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Direct electron transfer from graphite and functionalized gold electrodes to T1and T2T3 copper centers of bilirubin oxidase

Pablo Ramiacuterez a Nicolas Mano b Rafael Andreu a Tautgirdas Ruzgas c Adam Heller dLo Gorton e Sergey Shleev cf e

a Departamento de Quiacutemica Fiacutesica Universidad de Sevilla 41012 Sevilla Spainb Centre de Recherche Paul Pascal (CRPP) Universiteacute Bordeaux I UPR 8641 Avenue Albert Schweitzer 33600 Pessac Francec Biomedical Laboratory Science Malmouml University 20506 Malmouml Swedend Department of Chemical Engineering and The Texas Material Institute The University of Texas 78712 Austin USAe Department of Analytical Chemistry Lund University 221 00 Lund Swedenf Laboratory of Chemical Enzymology Institute of Biochemistry 119071 Moscow Russia

a b s t r a c ta r t i c l e i n f o

Article history

Received 8 May 2008Received in revised form 11 June 2008Accepted 17 June 2008Available online 28 June 2008

Keywords

Bilirubin oxidaseBioelectrocatalysisO2-electroreductionElectron transfer kinetics

Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modi1047297ed gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied underanaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry On cyclic voltammogramsnonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observedcorresponding to redox transformations of the T1 site and the T2T3 cluster of the enzyme respectively Theimmobilized BOD was differently oriented on the two electrodes and its catalysis of O 2-electroreduction wasalso massively different On SPGE where most of the enzyme was oriented with the T1 copper site proximalto the carbon with a quite slow ET process well-pronounced DET-bioelectroreduction of O2 was observedstarting already at N700 mV vs NHE In contrast on MPA-gold most of the enzyme was oriented with its T2T3 copper cluster proximal to the metal Indeed there was little DET-based catalysis of O2-electroreductioneven though the ET between the MPA-gold and the T2T3 copper cluster of BOD was similar to that observed

for the T1 site at SPGE When BOD actively catalyzes the O2-electroreduction the redox potential of its T1 siteis 690 mV vs NHE and that of one of its T2T3 copper centers is 390 mV vs NHE The redox potential of theT2T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs NHE These valuescombined with the observed biocatalytic behavior strongly suggest an uphill intra-molecular electrontransfer from the T1 site to the T2T3 cluster during the catalytic turnover of the enzyme

copy 2008 Elsevier BV All rights reserved

1 Introduction

Bilirubin oxidase (bilirubinoxygen oxidoreductase EC 1335) is amulticopper oxidase catalyzing the oxidation of bilirubin to biliverdinas well as the oxidation of other tetrapyrroles diphenols and aryldiamines with the concomitant reduction of molecular O

2 to H

2O [1]

Although the crystal structure of BOD is unknown the structures of itstwo catalytic centers are well understood from biochemical spectraland kinetic investigations of the wild-type and of mutated forms of the enzymes [2ndash8] The two centers one binding and oxidizing theorganic substrate the other binding and reducing O2 comprise fourcopper ions In analogy with other blue multicopper oxidases such aslaccase and ascorbate oxidase the organic substrate binding centercomprises one copper ion and is denoted T1 the O2-binding centercomprises a cluster of three copper centers denoted T2 and T3 [79ndash

11] The T2T3 copper cluster is one of the few sites encountered innature which effectively catalyzes the four-electron reduction of O2 toH2O [1213] The T1 Cu2+-center accepts electrons not only from the

Biochimica et Biophysica Acta 1777 (2008) 1364ndash1369

Abbreviations BOD bilirubin oxidase NI native intermediate PI peroxy inter-mediate FR fully reduced BOD RF resting form DET direct electron transfer ET

electron transfer IET intra-molecular electron transfer CV cyclic voltammogram E mmidpoint redox potential E T1 E T2 E T3 and E T2T3 redox potentials of the T1 T2 T3 sitesand the T2T3 cluster respectively ΔE p peak separation between anodic and cathodicpeaks k0 standard electron transfer rate constant kDET heterogeneous DET rateconstant Γ surface concentration jcat biocatalytic current density jcat

max maximumbiocatalytic current densityΔ jcat(+F minus) differences in biocatalytic current densities intheabsence and presence of Fminus EDC N -(3-dimethylaminopropyl)-N prime-ethylcarbodiimidehydrochloride NHS N -hydroxysuccinimide AMTP 4-aminothiophenol MHOL 6-mercapto-1-hexanol MPA 3-mercaptopropionic acid DT 1-decanethiol SPGEspectrographic graphite electrode MPA-gold 3-mercaptopropionic acid-modi1047297edgold electrode

Corresponding author Biomedical Laboratory Science Malmouml University 20506Malmouml Sweden

E-mail address sergeyshleevmahse (S Shleev)URL wwwmahseshleev (S Shleev)

0005-2728$ ndash see front matter copy 2008 Elsevier BV All rights reserved

doi101016jbbabio200806010

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e w w w e l s ev i e r c o m l o c a t e b b a b i o

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natural substrates of BOD butalso from arti1047297cial electron donors suchas redox mediators in homogeneous catalysis or electrodes poised atsuf 1047297ciently reducing potentials in electrocatalysis The acceptedelectrons are transferred to the T2T3 copper cluster where theyreduce O2 [10] Interestingly and signi1047297cantly when BOD or laccaseare embedded (ldquowiredrdquo) in an electron-conducting redox hydrogelwhere electron donors envelope the enzyme such that electrons 1047298owto their T1 centers irrespective of orientation the copper-enzymes are

greatly superior to platinum as electrocatalysts of the four-electronreduction of O2 to H2O [14] Here we illuminate some aspects of themechanism of this unique electrocatalytic process

Electrodes poised at suf 1047297ciently reducing potentials transferelectrons directly to fungal BODs from Myrothecium verrucaria andTrachyderma tsunodae [15ndash17] Even though M verrucaria BOD isreadily O2-oxidized when adsorbed on graphite electron transfer (ET)from graphiteto the adsorbed BOD is observed only at 515 mV vsNHEat pH 74 about 025 V more negative than the reversible potential of the O2H2O redox couple at a low current density [15] Spectro-electrochemical measurements show that this potentialis far from theredox potential of the T1 site (E T1) which is near 670 mV vs NHE atpH 70 [17] In contrast M verrucaria BOD adsorbed on carbonnanotubes is electroreduced already at 685 mV at pH 74 [18] but thereduced Cu2+ centers were believed to be those of the T2T3 clusternot the T1 site On gold very slow DET was observed [17] Here wereport DET results for T tsunodae BOD adsorbed on graphite and onmodi1047297ed gold electrodes Cyclic voltammograms (CV) exhibit clearlyvisible nonturnover waves with midpoint potentials (E m) of about700 mV and 400 mV corresponding to redox transformations of theT1 site and the T2T3 cluster respectively This is the 1047297rst report onwell-pronounced nonturnover signals of electron accepting andoxygen reducing sites of a blue multicopper oxidase coinciding withcorresponding catalytic waves of O2 bioelectroreduction Importantlyour data strongly suggest an uphill intra-molecular electron transferfrom the T1 site to the T2T3 cluster during the catalytic turnover of the enzyme

2 Experimental section

21 Reagents

Na2HPO4 KH2PO4 KCl NaCl H2O2 H2SO4 and NaF were obtainedfrom Merck (Darmstadt Germany) Citric acid 22prime-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and N -(3-dimethylamino-propyl)-N prime-ethylcarbodiimide hydrochloride (EDC) were from Sigma(St LouisMO USA) N -hydroxysuccinimide (NHS) 4-aminothiophenol(AMTP) and 6-mercapto-1-hexanol (MHOL) were from Aldrich (StLouis MO USA) 3-Mercaptopropionic acid (MPA) and NaIO4 werepurchased from Janssen Chimica (Geel Belgium) Absolute ethanol(997) was from Solveco Chemicals AB (Taumlby Sweden) 1-Decanethiol(DT) and K3[Fe(CN)6] were from Fluka (Buchs Switzerland) Allchemicals were of analytical grade Buffers were prepared with water

(18 MΩ) puri1047297ed with a Milli-Q system (Millipore Milford CT USA)Anaerobic and aerobic conditions were established using nitrogen

(N2) or oxygen(O2) gases from AGA Gas AB (SundbybergSweden) thatwere bubbled through the working solutions

22 Enzymes

BOD from Trachyderma tsunodae was from Amano Enzyme Inc(Elgin IL USA) Preparations of BOD were stored at minus18 degC Theconcentration of BOD in the stock solution was determined by theestablished method of Ehresmann [19] The turnover number of BODtowards K3[Fe(CN)6] was determined spectrophotometrically using anUvikon 930 spectrophotometer (Kontron Instruments Everett MAUSA) and was found to be 380 sminus1 in good agreement with a

previously reported value for a wild-type M verrucaria BOD [6]

23 Electrochemical measurements

Electrochemical measurements were performed using a threeelectrode potentiostat (CV-50W Bioanalytical Systems BAS WestLafayette IN USA) The reference electrode was a Hg|Hg2Cl2|KClsatelectrode K401 (SCE 242 mV vs NHE) from Radiometer (CopenhagenDenmark) and the counter electrode was a platinum wire Thesupporting electrolyte consisted of a 01 M phosphate buffer solution

at pH 70

231 Roughness factor and cleaning of the working electrodes

The gold working electrode model MF-2014 was purchased fromBAS its geometrical area was 002 cm2 Its microscopic roughnessfactor was calculated from the charge (qreal) associated with the goldoxide reduction process obtained when running a CV from 0 to1900 mV in 05 M H2SO4 The theoretical charge density (σ t)associated with the reduction of the gold oxide is 390plusmn10 μ C cmminus2

[20] The microscopic area was obtained using the ratio of themeasured charge of gold oxide electroreduction qreal and thecalculated theoretical charge for the 02 cm2 electrode if it weresmoothσ t ( Areal= qreal σ t) The microscopic roughness factor was thusestimated to be 14plusmn01

The gold electrode was pre-cleaned by CV scans at a 100 mVsbetween minus60 and minus1360 mV vs NHE in 05 M NaOH then polishedwith a DP-suspension (high performance diamond product) and analumina de-agglomerated polishing suspension (1 μ m and 01 μ mStruers Copenhagen Denmark) rinsed with Millipore H2O andsonicated in after each polishing step for 10 min The electrode wasthen cleaned by a series of CV scans at a 100 mVs scan rate betweenminus60 and+1790 mV vs NHE in 05 M H2SO4 and kept in concentrated(96) H2SO4 till use Immediately before its use it was rinsedthoroughly with H2O

The spectrographic graphite electrode (SPGE Ringsdorff WerkeGmbH Bonn Germany type RW001 305 mm diameter 13porosity) was polished with wet 1047297ne emery paper (Tufback DuriteP1200) rinsed thoroughly with H2O and allowed to dry Theadsorptive roughness factor of such an electrode was estimated to

be about 5 [21]

232 Thiol self-assembled monolayers and BOD deposition on gold

The procedures for physical adsorption of BOD on gold and forcovalent binding of BOD to AMTP modi1047297ed gold electrodes werereported earlier [22] To form DTand MHOL SAMs the electrodeswererespectively immersed in 1 mM (2080 vv H2Oethanol) or a 5 mM(absolute ethanol) solution of DT or MHOL and the monolayers wereallowed to assemble overnight at room temperature To adsorb BODon bare or modi1047297ed gold surfaces the electrode was mounted with itssurface facing up an aliquot of 5 μ l of the BOD solution (10 mgml) wasplaced on it and was allowed to react for 3 h while every 20 min 5 μ lof H2O were added to avoid complete drying of the BOD solution

To form the MPA monolayer the gold electrode was placed in a

5 mM solution (2575 vv H2Oethanol) of MPA overnight Toimmobilize on it the BOD the procedure of Ruumldiger et al [23] appliedearlier for the immobilization of Desulfovibrio gigas hydrogenase wasused The electrodewas placed with itssurface facing up and 6 μ l of ca25 μ M of the BOD solution in 10 mM phosphate buffer pH 70 wasadded Then 45 μ l of 18 mM NHS and 55 μ l of 36 mM EDC were alsoadded After about 90 min the electrode was washed in pH 70 01 Mphosphate buffer and was promptly used

233 Adsorption of BOD on graphite electrodesA volume of 10 μ l of BOD solution (25 mgml) was placed on the

electrode surface allowed to adsorb and after 15 min the SPGE wasrinsed with H2O

The current densities and enzyme surface concentrations were

estimated using the real area of the electrodes calculated by

1365P Ramiacuterez et al Biochimica et Biophysica Acta 1777 (2008) 1364 ndash1369

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multiplying the geometrical area by the roughness factor All reportedpotentials are vs NHE

3 Results

31 Electrochemistry of BOD adsorbed on graphite

In the absence of O2 tworedox processes were observed in therawCVs of T tsunodae BOD on SPGE (Fig 1A) and after subtracting the

background current (Fig 1A curve 2) their voltammetric waves weresymmetrical and well-de1047297ned One wave had an E m of 690 mV and apeak separation (ΔE p) of 155 mV another had an E m of 390 mV and aΔE p of 73 mV Calculation of the surface concentration of electroactivespecies (Γ ) from the charge associated with the waves and the area of theelectrodes provideda coverage of 83pmol cmminus2 forthe 1047297rst and of 52 pmol cmminus2 for the second assuming an exchange of one electronper electroactive molecule The waves 1047297t those expected from theMarcus-DOS theory of ET [24] The 1047297tting was computed assumingreorganization energy (λ) values within the 04ndash08 eV range asreported for small blue redox proteins containing one T1 site such asazurin [25] and plastocyanine [26] The calculated DET standard rateconstant (k0) values were 04 sminus1 and 13 sminus1 for the E m=690 mV andE m=390 mV redox processes respectively These values are indepen-

dent of λ within the above indicated range whereas the cathodic rate

constant (kDET) value at 200 mV (the cathodic limit of the usefulelectrocatalytic potential range) strongly depends on λ and for theE m=690 mV it takes values up to 74 sminus1 and 437 sminus1 for λ of 04 and08 eV respectively ie in the order of 100 sminus1

While O2 was not electroreduced on bare SPGE even at a potentialas reducing as 100 mV it was catalytically reduced on T tsunodaeBOD-modi1047297ed SPGE resembling in this respect M verrucaria BOD-modi1047297ed SPGE [15] The catalytic wave (Fig 1B) started already atN

700 mV (Fig 1B) Moreover the E m of the 1047297

rst voltammetric waveobserved in the absence of O2 (Fig1A) and the mid-wave potential of the CV under O2 coincide well and they are close to 690 mV (Fig 2)The potentials depended quite weakly on the O2 partial pressure asexpected for a 4-electron reduction process The maximum bioelec-trocatalytic current density ( jmax

cat ) of O2 electroreduction at the BOD-modi1047297ed SPGE was sim40 μ A cmminus2 (Fig1B) based on the true area of theelectrode ie the geometrical area of 0073 cm2 multiplied by theroughness factor of 5 [1521] The engineering current density wastherefore 200 μ A cmminus2 The BOD-SPGE CVs under O2 were indepen-dent of the scan rate between 10 and 100 mV sminus1 in the 730ndash600 mV potential range but did depend on the scan rate at more negativepotential values (Fig 1B)

The presence of K3[Fe(CN)6] or ABTS did not signi1047297cantly affect theO2-electroreduction current When 10 mM Fminus a known inhibitor of BOD [4] was added the true O2-electroreduction current densityobservedat 275mV and 10 mVsminus1 scan rate decreased by 17 μ A cmminus2

32 Electrochemistry of BOD on gold

Attempts to adsorb BOD on bare gold electrode and gold modi1047297edwith thiol monolayers providing hydrophobic neutral hydrophilic orcationic-hydrophilic surfaces failed This was the case when the goldsurface was modi1047297ed with monolayers of decanethiol 6-mercapto-1-hexanol or 4-aminothiophenol Covalent binding of BOD to the anionic-hydrophilic gold surface by forming amides of chemisorbed 3-mercaptopropionic acid (MPA) and BOD-amines was however success-ful Fig 3A shows theCVs of an MPA-gold electrode before (curve 1) andafter (curve 2) covalent linking of BOD in the absence of O2 A broad

single redox process was observed with an E m of 360 mV and a 56 mV peak separation The area under the anodic peak was somewhat biggerthan the area under its cathodic counterpart giving an average BODsurface concentration of 10 pmol cmminus2 by assuming a one-electrontransfer process The voltammetric wave was also 1047297tted using theMarcus-DOS theory of ET withλ values within the 04ndash08 eV range Thedashndashdotted voltammogram in Fig 3A was computed by assuming a

Fig1 (A) Cyclic voltammogram of BOD adsorbed on SPGE in the absence of O 2 pH 7001 M phosphate buffer 100 mV sminus1 scan rate 1000mV starting-scan potentialCurve 1

uncorrected for the background current Curve 2 circles background currentsubtracted The broken line curverepresents the calculatedtheoretical voltammograms(B) Effect of the scanrate on the background-correctedvoltammograms underO2 Curve110 mV sminus1 Curve 2 100 mV sminus1

Fig 2 Cyclic voltammograms of SPGE covered with a perm selective membrane under1 atm O2 Curve 1 before BOD adsorption Curve 2 after adsorbing the Trachyderma

tsunodae BOD pH 70 01 M phosphate buffer scan rate 10 mV sminus1 starting-scan

potential 1000 mV second scan

1366 P Ramiacuterez et al Biochimica et Biophysica Acta 1777 (2008) 1364ndash1369

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standard potential value of 036 V k0=03 sminus1 and by inserting any λ

value within the above indicated range Moreover addition of Fminus hadvery littlein1047298uence on thevoltammetric wave under both anaerobicandaerobic conditions

Chronoamperometry under N2 of the MPA-modi1047297ed gold and theBOD-MPA-modi1047297ed gold (inset in Fig 3A) with the electrodes poisedat +500 mV showed a minute reduction current attributed in alllikelihood to the residual O2 The current density increased 1047297ve-fold

to 033 μ A cm

minus2

when O2 was bubbled through the solution anddropped to the initial small value when N2 was bubbled again Afteraddition of Fminus to a 1047297nal concentration of 10 mM the O2-electroreduction current density was observed to decrease from033 to 022 μ A cmminus2 (inset in Fig 3A) Added K3[Fe(CN)6] allowedmediated electron transport from the gold electrode to the BODirrespective of its orientation and electronic coupling to the goldsurface (Fig 3B) An increase in the peak-height of the voltammetricwave when O2 was bubbled (curve 2) instead of N2 (curve 1) indicatedthat the BOD retained its catalytic activity in the presence of the K3[Fe(CN)6]K4[Fe(CN)6] couple at the electrode surface When 10 mM Fminus

was added the K3[Fe(CN)6]K4[Fe(CN)6] current enhancement wasdrastically reduced at potentials more negative than 400 mV (Fig 3B)The difference in the mediated bioelectrocatalytic current densities(Δ jcat(+Fminus)) for O2-reduction in the presence and absence of Fminus at275 mV and a scan rate of 10 mVsminus1 averaged 14 μ A cmminus2 eg veryclose to the value obtained for BOD-modi1047297ed SPGE (17 μ A cmminus2) Itshould also be noted that O2 was electroreduced to H2O2 on thiol-modi1047297ed gold electrodes at potentials more negative than 200 mV

(Fig 3B) [22]

4 Discussion

BOD and other blue multicopper oxidases unlike peroxidase andnitric-oxide synthase reduce O2 without producing reactive inter-mediates like H2O2 or O2

middotminus The reversible potential of the O2H2O half-cell under physiological conditions (pH 74 37 degC air) is about 770 mVUnderstanding of the electrochemical mechanism of BOD-catalyzedreduction of O2 to H2O requires knowledge of the redox potential of theelectron-feeding T1 coppercenter (E T1) and of the potentials of theO2-binding T2T3 copper cluster (E T2T3)

The proposed mechanism for DET-based O2-electroreductioncatalyzed by the O2-reducing multicopper oxidases such as fungallaccases [27] BOD [15] ascorbate oxidase [28] and tree laccase [29] oncarbon electrodes [1030] is shown in the scheme of Fig 4 Brie1047298y thedistance between the electrode-contacting enzyme surface and the T1site at its closest approach is less than 10 Aring [3132] short enough forelectrons to be transferred from the electrode to the T1 site (Fig 4A)but only when the enzyme is uniquely oriented [33] The electron isthen intra-molecularly transferred by a Cys-2His pathway shown as abroken trace in Fig 4 to the T2T3 cluster across a distance of ~13 Aring[1] where it reduces the copper-cluster-bound O2 We saw thepreviously reported [101527] direct electroreduction of O2 ongraphite We also observed the absence of direct O2 electroreductionon MPA-gold electrodes which transfer electrons to the T2T3 clusternot to the T1 site because of the unique orientation of the BOD as waspreviously con1047297rmed for fungal laccases [223435] Most likely suchwas the case also for BOD and bare gold where in absence of covalent

bonding of BOD to the electrode the signal was just above the noiselevel[ [17] Nevertheless the observed fast DET to the T2T3 cluster of BOD from MPA-gold did not provide for electrocatalytic O2-reductionEven though the coverage of the MPA-gold surface with electroactiveBOD was as large as 7 pmol cmminus2 and even though the MPA-goldbound BOD was active in Fe(CN)6

3minus4minusmediated ET with similarinhibition as for BOD-modi1047297ed SPGE in DET mode only a minutecatalytic O2-reduction current was observed in the absence of themediator Notably also laccases directly adsorbed on gold orcovalently immobilized on SAM-gold fail to catalyze O2 electroreduc-tion [10223435]

The T1 electron-relaying site which contains only one coppercation can only have a single redox potential E T1 which ismeasurable by potentiometric redox titration under anaerobic

conditions Indeed the redox potentials of the T1 site (E T1) of M verrucaria and T tsunodae BODs measured by redox couple mediatedpotentiometric titrations were of ca 670 mV and N650 mV [17] TheCV of Fig 1A of T tsunodae BOD-SPGE shows under anaerobicconditions an E m of 690 mV attributed in light of the earlier studiesto the T1 site The O2-electroreduction half-wave potential of T tsu-

nodae BOD on SPGE has exactly the same value 690 mV (Fig 1)Moreover the 670 mV E T1 potential for M verrucaria BOD measuredby redox couple mediated potentiometric titration is consistent withthe 685 mV voltammetric wave of M verrucaria BOD-modi1047297edcarbon nanotubes on which O2 is electroreduced [18] The E mpotential for O2 electroreduction and the E T1 redox potential haveclosely similar values not only for BODs [15] but also for othermulticopper oxidases like ascorbate oxidase fungal and plant laccases

[102729] establishing that the feeding of electrons through the T1

Fig 3 BOD adsorbed on MPA-modi1047297ed gold electrodes in pH 70 01 M phosphatebuffer (A)Cyclicvoltammogramsin theabsence ofO2 without (curve1) andwith(curve2) adsorbed BOD The dashndashdotted line (curve 3) represents the calculated theoreticalvoltammogram using the Marcus-DOS theory of ET and the parameter values indicatedin the text Scan rate10 mV sminus1 starting-scan potential 650 mV Inset Chronoampero-metric response at +500 mV O2 or N2 were bubbled and Fminus was added at the indicatedtimes (B) Linear scan voltammograms of BOD-MPA-modi1047297ed gold electrodes in thepresence of 2 mM K3[Fe(CN)6] The voltammograms were recorded in N2 saturatedbuffer (curve 1) O2 saturated buffer (curve 2) and O2 saturated buffer containing

10 mM Fminus

(curve 3) Scan rate 1 mV sminus1

starting-scan potential 650 mV


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from electrodes resembles their feeding by dissolved substrates intheir homogeneous catalysis of O2-reduction

Prior to this work one of the redox potentials of the BOD T2T3cluster has been estimated [17] but not actually measured The earlierattempted measurement of E T2T3 of BOD by potentiometric titrationcould not provide potentials of the electrochemical states of thecatalytic cycle Several different redox potentials are expected for thestructurally different T2T3 cluster intermediates ie its native andperoxy intermediates [112] its fully reduced BOD (FR) [17] and itsresting form [36] For laccase molecules uniquely oriented for DET onelectrode surfaces the voltammetrically observed E T2T3 is about400 mV at pH 65 [223435] In this study E m values of 390 mV and

360 mV are respectively observed for T tsunodae BOD on SPGE (Fig1A) and on MPA-gold (Fig 3A)

One of the possible explanations for the observed ef 1047297cientmediated but not DET electrocatalysis of O2-electroreduction byBOD on MPA-gold is the formation of a catalytically inactive restingform of the enzyme during DET from gold directly to the T2T3copper cluster The redox couple mediated O2-electroreductioncatalyzing enzyme was normally inhibited by Fminus just like the BODon graphite that did catalyze the direct electroreduction of O2 Whenthe BOD was oriented for proximity of the T2T3 cluster to the MPA-gold which made it non-catalytic in DET mode the ET rate was asfast as the ET between the T1 copper site proximal to graphite (k0 are03 and 04 sminus1 respectively) in the enzyme catalyzing direct O2-electroreduction (Fig 1B)

The observed E T2T3-potential of 360ndash390 mV values of BOD (Fig1A) implies uphill IET from the 690 mV (E T1) over an 300 mV barrierbetween the T1 site andthe T2T3 cluster at least forone intermediateof the enzyme in the catalytic turnover It is certainly possible for theresting catalytically irrelevant form of BOD [36] which might have avery slow intra-molecular electron transfer rate in analogy withresting forms of laccases [123738] It has indeed been suggested forlaccases that the potential of the native intermediate T2T3 cluster ishigher than that of the resting enzyme the native intermediate T2T3cluster slowly decayingto themore stablerestingT2T3 resting cluster[37] We consider it nevertheless likely that there is an uphill andcatalytically relevant ET step in the electroreduction of O2 as seen inFig 1B a sigmoidal wave (marked with an arrow) appears in thevoltammogramsmeasured in the O2-saturated solution at 390mV the

potential of the E T2T3 cluster of T tsunodae BOD (Fig 1) Participation

of the T2T3 cluster in O2 bioelectroreduction could be detectable onlyunder conditions where substrate turnover is limited by the rate of the supply of electrons and should become effective as the appliedpotential approaches its formal potential E T2T3 Indeed the absenceof 1047298at-topped current-potential CVs their sensitivity to change in thepotential scan rate the starting potential of the bioelectrocatalysisvery close to the potential of the T1 site (Figs 1B and 2) all are verystrong pieces of evidence for DET limiting bioelectrocatalysisImportantly because of signi1047297cant DET limitation the standardbiocatalytic rate constant of the adsorbed BOD is suggested to bemuch higher than the calculated kDET eg NN74 sminus1 taking intoaccount the mechanism of enzyme function during DET-based

bioelectrocatalysis [3940] The turnover of BOD towards K4[Fe(CN)6]in homogeneous catalysis was measured to be 380 sminus1 Comparison of two biocatalytic rate constants obtained in heterogeneous andhomogeneous assays (NN74 sminus1 vs 380 sminus1 respectively) allows us toconclude that BOD adsorbed on SPGE is at least quasi-native andde1047297nitely catalytically active

Here for the 1047297rst time we suggest an endergonic tunneling fromthe T1 site to the T2T3 cluster during the biocatalytic reduction of O2

by blue multicopper oxidases It is widely held that many biological ETchains contain an uphill ET step and oxidoreductases are built soindividual ET rates do not have to be optimized [41] For example atthe 13 Aring distance (approximate distance between T1 and T2T3 in allblue multicopper oxidases [1]) with unremarkable values of otherfactors affecting the ET rate the tunneling rate can be as high as few

hundreds sminus

1 fora 03eV endergonic step [41] Interestingly this valuecoincides with the maximal reported turnover numbers of BODsdetermined in homogeneous catalysis (ca 400 sminus1) Indeed furtherstudies of BOD to understand the mechanism of enzyme function arein the scope of our investigations

Acknowledgments

We thank Prof Ulf Ryde at the Department of TheoreticalChemistry Lund University (Sweden) for helpful suggestions regard-ing the structures of the intermediates of blue multicopper oxidasesand functioning of the enzymes TR LG and SS thank the SwedishResearch Council AH thanks the US Of 1047297ce of Naval Research and theWelch Foundation NM thanks funding from a European Young

Investigator Award (EURYI) and la Reacutegion Aquitaine

Fig 4 Proposed mechanisms of DET from electrodes to BOD connected (A) via the T1 site and (B) via the T2T3 cluster


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References

[1] EI Solomon UM Sundaram TE Machonkin Multicopper oxidases andoxygenases Chem Rev 96 (1996) 2563ndash2605

[2] Y Gotoh Y Kondo H Kaji A Takeda T Samejima Characterization of copperatoms in bilirubin oxidase by spectroscopic analyses J Biochem (Tokyo) 106(1989) 621ndash626

[3] K Hiromi Y Yamaguchi Y Sugiura H Iwamoto J Hirose Bilirubin oxidase fromTrachyderma tsunodae K-2593 a multi-copper enzyme Biosci BiotechnolBiochem 56 (1992) 1349ndash1350

[4] J Hirose K Inoue H Sakuragi M Kikkawa M Minakami T Morikawa HIwamoto K Hiromi Anions binding to bilirubin oxidase from Trachydermatsunodae K-2593 Inorg Chim Acta 273 (1998) 204ndash212

[5] K Kataoka K Tanaka Y Sakai T Sakurai High-level expression of Myrotheciumverrucaria bilirubin oxidase in Pichia pastoris and its facile puri1047297cation andcharacterization Protein Expr Purif 41 (2005) 77ndash83

[6] A Shimizu T Sasaki JH Kwon A Odaka T Satoh N Sakurai T Sakurai SYamaguchi T Samejima Site-directed mutagenesis of a possible type 1 copperligand of bilirubin oxidase a Met467Gln mutant shows stellacyanin-like proper-ties J Biochem (Tokyo) 125 (1999) 662ndash668

[7] A Shimizu JH Kwon T Sasaki T Satoh N Sakurai T Sakurai S Yamaguchi TSamejima Myrothecium verrucaria bilirubin oxidase and its mutants for potentialcopper ligands Biochemistry 38 (1999) 3034ndash3042

[8] A Shimizu T Samejima S Hirota S Yamaguchi N Sakurai T Sakurai Type III Cumutants of Myrothecium verrucaria bilirubin oxidase J Biochem (Tokyo) 133(2003) 767ndash772

[9] S Koikeda K Ando H Kaji T Inoue S Murao K Takeuchi TSamejima Molecularcloning of the gene for bilirubin oxidase from Myrothecium verrucaria and itsexpression in yeast J Biol Chem 268 (1993) 18801ndash18809

[10] S Shleev J Tkac A Christenson T Ruzgas AI Yaropolov JW Whittaker LGorton Direct electron transfer between copper-containing proteins and electro-des Biosens Bioelectron 20 (2005) 2517ndash2554

[11] T Sakurai K Kataoka Structure and function of type 1 copper in multicopperoxidases Cell Mol Life Sci 64 (2007) 2642ndash2656

[12] L Rulisek EI Solomon U Ryde A combined quantum and molecular mechanicalstudy of the O2 reductive cleavage in the catalytic cycle of multicopper oxidasesInorg Chem 44 (2005) 5612ndash5628

[13] I BentoMA Carrondo PF Lindley Reduction of dioxygen by enzymes containingcopper J Biol Inorg Chem 11 (2006) 539ndash547

[14] N Mano JL Fernandez Y Kim W Shin AJ Bard A Heller Oxygen iselectroreduced to water on a ldquowiredrdquo enzyme electrode at a lesser overpotentialthan on platinum J Am Chem Soc 125 (2003) 15290ndash15291

[15] S Shleev A El Kasmi T Ruzgas L Gorton Direct heterogeneous electron transferreactions of bilirubin oxidase at a spectrographic graphite electrode ElectrochemCommun 6 (2004) 934ndash939

[16] S Tsujimura T Nakagawa K Kano T Ikeda Kinetic study of direct bioelec-trocatalysis of dioxygen reduction with bilirubin oxidase at carbon electrodesElectrochemistry (Tokyo) 72 (2004) 437ndash439

[17] A Christenson S Shleev N Mano A Heller L Gorton Redox potentials of theblue copper sites of bilirubin oxidases Biochim Biophys Acta 1757 (2006)1634ndash1641

[18] M Weigel E Tritscher F Lisdat Direct electrochemical conversion of bilirubinoxidase at carbon nanotube-modi1047297ed glassy carbon electrodes ElectrochemCommun 9 (2007) 689ndash693

[19] B Ehresmann P Imbault JH Weil Spectrophotometric determination of proteinconcentration in cell extracts containing tRNA and rRNA Anal Biochem 54 (1973)454ndash463

[20] S Trasatti OA Petrii Real surface area measurements in electrochemistry PureAppl Chem 63 (1991) 711ndash734

[21] H Jaegfeldt T Kuwana G Johansson Electrochemical stability of catechols with apyrene side chain strongly adsorbed on graphite electrodes for catalytic oxidation

of dihydronicotinamide adenine dinucleotide J Am Chem Soc 105 (1983)1805ndash1814

[22] M Pita S Shleev T Ruzgas VM Fernandez AI Yaropolov L Gorton Directheterogeneous electron transfer reactions of fungal laccases at bare and thiol-modi1047297ed gold electrodes Electrochem Commun 8 (2006) 747ndash753

[23] O Ruumldiger JM Abad EC Hatchikian VM Fernandez AL De Lacey Orientedimmobilization of Desulfovibrio gigas hydrogenase onto carbon electrodes bycovalent bonds for nonmediated oxidation of H2 J Am Chem Soc 127 (2005)16008ndash16009

[24] K Weber SE Creager Voltammetry of redox-active groups irreversibly adsorbedonto electrodes Treatment using the Marcus relation between rate and

overpotential Anal Chem 66 (1994) 3164ndash

3172[25] SJ Corni The reorganization energy of azurin in bulk solution and in theelectrochemical scanning tunneling microscopy setup J Phys Chem B 109 (2005)3423ndash3430

[26] U Ryde MHM Olsson Structure strain and reorganization energy of bluecopper models in the protein Inter J Quant Chem 81 (2001) 335ndash347

[27] S Shleev A Jarosz-Wilkolazka A Khalunina O Morozova A Yaropolov T RuzgasL Gorton Direct electron transfer reactions of laccases from different origins oncarbon electrodes Bioelectrochemistry 67 (2005) 115ndash124

[28] R Santucci T Ferri L Morpurgo I Savini L AviglianoUnmediatedheterogeneouselectron transfer reaction of ascorbate oxidase and laccase at a gold electrodeBiochem J 332 (1998) 611ndash615

[29] AI Yaropolov AN Kharybin J Emneus G Marko-Varga L Gorton Electro-chemical properties of some copper-containing oxidases BioelectrochemBioenerg 40 (1996) 49ndash57

[30] A Christenson N Dimcheva E Ferapontova L Gorton T Ruzgas L StoicaS ShleevA Yaropolov D Haltrich R Thorneley S Aust Direct electron transfer betweenligninolytic redox enzymes and electrodes Electroanalysis 16 (2004) 1074ndash1092

[31] A Messerschmidt R Ladenstein R Huber M Bolognesi L Avigliano R

Petruzzelli A Rossi A Finazzi-Agro Re1047297ned crystal structure of ascorbateoxidase at 19 Aring resolution J Mol Biol 224 (1992) 179ndash205

[32] K Piontek M Antorini T Choinowski Crystal structure of a laccase from thefungus Trametes versicolor at 190 Aring resolution containing a full complement of coppers J Biol Chem 277 (2002) 37663ndash37669

[33] HBGray JRWinklerElectrontunneling through proteins Quart Rev Biophys 36(2003) 341ndash372

[34] S Shleev A Christenson V Serezhenkov D Burbaev A Yaropolov L Gorton TRuzgas Electrochemical redox transformations of T1 and T2 copper sites in nativeTrametes hirsuta laccase at gold electrode Biochem J 385 (2005) 745ndash754

[35] S Shleev M Pita AI Yaropolov T Ruzgas L Gorton Direct heterogeneouselectron transfer reactions of Trametes hirsuta laccase at bare and thiol-modi1047297edgold electrodes Electroanalysis 18 (2006) 1901ndash1908

[36] T Sakurai L Zhan T Fujita K Kataoka A Shimizu T Samejima S YamaguchiAuthentic and recombinant bilirubin oxidases are in different resting formsBiosci Biotechnol Biochem 67 (2003) 1157ndash1159

[37] S-K Lee S DeBeer George WE Antholine B Hedman KO Hodgson EISolomon Nature of the intermediate formed in the reduction of O 2 to H2O2 at thetrinuclear copper cluster active site in native laccase J Am Chem Soc 124 (2002)6180ndash6193

[38] J Yoon BD Liboiron R Sarangi KO Hodgson B Hedman EI Solomon The twooxidized forms of the trinuclear Cu cluster in the multicopper oxidases andmechanism for the decay of the native intermediate Proc Natl Acad Sci USA 104(2007) 13609ndash13614

[39] A Sucheta R Cammack J Weiner FA Armstrong Reversible electrochemistry of fumarate reductase immobilized on an electrode surface Direct voltammetricobservations of redox centers and their participation in rapid catalytic electrontransport Biochemistry 32 (1993) 5455ndash5465

[40] KA Vincent A Parkin FA Armstrong Investigating and exploiting theelectrocatalytic properties of hydrogenases Chem Rev 107 (2007) 4366ndash4413

[41] CC Page CC Moser X Chen PL Dutton Natural engineering principles of electron tunneling in biological oxidation-reduction Nature 402 (1999) 47ndash52


8132019 1-s20-S0005272808006373-main


natural substrates of BOD butalso from arti1047297cial electron donors suchas redox mediators in homogeneous catalysis or electrodes poised atsuf 1047297ciently reducing potentials in electrocatalysis The acceptedelectrons are transferred to the T2T3 copper cluster where theyreduce O2 [10] Interestingly and signi1047297cantly when BOD or laccaseare embedded (ldquowiredrdquo) in an electron-conducting redox hydrogelwhere electron donors envelope the enzyme such that electrons 1047298owto their T1 centers irrespective of orientation the copper-enzymes are

greatly superior to platinum as electrocatalysts of the four-electronreduction of O2 to H2O [14] Here we illuminate some aspects of themechanism of this unique electrocatalytic process

Electrodes poised at suf 1047297ciently reducing potentials transferelectrons directly to fungal BODs from Myrothecium verrucaria andTrachyderma tsunodae [15ndash17] Even though M verrucaria BOD isreadily O2-oxidized when adsorbed on graphite electron transfer (ET)from graphiteto the adsorbed BOD is observed only at 515 mV vsNHEat pH 74 about 025 V more negative than the reversible potential of the O2H2O redox couple at a low current density [15] Spectro-electrochemical measurements show that this potentialis far from theredox potential of the T1 site (E T1) which is near 670 mV vs NHE atpH 70 [17] In contrast M verrucaria BOD adsorbed on carbonnanotubes is electroreduced already at 685 mV at pH 74 [18] but thereduced Cu2+ centers were believed to be those of the T2T3 clusternot the T1 site On gold very slow DET was observed [17] Here wereport DET results for T tsunodae BOD adsorbed on graphite and onmodi1047297ed gold electrodes Cyclic voltammograms (CV) exhibit clearlyvisible nonturnover waves with midpoint potentials (E m) of about700 mV and 400 mV corresponding to redox transformations of theT1 site and the T2T3 cluster respectively This is the 1047297rst report onwell-pronounced nonturnover signals of electron accepting andoxygen reducing sites of a blue multicopper oxidase coinciding withcorresponding catalytic waves of O2 bioelectroreduction Importantlyour data strongly suggest an uphill intra-molecular electron transferfrom the T1 site to the T2T3 cluster during the catalytic turnover of the enzyme

2 Experimental section

21 Reagents

Na2HPO4 KH2PO4 KCl NaCl H2O2 H2SO4 and NaF were obtainedfrom Merck (Darmstadt Germany) Citric acid 22prime-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and N -(3-dimethylamino-propyl)-N prime-ethylcarbodiimide hydrochloride (EDC) were from Sigma(St LouisMO USA) N -hydroxysuccinimide (NHS) 4-aminothiophenol(AMTP) and 6-mercapto-1-hexanol (MHOL) were from Aldrich (StLouis MO USA) 3-Mercaptopropionic acid (MPA) and NaIO4 werepurchased from Janssen Chimica (Geel Belgium) Absolute ethanol(997) was from Solveco Chemicals AB (Taumlby Sweden) 1-Decanethiol(DT) and K3[Fe(CN)6] were from Fluka (Buchs Switzerland) Allchemicals were of analytical grade Buffers were prepared with water

(18 MΩ) puri1047297ed with a Milli-Q system (Millipore Milford CT USA)Anaerobic and aerobic conditions were established using nitrogen

(N2) or oxygen(O2) gases from AGA Gas AB (SundbybergSweden) thatwere bubbled through the working solutions

22 Enzymes

BOD from Trachyderma tsunodae was from Amano Enzyme Inc(Elgin IL USA) Preparations of BOD were stored at minus18 degC Theconcentration of BOD in the stock solution was determined by theestablished method of Ehresmann [19] The turnover number of BODtowards K3[Fe(CN)6] was determined spectrophotometrically using anUvikon 930 spectrophotometer (Kontron Instruments Everett MAUSA) and was found to be 380 sminus1 in good agreement with a

previously reported value for a wild-type M verrucaria BOD [6]

23 Electrochemical measurements

Electrochemical measurements were performed using a threeelectrode potentiostat (CV-50W Bioanalytical Systems BAS WestLafayette IN USA) The reference electrode was a Hg|Hg2Cl2|KClsatelectrode K401 (SCE 242 mV vs NHE) from Radiometer (CopenhagenDenmark) and the counter electrode was a platinum wire Thesupporting electrolyte consisted of a 01 M phosphate buffer solution

at pH 70

231 Roughness factor and cleaning of the working electrodes

The gold working electrode model MF-2014 was purchased fromBAS its geometrical area was 002 cm2 Its microscopic roughnessfactor was calculated from the charge (qreal) associated with the goldoxide reduction process obtained when running a CV from 0 to1900 mV in 05 M H2SO4 The theoretical charge density (σ t)associated with the reduction of the gold oxide is 390plusmn10 μ C cmminus2

[20] The microscopic area was obtained using the ratio of themeasured charge of gold oxide electroreduction qreal and thecalculated theoretical charge for the 02 cm2 electrode if it weresmoothσ t ( Areal= qreal σ t) The microscopic roughness factor was thusestimated to be 14plusmn01

The gold electrode was pre-cleaned by CV scans at a 100 mVsbetween minus60 and minus1360 mV vs NHE in 05 M NaOH then polishedwith a DP-suspension (high performance diamond product) and analumina de-agglomerated polishing suspension (1 μ m and 01 μ mStruers Copenhagen Denmark) rinsed with Millipore H2O andsonicated in after each polishing step for 10 min The electrode wasthen cleaned by a series of CV scans at a 100 mVs scan rate betweenminus60 and+1790 mV vs NHE in 05 M H2SO4 and kept in concentrated(96) H2SO4 till use Immediately before its use it was rinsedthoroughly with H2O

The spectrographic graphite electrode (SPGE Ringsdorff WerkeGmbH Bonn Germany type RW001 305 mm diameter 13porosity) was polished with wet 1047297ne emery paper (Tufback DuriteP1200) rinsed thoroughly with H2O and allowed to dry Theadsorptive roughness factor of such an electrode was estimated to

be about 5 [21]

232 Thiol self-assembled monolayers and BOD deposition on gold

The procedures for physical adsorption of BOD on gold and forcovalent binding of BOD to AMTP modi1047297ed gold electrodes werereported earlier [22] To form DTand MHOL SAMs the electrodeswererespectively immersed in 1 mM (2080 vv H2Oethanol) or a 5 mM(absolute ethanol) solution of DT or MHOL and the monolayers wereallowed to assemble overnight at room temperature To adsorb BODon bare or modi1047297ed gold surfaces the electrode was mounted with itssurface facing up an aliquot of 5 μ l of the BOD solution (10 mgml) wasplaced on it and was allowed to react for 3 h while every 20 min 5 μ lof H2O were added to avoid complete drying of the BOD solution

To form the MPA monolayer the gold electrode was placed in a

5 mM solution (2575 vv H2Oethanol) of MPA overnight Toimmobilize on it the BOD the procedure of Ruumldiger et al [23] appliedearlier for the immobilization of Desulfovibrio gigas hydrogenase wasused The electrodewas placed with itssurface facing up and 6 μ l of ca25 μ M of the BOD solution in 10 mM phosphate buffer pH 70 wasadded Then 45 μ l of 18 mM NHS and 55 μ l of 36 mM EDC were alsoadded After about 90 min the electrode was washed in pH 70 01 Mphosphate buffer and was promptly used

233 Adsorption of BOD on graphite electrodesA volume of 10 μ l of BOD solution (25 mgml) was placed on the

electrode surface allowed to adsorb and after 15 min the SPGE wasrinsed with H2O

The current densities and enzyme surface concentrations were

estimated using the real area of the electrodes calculated by


8132019 1-s20-S0005272808006373-main



3 Results




















8132019 1-s20-S0005272808006373-main





to 033 μ A cm

minus2



(Fig 3B) [22]

4 Discussion











10 mM Fminus




8132019 1-s20-S0005272808006373-main












hundreds sminus


Acknowledgments





8132019 1-s20-S0005272808006373-main


References














































8132019 1-s20-S0005272808006373-main



3 Results




















8132019 1-s20-S0005272808006373-main





to 033 μ A cm

minus2



(Fig 3B) [22]

4 Discussion











10 mM Fminus




8132019 1-s20-S0005272808006373-main












hundreds sminus


Acknowledgments





8132019 1-s20-S0005272808006373-main


References














































8132019 1-s20-S0005272808006373-main





to 033 μ A cm

minus2



(Fig 3B) [22]

4 Discussion











10 mM Fminus




8132019 1-s20-S0005272808006373-main












hundreds sminus


Acknowledgments





8132019 1-s20-S0005272808006373-main


References














































8132019 1-s20-S0005272808006373-main












hundreds sminus


Acknowledgments





8132019 1-s20-S0005272808006373-main


References














































8132019 1-s20-S0005272808006373-main


References














































Documents

1-s2.0-S0005272808006373-main