Electrochemical studies on c type cytochromes at microelectrodes

Journal of Electroanalytical Chemistry 464 (1999) 76–84

Electrochemical studies on c-type cytochromes at microelectrodes

M.M. Correia dos Santos a,*, P.M. Paes de Sousa a, M.L. Simoes Goncalves a, H. Lopes b,I. Moura b, J.J.G. Moura b

a Centro de Quımica Estrutural, Instituto Superior Tecnico, A6. Ro6isco Pais, 1096 Lisboa Codex, Portugalb Departamento de Quımica, Centro de Quımica Fina e Biotecnologia, Faculdade de Ciencias e Tecnologia, Uni6ersidade No6a de Lisboa,

2825 Monte da Caparica, Portugal

Received 23 July 1998; received in revised form 19 October 1998

Abstract

The aim of this work is to use microelectrodes as a current approach for the study of unmediated electrochemistry of redoxproteins. An electrochemical study of monohemic cytochromes c552 from Pseudomonas nautica 617, cytochrome c553 fromDesulfo6ibrio 6ulgaris and horse heart cytochrome c is presented at inlaid disk microelectrodes of platinum, gold and carbon.Different electrochemical techniques were used such as linear scan, differential pulse and square wave voltammetry. Theelectrochemical response was also analysed at conventional size (macro) electrodes for comparison. In all situations a promoterwas used. The electrochemical behaviour was evaluated in terms of kinetics of the electrode processes and the formal potentialsdetermined. Diffusion coefficients were also calculated from the voltammetric data. A critical comparison of the results obtainedis carried out and the advantages of microelectrodes for electrochemical studies of metalloproteins are pointed out. © 1999Elsevier Science S.A. All rights reserved.

Keywords: Cytochrome c552; Cytochrome c553; Horse heart cytochrome c ; Electrochemistry; Macro and microelectrodes

1. Introduction

The investigation of electrode reactions of redoxproteins has attracted widespread interest over pastyears since they constitute a good approach to redoxprocesses taking place in vivo [1,2]. It is now possible toachieve direct electrochemistry of redox proteins with-out the need of a mediator between the redox center ofthe protein and the electrode. In many circumstances,the presence of a promoter may be required, but al-though such a compound encourages electron transferwith the protein to proceed, it does not take part in theelectron transfer process. Under these conditions, awide range of information can thus be obtained includ-ing determination of redox potentials, evaluation of thenumber of electrons involved and estimation of the rateconstants for electron transfer.

Several c-type cytochromes are among the metallo-proteins that have been investigated by electrochemicalmethods, since Eddowes and Hill found that essentiallyreversible cyclic voltammetry of horse heart cytochromec could be observed at a 4,4%-dipyridyl modified goldelectrode [3,4]. More recently, direct electrochemistry ofhorse heart cytochrome c has been investigated in thepresence of various amino acids [5] and at polymer [6]and cysteine [7] modified gold electrodes. However thequestion of how cytochromes function as electrontransfer proteins is still an up to date subject. Althoughthe question has been widely addressed by differentauthors, important issues related to molecular recogni-tion, electron transfer mechanisms and conformationalchanges coupled to proton and electron transfer are stilla main research topic [8]. Improvements in understand-ing such mechanisms may be expected from compara-tive studies of a major number of protein systems.

In most electrochemical studies of metalloproteins* Corresponding author. Fax: +351-1-846-44-55; e-mail: mcsantos

@alfa.ist.utl.pt.

0022-0728/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.PII: S 0 0 2 2 -0728 (98 )00474 -4

M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–84 77

conventional size electrodes, metal (e.g. platinum, gold)as well as non-metal (e.g. carbon), have been used.However, electrochemistry at microelectrodes (criticaldimension in the range 0.1–50 mm) may present someadvantages over electrodes of conventional size [9]. Oneof them is the high rate of steady state diffusion due toenhanced mass transportation by nonplanar diffusion.So, steady state currents are readily attained and kineticparameters may be determined without capacitive cur-rents [10,11]. Moreover, due to their dimensions it ispossible to work with minimal size samples, an impor-tant feature when working with proteins.

The aim of this work is to investigate whethervoltammetric methods using microelectrodes can be aneffective approach for the study of unmediated electro-chemistry of redox proteins. Three c-type cytochromes,all with the same type of axial ligands at the iron atom,i.e. methionyl and histidinyl residues, were chosen: (i)horse heart cytochrome c, whose electrochemical be-haviour has been widely studied at macroelectrodes[12–16]. This system was recently the subject of acomparative study at macro and microelectrodes [17]and its redox behaviour at a large assembly of mi-croelectrodes was also reported [18–20]; (ii) cytochromec553 from Desulfo6ibrio 6ulgaris previously investigatedby cyclic voltammetry and differential pulse voltamme-try at macroelectrodes [21] and (iii) cytochrome c552

from Pseudomonas nautica 617 isolated from a marinesediment [22] for which no voltammetric data isavailable.

So, in this manuscript, we report the electrochemicalbehaviour of cytochrome c552 at inlaid disk macro andmicroelectrodes of gold, platinum and carbon usingdifferent techniques. Moreover, the electrochemistry ofcytochrome c553 from D. 6ulgaris and horse heart cy-tochrome c is revisited at microelectrodes of platinum,gold and carbon. The redox behaviour of cytochromec553 is also analysed at a gold macroelectrode. Since theelectrochemical behaviour of redox proteins at un-modified electrodes is complex and much dependentupon factors such as the electrode surface conditionsand traces of impurities [2,23,24] in all situationsthroughout this work a promoter is used. The electro-chemical response is evaluated analysing the reversibil-ity of the electrode processes and the formal potentialsdetermined. Diffusion coefficients are also calculatedfrom the voltammetric data. A critical comparison ofthe results obtained with macro and microelectrodes ispresented and the advantages of microelectrodes forelectrochemical studies of proteins are pointed out.

2. Experimental

Horse heart cytochrome c Type VI was obtainedfrom Sigma and used with no further purification. D.

6ulgaris cytochrome c553 and P. nautica cytochrome c552

were purified as described in Refs. [22,25], respectively.4,4%-Dipyridyl dihydrochloride and 4,4%-dithiodipyridinewere purchased from Sigma. All other chemicals usedwere pro-analysis grade and all the solutions were madeup with deionized water from a Milli-Q water purifica-tion system.

Protein solutions with concentrations in the range0.04–0.35 mM were prepared in 0.1 M NaNO3 and in10−2 M phosphate buffer (pH 7.0190.05). The con-centrations of the reduced forms of the proteins weredetermined spectrophotometrically using the followingmolar absorptivities: o550=29500 M−1 cm−1 for horseheart cytochrome c [26,27], o553=23400 M−1 cm−1 forD. 6ulgaris cytochrome c553 [28] and o552=19000 M−1

cm−1 for P. nautica cytochrome c552 [22]. All electro-chemical measurements were done in the presence ofpromoters (1 or 15 mM).

Voltammetric experiments were performed using apotentiostat/galvanostat AUTOLAB/PSTAT10 (withthe low current module ecd for measurements withmicroelectrodes) from Eco-Chemie and the data analy-sis processed by the General Purpose ElectrochemicalSystem GPES 3.2 software package also from Eco-chemie. Different electrochemical techniques were used:linear scan (LS) and cyclic voltammetry (CV), differen-tial pulse (DP) and square wave (SW) voltammetry. InLS scan rates of 2–10 mV s−1 were used while in CVthe scan rate varied between 10 and 500 mV s−1. In DPthe pulse amplitude, DE, was 50 mV while the pulseduration, tp, was 50 ms. In SW the square wave ampli-tude, ESW, was 50 mV, the step height, DESW, was 10mV while the frequency was varied in some experimentsbetween 8 and 50 Hz. In all experiments, the potentialwas varied between an initial value Ei and a final valueEf depending on the redox potential of the cytochromeunder study.

Several working electrodes were tested: Metrohmmacroelectrodes of platinum (Ref. 6.1204.010), gold(Ref. 6.1204.020) and carbon (Ref. 6.1204.000) withdiameters of 3.0 and 2.8 mm and a gold electrode with1.6 mm diameter from Bioanalytical Systems. All mi-croelectrodes used were purchased from BAS: platinum(Ref. NS-PT25), gold (Ref. NS-AU25) and carbon(Ref. MF-2007) with diameters of 22.0, 20.5 and 6.6mm, respectively. Before each experiment (or set ofexperiments) the electrodes were polished using finealumina suspension and washed with deionized water,followed by sonication. Immersion in 0.1 M HNO3 wasrequired, sometimes, in order to restore the backgroundsignal. The electrode radius was estimated before eachset of experiments using the ferro/ferricyanide couple(D=7.84×10−6 cm2 s−1 [29]) in 0.5 M KCl acidifiedto pH 3. Limiting currents at the rotating disk elec-trodes were measured for the calculation of themacroelectrode radius while steady state currents ob-

M.M. Correia dos Santos et al. / Journal of Electroanalytical Chemistry 464 (1999) 76–8478

Table 1Electrochemical behaviour of monohemic cytochromes at microelectrodesa

Gold CarbonProtein Electrode Platinum

DP LS SWTechnique LS DPSW DP LS SW

Parameter/mV

78P. nautica cytochrome c552 (E3/4−E1/4) or W1/2 20065 181 178 68.5 – 177 197– 283 249228234 233274 281 233E1/2 or EP

102 59D. 6ulgaris cytochrome c553 (E3/4−E1/4) or W1/2 – –56 121 101 57 12114 33 –49 –14 31 11E1/2 or EP

118 – –Horse heart cytochrome c (E3/4−E1/4) or W1/2 –57 126 96 70 137–– –270 273 290 265 279 282E1/2 or EP

a Tomes' criterion (E3/4−E1/4), peak width at half height (W1/2) and half wave (E1/2) and peak (EP) potentials (95 mV).

(D=7.84×10−6 cm2 s−1 [29]) in 0.5 M KCl acidifiedto pH 3. Limiting currents at the rotating disk electrodeswere measured for the calculation of the macroelectroderadius while steady state currents obtained in linear scanvoltammetry were used to estimate the radius of themicroelectrodes. The constancy of the values determinedsuggests that the polishing between experiments has onlya small effect on the electrode radius.

In all experiments the auxiliary electrode was a plat-inum wire and the reference electrode was a saturatedcalomel electrode (potential equal to 245 mV vs. SHE)or a silver � silver chloride electrode (potential equal to205 mV vs. SHE).

All measurements were done in deaerated solutionswith oxygen free U-type nitrogen and at T=2091°C.

3. Results and discussion

As is well known, the formal potentials of redoxproteins are very useful in order to understand thebiological reactions in which they may be involved aselectron carriers and voltammetric methods usingmacroelectrodes proved to be valuable tools in supplyingsuch data. However, meaningful thermodynamic poten-tials of metalloproteins require firstly a thorough analysisof the electrochemical results. Marked changes in poten-tial may occur due to, e.g. adsorption phenomena andthe degree of reversibility of the electrode processes.

Diagnostic criteria, when using macroelectrodes, arewell established [30]. The situation may be misleading atmicroelectrodes due to the contribution of sphericaldiffusion to mass transport. Steady state and transientvoltammograms are obtained with microelectrodes onlyat both extremes of short and long time scales dependingon the dimension of the electrode [9].

The reduction of cytochrome c552 from P. nautica 617,cytochrome c553 from D. 6ulgaris and horse heart cy-tochrome c at the different type of microelectrodes was

analysed using different voltammetric methods such aslinear scan, square wave and differential pulse voltamme-try. In all experiments the promoter 4,4%-dipyridyl wasused in a concentration of 15 mM.

Half wave potentials values, E1/2, of LS voltam-mograms and of peak potentials, EP, of SW and DPvoltammograms are summarized in Table 1 for thereduction of the heme proteins at microelectrodes (allvalues referred to the SHE). In Fig. 1, LS and SWvoltammograms are shown for the reduction of cy-tochromes c552 and c553 at a platinum microelectrode.Due to the time regime operating at a microelectrode asigmoidal curve characterised by a steady state current,ISS, is obtained in LS as long as the potential is scanned

Fig. 1. (A) Linear scan voltammograms (6=5 mV s−1) and (B)square wave voltammograms ( f=8 Hz) for the reduction of (I) 0.22mM cytochrome c553 and (II) 0.18 mM cytochrome c552, at a plat-inum microelectrode with r=11 mm. Medium: phosphate buffer 0.01M (pH 7.0)+0.1 M NaNO3+ 0.015 M 4,4%-dipyridyl.


Table 2W1/2 values of differential pulse voltammograms at macro and microelectrodesa

W1/2/mV95

bHorse heartP. nautica D. 6ulgarisProteincytochrome c553 cytochrome ccytochrome c552

ElectrodePt

195 –Macro 9196101Micro 178

Au10594Macro 190

177 102Micro 118

C––Macro 175

– –Micro 197

a Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M.b From [17].

slowly enough [31]. Depending on the radius of theelectrode the sweep rate must be as slow as possible,without encountering the irreproducibility that convec-tive interference causes, but in order to avoid mixed masstransport. Fortunately experimental voltammograms re-main virtually steady state even when the sweep rate isincreased to values in excess of those one may computetaking into account the electrode dimensions [31]. Thiscan be easily checked if during the backward scan thecurrent almost retraces that of the forward scan. Thisretracing feature is characteristic of the steady state andis independent of the kinetics of the electrode process. InSW voltammetry a bell shaped curve is obtained for thenet current no matter what the electrode size [32]. Thesame seems to be true for differential pulse voltammetry.

3.1. Re6ersibility analysis

Steady state voltammograms were analysed in termsof the Tomes' criterion [33]. As can be seen from Table1 the reduction of cytochrome c553 at Pt, Au and Cmicroelectrodes is reversible. The values found for theTomes' criterion are in quite good agreement with thetheoretical ones for a reversible system (56 mV, forT=20°C [30]). The same behaviour was previouslyfound for horse heart cytochrome c at a platinummicroelectrode though the reduction at a gold microelec-trode occurs at the boundary of reversibility [17]. In thereduction of c552 from P. nautica the analysis of steadystate voltammograms also suggests quasi-reversible be-haviour.

Square wave voltammetry at microelectrodes seems tocombine the advantages of both approaches. In particu-lar, in sharp contrast with linear scan voltammetry, thevoltammograms are basically invariant in shape over alarge range of the dimensionless parameter p=4Dt/r2 (D

is the diffusion coefficient, t the square wave period andr the radius of the electrode) which characterises theresponse [32]. Thus they may be well-suited for measure-ments of formal potentials.

Although the net current profiles are bell shaped andsymmetrical for several values of p, the dimensionlessnormalised net current, DCP, is a function of p [32]. Theconstant value obtained for reversible processes in con-ditions of semi-infinite linear diffusion is retained onlyfor small values of p (large radii or high frequencies). Sothe simplest criterion for the reversible square waveresponse at both macro and microelectrodes is given bythe peak width at the half height, W1/2, expressed by [34]:

W1/2= (RT/nF){3.53+3.46jSW2 /jSW+8.1} (1)

where jSW=nFESW/RT, ESW being the square waveamplitude. Experimental values of W1/2, shown in Table1, for the reduction of cytochrome c553 from D. 6ulgarisat the Pt and Au microelectrodes and for horse heartcytochrome c at the Pt microelectrode, agree with thetheoretical one (123 mV for T=20°C) within the exper-imental errors (95 mV). The same is not true for thereduction of cytochrome c552 from P. nautica wherelarger values are observed, as can be seen in Fig. 1. Thepeak due to the reduction of horse heart cytochrome cat the Au electrode is also slightly larger than expectedaccording to a fully reversible process.

Differential pulse voltammograms for the reduction ofthe monohemic cytochromes at macro and microelec-trodes were also analysed in terms of W1/2. From Table2 where W1/2 values of differential pulse voltammogramsat both types of electrodes are shown, one may envisagethat, although no theory has been developed for DPVwith microelectrodes, W1/2 measured with these sensorsmay give us some insight into the degree of revers-ibility as happens with macroelectrodes. Wider peaks


are obtained at both macro and microelectrodesfor irreversible electrochemical reactions, as previouslychecked by LS and SW voltammetry. On the otherhand W1/2 values close to the width of 91 mV for areversible one-electron reduction process [30] are ob-tained for the reduction of cytochrome c553 at Au andPt microelectrodes and horse heart cytochrome c at thePt electrodes. The above behaviour can also be ob-served in Fig. 2 where differential pulse voltam-mograms at macro and microelectrodes are shown forcytochromes c and c552.

Electrochemical studies using a gold macroelectrodewere also performed in D. 6ulgaris cytochrome c553. Themain results obtained at pH 7 are: CV measurementsindicate a reversible behaviour up to scan rates of 500mV s−1 with E0%=E1/2=4 mV; square wave determi-nations lead to E0%=EP=30 mV and W1/2=121 mVwhile in DPV E0% E1/2=EP+DE/2=26 mV andW1/2=94 mV were obtained. So this cytochrome has areversible behaviour both at macro and micro-electrodes.

As was previously mentioned the ionic medium waskept constant throughout the experiments, includingthe presence of the promoter. So one must bear in mindthat the experimental values reported are for theprotein–promoter–electrode interaction. From thevoltammetric behaviour analysed so far, one may saythat 4,4%-dipyridyl is as good in promoting electrontransfer at inlaid disk microelectrodes as at macroelec-trodes of identical material. The same may be true forother substances used as surface modifiers for the pro-motion of direct electrochemistry of redox proteins [16].

Fig. 3. Cyclic voltammograms at a Au macroelectrode with r=1.4mm for the reduction of 0.034 mM cytochrome c552. Scan rate (mVs−1): (a) 10; (b) 25; (c) 50; (d) 75; (e) 100. Medium: phosphate buffer0.01 M (pH 7.0)+0.1 M NaNO3+0.015 M 4,4%-dipyridyl.

3.2. Determination of the heterogeneous rate constant

For those processes that are not reversible the elec-trochemical response contains information regardingthe kinetics of the charge transfer reaction. Recently, asimple analysis of quasi-reversible steady state voltam-mograms was proposed [35] where the heterogeneouscharge transfer rate constant, kS, can be found directlyfrom the values of two easily accessible experimentalparameters (E1/4−E1/2) and (E1/2−E3/4), where E1/2 isthe experimental half-wave potential and E1/4 and E3/4

are voltammetric quartile potentials. The procedurealso yields the formal potential E0%.

The above methodology was used to extract kineticinformation from the steady state voltammograms ob-tained at microdisk electrodes. Cyclic voltammetry atconventional size electrodes was also carried out forcomparison since, as is well known, for a quasi-reversible system, kinetic data can be easily determinedusing Nicholson’s treatment where kS is estimated fromthe peak potentials separation with increasing scanrates [36]. In Fig. 3 cyclic voltammograms obtained ata gold electrode are shown for cytochrome c552. Thereduction of this protein at Pt, Au and C macro-electrodes showed behaviour consistent with quasi-reversible electrochemistry: plots of peak current versussquare root of scan rate, 6, were linear up to a certainvalue after which deviations from linearity became ap-parent, while the peak potential separation increasedwith 6 (Fig. 3).

In Table 3 a summary of the values obtained for theheterogeneous charge transfer rate constant for cy-tochrome c552, together with previous results obtainedfor horse heart cytochrome c [17], is shown and goodagreement exists between kS values determined at bothmacro and microelectrodes. So steady state voltam-mograms recorded using inlaid disk microelectrodesalso provide a practical and convenient means to deter-

Fig. 2. Differential pulse voltammograms (tP=50 ms) for the reduc-tion of (I) 0.20 mM cytochrome c and (II) 0.20 mM cytochrome c552

at (A) a platinum macroelectrode with r=1.5 mm and (B) a platinummicroelectrode with r=11 mm. Medium: phosphate buffer 0.01 M(pH 7.0)+0.1 M NaNO3+0.015 M 4,4%-dipyridyl.


Table 3Values of the heterogeneous rate constants, kS, at macro and mi-croelectrodes*

104 kS/cm s−1

Protein P. nautica a Horse heartcytochrome c552 cytochrome c

ElectrodeAu

891 13090.3MacroMicro 491 Not accessible

PtReversible691Macro

491 ReversibleMicro

C–5091Macro–Micro 2091

a From [17].* Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and

4,4%-dipyridyl 0.015 M.

agreement with our result.

3.3. Calculation of diffusion coefficients

Diffusion coefficients, D, of the monohemic cy-tochromes were also evaluated from the voltammetricdata at microelectrodes. Irrespective of the degree ofreversibility of the electrode reactions steady state cur-rents, ISS, are always achieved in linear scan voltamme-try (as long as 6 is small enough as previouslydiscussed) that are proportional to D [33]:

ISS=4nFDcr (2)

In square wave voltammetry the situation is not sostraightforward since the net current, DIP, is a functionof p=4Dt/r2 as well as of the kinetics of the electrodereaction. For reversible systems, D can be calculatednumerically once DIP is known, finding the minimum ofthe function (DIp

th−DIp) where the theoretical net cur-rent DIP

th is given by:

DIpth=nFAc

' Dptp

DCp (3)

and the dimensionless net current DCP is a function ofp according to

DCP=0.846 p1/2+1.06+0.25 exp (−0.8 p1/2) (4)

valid for nDE=10 mV and nESW=50 mV [32].The values thus computed for the diffusion coeffi-

cients using this technique, together with those fromsteady state voltammograms are shown in Table 4.Emphasis should be put on the good agreement amongthe values within the experimental error of 5–10%, evenin the situations where full reversibility was assumed inthe calculations. This was the case for the reduction ofhorse heart cytochrome c at a gold microelectrode andcytochrome c552 at a carbon microelectrode wheresquare wave net currents for the lowest frequenciesused produced meaningful values of D.

So, for horse heart cytochrome c voltammetric dataat microelectrodes lead to D= (1.790.4)×10−6 cm2

s−1, in good agreement with values determined from

mine kinetic parameters of quasi-reversible reactions ofproteins.

As to the accessible range of kS values in transienttechniques, difficulties arising from charging currentand ohmic polarization impose the upper limit. In thesteady state there is no charging current whatsoever,but a upper limit for the accessible kS values is depen-dent upon the dimensions of the electrode. So as to thereduction of the horse heart cytochrome c at the goldmicroelectrode used, according to the mass transferparameter m=D/r�10−3 cm s−1 (with D�10−6 cm2

s−1), values of kS\10−3 cm s−1 are not accessible forthe experimental conditions used, unless a smaller elec-trode is available. This also agrees with the value(kS�10−2 cm s−1) determined by cyclic voltammetry,thus showing that the electrode reaction is nearly re-versible. Previous electrochemical studies of horse heartcytochrome c at a gold macroelectrode in the presenceof 4,4%-dipyridyl showed that the electrode reaction wasalmost reversible with kS= (1.4 to 1.9)×10−2 cm s−1

as determined by ac impedance measurements [37] in

Table 4Diffusion coefficients, D, for monohemic cytochromes estimated from voltammetric techniques with microelectrodesa

106 D/cm2 s−1

Electrode GoldPlatinum Carbon

LS SWSWTechnique LSLS SW

Protein1.0P. nautica cytochrome c552 – 1.0 – 0.8 0.7

1.02.11.41.32.0 –D. 6ulgaris cytochrome c553

1.2 2.0 1.4 2.0 – –Horse heart cytochrome c

a Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M


Table 5Comparison of formal potentials values, E0% versus SHE, for monohemic cytochromes estimated from voltammetric techniques withmicroelectrodesa

E0%/mV95

Gold CarbonElectrode Platinum

LS DP LSTechnique SWLS SW DP

Protein235––P. nautica cytochrome c552 265270 – –

14 8D. 6ulgaris cytochrome c553 9 14 6 11 4273 257Horse heart cytochrome c 270 273 265 265 -

a Medium: phosphate buffer 0.01 M (pH 7.0)+NaNO3 0.10 M and 4,4%-dipyridyl 0.015 M.

voltammetric data at macroelectrodes as well as byother methods, D=1.14×10−6 cm2 s−1 [38]. For cy-tochromes c553 and c552 diffusion coefficients of (1.690.5)×10−6 cm2 s−1 and (0.990.2)×10−6 cm2 s−1

were determined. These values also agree quite wellwith those determined from CV when a gold macroelec-trode was used: from the slope of the linear region ofthe plot peak current versus square root of the scan rateD=1.5×10−6 cm2 s−1 and D=1.1×10−6 cm2 s−1

were obtained for cytochrome c553 and c552, respec-tively. No literature values are known for comparison,but since the molar masses of all the three cytochromesare alike, similar diffusion coefficients were expected.

3.4. Formal potentials E0%

In the light of the above results the formal potentialsof the monohemic cytochromes were then estimatedfrom the voltammetric data at microelectrodes. For thereversible processes, and assuming the similarity of thediffusion coefficients of both oxidized and reducedforms, E1/2 values from steady state voltammograms, aswell as EP values from square wave voltammograms,are a direct measure of E0% [31,32,39]. As far as DPvoltammograms are concerned it looks like that therelation valid for macroelectrodes E1/2=EP+DE/2 [30]still holds for reversible processes at microelectrodes.This can be seen in Table 5 where a summary of the E0%results obtained in this work is shown. From the goodagreement in all situations between E0% values estimatedusing either EP or E1/2 data at microelectrodes for horseheart cytochrome c and cytochrome c553 from D. 6ul-garis, average values of 26594 mV versus SHE and994 mV versus SHE were computed, respectively.These values are in excellent agreement with previouslydetermined ones of 25595 mV versus SHE for horseheart cytochrome c [4] and 20910 mV versus SHE forc553 [21] and also with the values reported in this workfor c553 at a gold macroelectrode (20914 mV versusSHE).

It is worthwhile to point out that the use of eithersquare wave or differential pulse voltammetry at mi-croelectrodes to estimate formal potentials may be theonly chance at very low concentrations of proteins, dueto the low detection limit achieved by both techniques.Additionally, square wave voltammetry is a faster tech-nique than DPV.

For the reduction of cytochrome c552 from P. nauticathat occurs as a quasi-reversible process for the experi-mental conditions used throughout this work either E1/2

or EP potentials are affected by the kinetics of theelectrode reaction. However, the procedure followed tocompute kS from steady state voltammograms alsoyields the formal potential E0%=E1/2+DE0%, being thecorrection term DE0% also found directly from the dif-ferences (E1/4−E1/2) and (E1/2−E3/4) [35]. From thedata shown in Table 5 an average value of E0%=257mV versus SHE was computed. The electrochemicalresponses of P. nautica cytochrome c552 and D. 6ulgarisc553 were studied in detail in a wide pH range (5–11) bycyclic voltammetry at a gold macroelectrode in thepresence of 4,4%-dithiodipyridine. As indicated in Fig. 4a single pKox for c552 and c553 was determined: 10.60and 10.75, respectively.

A wide range of redox potentials were observed forthe heme proteins studied in this work (Table 5). Cy-tochrome c553 is a member of the cytochrome c super-family. The redox potential of this heme protein ismuch more negative than those of other members ofthis class. The potential of other cytochromes present insulfate reducing bacteria, such as multiheme cy-tochromes (with bis-histidinyl axial ligation) are evenmore negative [25]. The three cytochromes studied herehave the same axial ligands (methionine-histidine). The3D X-ray structure of D. 6ulgaris Miyazaki cytochromec553 was solved at 1.3 A [40]. The structure of ferrocy-tochrome c553 from D. 6ulgaris Hildenborough waselucidated by NMR [41]. The heme axial methionylligand has the same orientation in D. 6ulgaris Miyazakiferricytochrome c553 [25], in D. 6ulgaris Hildenboroughferrocytochrome c553 [42], in P. nautica ferrocytochrome


c552 [22] and in horse heart ferrocytochrome c [43]. Theoverall structures of both redox states of D. 6ulgariscytochrome c553 are similar, but no 3D structure is yetavailable for P. nautica cytochrome c552 (in progress, incollaboration with C. Canbillau). Since the redox po-tential of a cytochrome is the result of multiple contri-butions (electrostatic and hydrophobic environment ofthe heme pocket, hydrogen bonding, heme axial ligandsgeometry, solvent exposure, etc,…) and assuming thatthe main overall structural features are maintainedwithin these cases, we may be observing the result of avery fine tuning of the heme environment in the redoxproperties. Moore and Williams [44] proposed that theNMR chemical shifts of the methionine methyl reso-nance in ferrocytochromes could be used as an indica-tor of the Fe–S bond length (and subtle alterations ofthis parameter). As the bond length shortened, themethyl protons should move further into the cloud ofthe heme group experiencing greater shielding and anupper field shift. A correlation was then proposed withthe redox potential of monoheme cytochromes withmethionine–histidine coordination. The greater theshift (implying shorter Fe–S bond length) the morenegative is the redox potential. However this proposalseems not to have been further supported by furtherNMR work [45] and the differences in Fe–S bondlength may not be the determining factor. The trenddoes not seem to apply in this case (chemical shifts ofmethionine methyl group −3.62 ppm c553, −3.01 ppmhorse heart and −3.50 ppm c552). The redox control isa problem not fully understood yet. However it isinteresting to note that the main event upon altering the

pH values of the medium is a pK transition associatedwith an alkaline transition, most probably related tothe exchange of the methionine axial ligand [42] forboth cytochromes c552 and c553, as can be inferred fromthe pKox values.

4. Conclusions

From the above results microelectrodes proved to beefficient tools to study the unmediated electrochemicalbehaviour of redox proteins. Meaningful values for theformal potentials and diffusion coefficients of theproteins under study were obtained in a variety ofsituations: different microelectrodes were used as wellas different electrochemical techniques.

Steady state currents readily attained due to the verysmall dimensions of the electrodes make the measure-ment of electrochemical kinetics and transport parame-ters straightforward procedures. Namely diffusioncoefficients determined by classical electrochemicalmethods are more susceptible to errors since they leadto a determination of D1/2 and any error in r, c or I willbe squared when used to calculate the diffusion coeffi-cients. On the other hand the use of steady statevoltammetry for determining kinetic parameters over-comes difficulties arising from charging currents andohmic polarization which often plague transient tech-niques. This is particularly true for the quasi-reversiblereactions for which kS can be easily computed withcommercially available microelectrodes. Moreover, themethodology followed to extract kinetic informationfrom steady state voltammograms also provides thedetermination of the formal potentials. Evaluation ofthese parameters are based on the values of the quartilepotentials that are independent of the electrode surfacearea and the concentration of the protein.

The benefits resulting from the use of pulse tech-niques (differential pulse and square wave voltamme-try) are not only the low detection limits achieved. Forreversible reactions the formal potentials are also read-ily accessible by measuring peak potentials. Specialemphasis is given to the results obtained by differentialpulse voltammetry since square wave voltammetry maynot be available in older equipment. From the discus-sion of the results it is apparent that although no theoryhas been developed for DPV with microelectrodes, wellknown procedures to analyse data obtained withmacroelectrodes can be applied to microelectrodes. Fornon reversible processes, due to the mixed regime oper-ating at microelectrodes, evaluation of kinetic parame-ters and/or correction factors is not straightforward forpulse techniques. However, depending on the electrodesize conditions close to reversibility may be achieved,which are the best conditions for the determination ofE0%.

Fig. 4. pH dependence of the redox potentials of D. 6ulgaris cy-tochrome c553 (upper panel) and P. nautica cytochrome c552 (lowpanel), as determined by cyclic voltammetry measurements at a goldmacroelectrode (r=1.6 mm). Medium: 0.1 M NaNO3 and 0.001 M4,4%-dithiodipyridine.


Obviously, due to the small dimensions of microelec-trodes, electrochemistry in ml of solutions is also feasi-ble which is an important aspect when dealing withproteins.

Acknowledgements

This work is within the context of Research ProjectPraxis 2/2.1/QUI/312/94.

References

[1] H.A.O. Hill, N.I. Hunt, Methods in Enzymology, chapter 19,vol. 227, Academic Press, NY, 1993.

[2] F.A. Armstrong, J.N. Butt, A. Sucheta, Methods in Enzymol-ogy, Chapter 18, vol. 227, Academic Press, NY, 1993.

[3] M.J. Eddowes, H.A.O. Hill, J. Chem. Soc., Chem. Commun. 21(1977) 771.

[4] M.J. Eddowes, H.A.O. Hill, J. Am. Soc. 101 (1979) 4461.[5] Z.X. Huang, M. Feng, Y.H. Wang, J. Cui, D.S. Zou, J. Elec-

troanal. Chem. 416 (1996) 31.[6] X. Qu, T. Lu, S. Dong, J. Mol. Catal, A: Chem. 102 (1995) 111.[7] W. Qian, J. Zhuang, Y. Wang, Z. Huang, J. Electroanal. Chem.

447 (1998) 187.[8] F.A. Armstrong, H.A.O. Hill, N.J. Walton, Acc. Chem. Res. 21

(1988) 407.[9] M.I. Montenegro, M.A. Queiros, J.L. Daschbach, Microelec-

trodes: Theory and Applications, NATO ASI Series, vol. 197,Kluwer, Dordrecht, 1991.

[10] K.B. Oldham, C.G. Zoski, A.M. Bond, D.A. Sweigart, J. Elec-troanal. Chem. 248 (1988) 467.

[11] K.B. Oldham, J.C. Myland, C.G. Zoski, A.M. Bond, J. Elec-troanal. Chem. 270 (1989) 79.

[12] W.J. Albery, M.J. Eddowes, H.A.O. Hill, A.R. Hillman, J. Am.Chem. Soc. 103 (1981) 3904.

[13] I. Taniguchi, T. Murakami, K. Toyosawa, H. Yamaguchi, K.Yasukouchi, J. Electroanal. Chem. 131 (1982) 397.

[14] I. Taniguchi, M. Iseki, K. Toyosawa, H. Yamaguchi, K. Yasuk-ouchi, J. Electroanal. Chem. 164 (1984) 385.

[15] M.A. Harmer, H.A. Hill, J. Electroanal. Chem. 170 (1984) 369.[16] P.M. Allen, H.A.O. Hill, N.J. Walton, J. Electroanal. Chem. 178

(1984) 69.[17] P.M.P. Sousa, M.M. Correia dos Santos, M.L. Simoes

Goncalves, Port. Electrochim. Acta 15 (1997) 407.

[18] C. Cai, J. Electroanal. Chem. 393 (1995) 119.[19] P. Bianco, C. Lattuca, Anal. Chim. Acta 353 (1997) 53.[20] P. Bianco, J. Haladjian, S. Fletcher, Eletroanalysis 9 (1997) 307.[21] P. Bianco, J. Haladjian, R. Pilard, J. Electroanal. Chem. 136

(1982) 291.[22] L.M. Saraiva, G. Fauque, S. Besson, I. Moura, Eur. J. Biochem.

224 (1994) 1011.[23] F.N. Buchi, A.M. Bond, J. Electroanal. Chem. 314 (1991) 191.[24] H. Allen, O. Hill, N.I. Hunte, A.M. Bond, J. Electroanal. Chem.

436 (1997) 17.[25] T. Yagi, in: H.D. Peck Jr, J. LeGall (Ed.), Methods in Enzymol-

ogy, vol. 243, Academic Press, New York, 1994, Chap. 8, pp.104.

[26] K.J.H. Van Buuren, B.F. Van Gelder, J. Wilting, R. Braams,Biochim. Biophys. Acta 333 (1974) 421.

[27] E.F. Bowden, F.M. Hawkridge, J.F. Chlebowski, E.E. Bancroft,C. Thorpe, H.N. Blount, J. Am. Chem. Soc. 104 (1982) 7641.

[28] G. Fauque, J.J.G. Moura, S. Besson, L.M. Saraiva, I. Moura,Oceanis 18 (1992) 211.

[29] M. Kakihana, H. Ikenchi, G.P. Sato, K. Tokudo, J. Electroanal.Chem. 108 (1980) 38.

[30] A.J. Bard, L.R. Faulkner, Electrochemical Methods, Fundamen-tals and Applications, Wiley, NY, 1980.

[31] C.G. Zoski, A.M. Bond, C.L. Colyer, J.C. Myland, K.B. Old-ham, J. Electroanal. Chem. 263 (1989) 1.

[32] D.P. Whelan, J.J. O’Dea, J. Osteryoung, K. Aoki, J. Electroanal.Chem. 202 (1986) 23.

[33] A.M. Bond, K.B. Oldham, C.G. Zoski, Anal. Chim. Acta 216(1989) 177.

[34] K. Aoki, K. Maeda, J. Osteryoung, J. Electroanal. Chem. 272(1989) 17.

[35] M.V. Mirkin, A.J. Bard, Anal. Chem. 64 (1992) 2293.[36] R. Nicholson, Anal. Chem. 37 (1965) 1351.[37] M.J. Eddowes, H.A.O. Hill, K. Uosaki, J. Electroanal. Chem.

116 (1980) 527.[38] A. Ehrenberg, Acta Chem. Scand. 11 (1957) 1257.[39] J. Osteryoung, J.J. O’Dea, in: A.J. Bard (Ed.), Electroanalytical

Chemistry, vol. 14, Marcel Dekker, NY, 1986.[40] A. Nakagawa, Y. Higuchi, N. Yasuoka, Y. Katsube, T. Yagi, J.

Biochem. (Tokyo) 108 (1990) 701.[41] D. Marion, F. Guerlesquin, Biochemistry 31 (1992) 8171.[42] H. Senn, F. Guerlesquin, M. Bruschi and K. Wutrich, Biochim.

Biophys. Acta 748 (1983) 194.[43] G.R. Moore, G. Pettigrew, Cytochrome c, vol. 2, Springer

Verlag, Germany, 1990.[44] G.R. Moore, R.J.P. Williams, FEBS Lett. 79 (1977) 229.[45] G.R. Moore, G. Pettigrew, R.C. Pitt, R.J.P. Williams, Biochim.

Biophys. Acta 590 (1980) 261.

..

Documents

Electrochemical studies on c type cytochromes at microelectrodes