 obtained with CYP101 (Lvov et al., 1998; Zu etal., 1999). Assays carried out with CYP101 and CYP1A2 inconventional solution reactions with electron donors and re-ductase showed turnover rates that lies at 10 h−1 for CYP101and 17 h−1 for CYP1A2.

In addition to CYP101 and CYP1A2, CYP2E1 hasalso been studied with phospholipid modified GC and Auelectrodes. Reversible electrochemistry was observed bothwith DDAB and poly(diallyldimethylammonium chloride)(PDDA) modified GC electrodes with anE◦′ of −329 mVversus SCE. The electron transfer rate was 2 s−1 for DDABand 1 s−1 for PDDA, which is lower than the similar studydone by Zhang and co-workers. The authors have evenexamined the direct electrochemistry of CYP2E1 usingAu electrodes modified with thiol and/or PDDA. Using a

ersus SCE to−291 mV versus SCE for DDAB, whereashe protein in solution the redox potential changes from−547o −394 mV versus SCE (Zhang et al., 1997; Jefferson anriffin, 1972). These results indicate that electron transfe

olves the heme Fe3+/2+ couple of the enzyme. The heteroeous electron transfer rate (ks) was calculated in this casee 25–26 s−1. The authors have obtained catalytic respoith 50 mM trichloroacetic acid in apparently oxygen-feasuring buffer.

.4. Electrodes modified with multilayer films

In order to improve the direct electron transfer betwu electrodes and heme proteins like CYP, Ruslingo-workers have investigated the direct electrochemistYP101 using layer-by-layer films of CYP101 with polyio

Lvov et al., 1998; Munge et al., 2003). In these studies And pyrolytic graphite (PG) electrodes were modified byuential adsorption of poly(styrenesulfonate) (PSS) anranched poly(ethyleneimine) (PEI) and CYP101 thusting CYP101-multilayer films. Quartz crystal microbalaQCM) investigation has revealed regular and reproduayer formation. The thickness and the amount of CYPor bilayers on Au were 15 nm and 0.1 nmol cm−2. The asociation of CYP101 with the cationic PEI and the anioSS was possible due to the distribution of positivelyegatively charged amino acids on the surface of the proirect electron transfer between the heme group of CYPnd the Au electrode was observed in both cases, altholtammetric peaks were larger with PSS than with PEI.


mercaptopropionic acid (MPA)-PDDA modified Au elec-trode anE◦′ of −530 mV versus SCE and aks of 2 s−1 wereobtained, which with a cystamine-maleimide modified Auelectrode theE◦′ and ks were −421 mV versus SCE and10 s−1. Although the electron transfer rate is faster when acystamine-maleimide modified Au electrode than the otherAu modified electrodes was used, it is still not as fast as theelectron transfer rate obtained with GC modified electrodes.

Besides catalyzing styrene and benzaldehyde, CYP en-zymes play an important role in the metabolism of endoge-nous compounds as well as in pharmacokinetics and toxicoki-netics. That’s why Joseph have developed a biosensor withhuman CYP3A4 as a novel drug-screening tool (Joseph etal., 2003). It was constructed by assembling enzyme films onAu electrodes by alternate adsorption of a layer of CYP3A4on top of a layer of PDDA. The biosensor was applied tothe detection of verapamil, midazolam, quinidine and pro-gesterone. QCM monitoring showed that the protein concen-tration on the surface of the biosensor was 27 pmol cm−2.Electrochemical investigation of the enzyme-bound film re-vealed well-defined anodic and cathodic redox peaks withanE◦′ of −146 mV versus SCE, which indicates reversibleoxidation and reduction of the heme group. Furthermore, theenzyme was catalytically active as indicated by the concentra-tion dependent catalytic responses obtained using verapamil,midazolam, quinidine and progesterone. Product analysis af-t oft ithv -p idinew onset ately1

etineu Thee− tionsosA it ofd ta

4

lec-t msa -d ined( tu-r Hvei desO n thee

the first case a quasi-reversible reduction and oxidation wasobserved with anE◦′ of −250 mV versus Ag/AgCl and a peakseperation (�E) of 90 mV at a scan rate of 0.2 V s−1. TheE◦′at 80◦C was shifted to−50 mV versus Ag/AgCl and the�Elied at 30 mV. This result clearly shows that CYP119 is re-dox active even at higher temperatures, whereas the ordinaryCYPs have a relatively low thermostability.

Dehalogenation of hepatotoxic and carcinogenic solventslike chloroform and methyl chloride have been electrocatalyt-ically induced using this thermophilic enzyme (Blair et al.,2004). Blair and co-workers have immobilized CYP119 in amethyltriethoxysilane sol–gel film on graphite and also ad-sorbed in dimethyldidodecylammonium poly(p-styrene sul-fonate) (DDABPSS) modified graphite electrode. The en-zyme retains about 93% of its activity when adsorbed inDDABPSS at 30◦C with only a moderate loss at tempera-tures between 80 and 90◦C.E◦′ was−220 mV versus SCE at30◦C and−285 mV versus SCE at 80◦C. Catalytic currentswere observed with 10 mM CH2Cl2, CHCl3 and CCl4 withelectron turnover ranging from 4.5 s−1 (CH2Cl2) to 52.1 s−1

(CCl4).Mutagenesis studies have shown that catalysis with

CYP119 was improved when the side chain of Thr214, whichalthough not highly conserved in other CYP systems is nextto the highly conserved and catalytically important Thr213,was replaced by Val (Koo et al., 2002). In particular,k forltw par-ta enceo wasa0

4r

rodea ll nots na-t em-i ac-c ctro-e eenc wee istryo andp xidee weent weret r fort nves-t ator-f tein

er the electrolysis also confirmed the catalytic activityhe enzyme. A linearity of calibration up to 2.85 mM werapamil was obtained.Kappm values calculated from amerometric data for midazolam, progesterone and quinere 0.547, 0.271 and 1.082 mM, respectively. The resp

ime to reach 95% of the steady state was approxim5–25 s.

Catalytic response has also been obtained with fluoxsing CYP2D6 on a polyaniline-doped GC electrode.nzyme exhibited reversible electrochemsitry with aE◦′ of120 mV versus SCE, which upon increasing concentraf fluoxetine a cathodic shift was observed up to 350�M ofubstrate. Linearity was however observed only until 1.0�M.t higher concentration saturation was attained. The limetection was 1nM and theKappm value 3.7�M (Iwuoha el., 2004).

.5. CYP bionsensing at high temperatures

An interesting aspect of CYP electrochemistry is the eron transfer ability of a CYP from thermophilic organist high temperatures. CYP119 is found inSulfolobus tokoaii strain 7 and its crystal structure was recently determOku et al., 2004). CYP119 has an unusually high denaation point of around 90◦C and tolerance for extreme palues and pressures for extended periods in solution (Yanot al., 2000; Koo et al., 2000, 2002; Puchkaev et al., 2002). Us-

ng DDAB modified plastic formed carbon (PFC) electroku and co-workers adsorbed CYP119 and have showlectrochemistry at 20◦C and at 80◦C (Oku et al., 2004). In

catauric acid hydroxylation, increased from 0.36 min−1 (wildype) to 2.08 min−1 (T214V mutant) and even to 8.80 min−1

hen the additional D77R mutation was introduced. Asic acid (D) is also near the active site of CYP119 (Koo etl., 2000). Measurements were carried out in the presf Pdx, Pdx reductase and NADH. Styrene epoxidationlso improved in the same study withVmax increasing from.05 min−1 (wild type) to 0.20 min−1 (T214V).

.6. Spectroelectrochemistry and surface plasmonesonance of CYP

Although direct electron transfer between the electnd CYP has been established, one important but stiolved question is whether cytochromes CYP remainsive when interacting with electrodes. Spectroelectrochstry allows getting an insight into the structural changesompanied with the electrochemical redox cycling. Spelectrochemistry of wild type high-spin CYP101 has barried out by using mediator (Reipa et al., 1997; Mayhet al., 2000; Reipa et al., 2002) and mediator-free (Bistolast al., 2004) electrode system. The spectroelectrochemf CYP101 in the presence of mediators such as Pdxhenosafranine was studied using antimony-doped tin olectrode. Although electron transfer was observed bet

he electrode, via the mediator to CYP101, no spectraaken in the presence of CO, which is the best indicatohe presence of the protein’s native state. We therefore iigated CYP101 with spectroelectrochemistry on a mediree environment with the purpose to indicate that the pro


retains its native state in the electrochemical cell during elec-trolysis. Reversible oxidation and reduction was observedusing both 4,4′-dithiodipyridin and sodium dithionite mod-ified Au capillary electrodes (Bistolas et al., 2004). An E◦′of −380 mV versus SCE was determined, which is similar tothe CV studies of CYP101 (Lei et al., 2000) and the redoxpotential of high-spin CYP101−394 mV versus SCE. Thespectra of CYP101 in the presence of either carbon monox-ide or metyrapone, both being inhibitors of CYP101 cataly-sis, displayed the spectroscopic pattern that is characteristicof the binding of these two ligands to the heme center of theenzyme. This is the first spectroscopic proof that the CYP101remains in the native state during electrolysis.

In order to elucidate the mechanisms whereby interproteinelectron transfer takes place in CYP systems and how the sys-tem is operated, Ivanov and Archakov have constructed anoptical biosensor to study binary and ternary complex for-mation of CYP2B4, CYP101 and CYP11A1 with their re-spective redox partners (Ivanov et al., 1999, 2000, 2001a,2001b, 2001c). The method was based on the design of anoptical evanescent sensor, a resonant mirror (Ivanov et al.,2001a). Proteins were immobilized on the carboxymethy-lated dextran-coated cuvette. The results indicated that onone hand, the complex formation of CYP2B4 with its redoxpartners was demonstrated to occur due to hydrophobic in-teractions of protein fragments with the phospholipid layer.O rtantf ers.C erep timeo hish lec-t tantsf meb fus-s ncC om-pws onlyd ion.T s ont thef

5

perso i-d s att elec-t terizet toI er-

ing of the electrode surface for appropriately orienting the ter-minal oxidase towards the electrode have been successfullyadopted from cytochromec. In this respect, the electrostaticattraction on molecular level has been used. However, dueto the well-known low conformational stability pronouncedstructural changes are plausible in the process of embeddingin the matrix at the electrode. These deviations from the be-haviour in solution are obviously reflected by the anodic shiftof the structure-free protein and it is reflected by the smalleranodic peak in CVs.

The electron transfer at electrodes modified with mono-layers of phospholipids or mercaptolalkanol/acids should re-semble in analogy to cytochromec—the other sphere type.The mode of electron transfer via conducting polymers ormultilayers like clay nanoparticles or polyelectrolytes seemswidely unclear.

For the fast heterogenous electron transfer with glucoseoxidase – an intrinsic redox enzyme with the prosthetic groupburied deep within the protein fabric – the coupling of redoxrelays to the protein has been established (Degani and Heller,1987). This concept has been successfully transferred to theCYP electrochemistry by Archakov’s group by binding ri-boflavin to the protein surface (Shumyantseva et al., 2000).On the other hand, the concept of ‘redox wire’ (Willner andKatz, 2000) transferring the electrons via immobilized me-diators from the electrode to the prosthetic group has not yetb ationb

atedb hilstt odico ros-t rode.T d ofC

( thef in-

them

6

lyze av recenty neer-i re-g neticl facec ont redoxc nsfer.

n the other hand electrostatic interactions were impoor the complex formation of CYP101 with its redox partnomplex formation of CYP11A1 with its redox partners wroblematic to be seen due to the two orders higher lifef the binary complexes than the hydroxylation cycle. Tas ruled out the effective functioning of adrenodoxin as e

ron carrier in the complex. The associations rate consor the CYP2B4, NADPH-CYP reductase and cytochro5 complexes formed were found to be close to the difion limit−0.5 to 4× 106 M−1 s−1—while their dissociatioonstants did not exceed 0.5 s−1 (Ivanov et al., 1999). ForYP101, putidaredoxin reductase and putidaredoxin clex the association rate constant was 0.34× 106 M−1 s−1,hile the dissociation constant was 0.047 s−1. It was alsohown that the interprotein electron transfer occurs noturing complex formation but also upon random collishis was due to hydrophobic and charged amino acid

he surface of the CYP, which play a dominant role inormation of productive electron transfer complexes.

. Summary and conclusions

Regarding protein electrochemistry the number of pan CYP ranks third after cytochromec and glucose oxase. The binding sites for electron transferring protein

he molecule surface which are essential for the fastron transfer and subsequent oxygen activation charache CYP family to the intrinsic redox proteins accordingkeda’s (Ikeda, 1992) classification. Therefore, the engine

een realized for the electrochemical substrate hydroxyly CYP.

Interpretations of the reaction mechanism are complicy the fact that substrate conversion requires oxygen w

he reduction of the CYP heme is mashed by the cathxygen reduction. Most probably, both reduction of the p

hetic group and of oxygen proceed in parallel at the electo unravel the complexity, the influence of catalase anYP inhibitors is a useful diagnostic criterion.

(i) Substrate conversion by the by-product H2O2 is sup-pressed by the addition of catalase.

ii) Substrate hydroxylation via the two-e-reduction ofiron–oxygen complex is effected by the presence ohibitors, e.g., metyrapone.

These criteria have not yet been strictly applied inajority of investigations.

. Future perspectives

In most cases biosensors based on CYP can cataariety of substrates, as has been described above. Inears profit has been taken from the use of protein enging. This implies the introduction of specific attachmentions and electron transfer regions to overcome the ki

imitations. Furthermore, engineering of the protein surould provide means of immobilizing the protein directlyhe electrode, thus shortening the distance between theenter and the electrode and achieving faster electron tra


Recent work indicates the potential of engineering sites forsurface binding and redox active dyes, de-novo designed re-dox systems and genetic chimeras (Gilardi et al., 2001, 2002;Wong and Schwaneberg, 2003).

Nature offers almost unique biocatalysis of the CYP fam-ily exhibiting both (i) almost absolute specificity for recog-nizing a given substrate also in mixtures of very similar com-pounds, e.g., steroid hormones, and (ii) conversion of highlydifferent compounds following the same reaction type, e.g.,demethylation of various drugs. Using genetic engineeringboth recognition types may be optimized for the analyticalsystem under investigation. In this respect, arrays carryingdifferent CYP isoforms will be developed for characterizingthe metabolism of drug converting organs, e.g., the liver ofdifferent species. Full interaction of specific substrate conver-sion and electrochemical signal transduction can be expectedin the next generation of electronic biochips.

Acknowledgments

The funding from the Fond der Chemische Industrie, Eu-ropean Community (Intellisens QLK-3-CT2000-01481) andGerman Ministry of Education and Research (BMBF 3i308-B) is greatly appreciated.

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Cytochrome P450 biosensors—a review