Journal of Electroanalytical Chemistry 447 (1998) 187–199

The effect of magnesium ion on the electrochemistry of cytochrome cand cytochrome b5 at a gold electrode modified with cysteine

Wen Qian, Ji-Hua Zhuang, Yun-Hua Wang, Zhong-Xian Huang *

Department of Chemistry, Fudan Uni6ersity, Shanghai 200433, People’s Republic of China

Received 14 October 1997; received in revised form 13 January 1998

Abstract

The direct electrochemistry of cytochrome c, cytochrome b5 and Cyt c/Cyt b5 complex has been studied by cyclic voltammetryat a cysteine-modified gold electrode. The results indicate that this functional electrode permits selectively a quasi-reversibleelectrochemical response of cytochrome c only. The near-reversible electrochemical response of cytochrome b5 was stimulated byMg2+ion. From titration experiments, we observed that either the Mg2+ion or cytochrome b5 could improve the cyclicvoltammetric response of cytochrome c in different ways, and proved that the 1:1 protein–protein complex formed in the presenceof Mg2+ion even at higher ionic strength. Meanwhile, we observed that the Mg2+ion and the ionic strength affected theelectrochemical behaviour of cytochrome c more strongly than cytochrome b5. Thus, we propose that there are differentinteraction models involving protein–electrolyte–electrode between cytochrome c, cytochrome b5 and the 1:1 protein complexwith the electrode. The mutual interaction between two proteins was favourable for the heterogeneous electron transfer reaction,and the assumed interaction patterns of the 1:1 protein complex with the electrode were also confirmed by potential-stepchronocoulometry. © 1998 Elsevier Science S.A. All rights reserved.

Keywords: Cytochrome c; Cytochrome b5; Cyt c/Cyt b5 complex; Cyclic voltammetry; Potential-step chronocoulometry

1. Introduction

The electron transfer reaction between cytochrome cand cytochrome b5, as an important system for investi-gating fundamentals regarding long range electrontransfer in biological systems, has attracted consider-able experimental and theoretical attention. Over nearly20 years, the electron transfer between cytochrome cand cytochrome b5 has been studied extensively usingseveral different techniques [1–4]. Several models basedon a protein–protein electron transfer percursor for thereaction have been widely accepted. It is generallyaccepted that a binary protein–protein complex couldbe formed under certain conditions through the saltbridge contributed by the interaction between theresidues of the surface of the two proteins [5,6]. There-

fore, ionic strength, [7,8] which is the crucial factor, hasgreatly influenced the combination between proteins. Inspite of great efforts made in the past decade, there stillremain several important questions not fully under-stood. One of them is the role of small amounts ofphysiological divalent cation on the electron transferbetween cytochrome c and cytochrome b5.

It is well known that when cytochrome c and cy-tochrome b5 take part in the metabolic processes inbiological systems, the couple of Fe(III)/Fe(II) in hemeis involved in the reversible redox reaction [9,10]. Cyclicvoltammetry, as an efficient method to investigate elec-tron transfer reactions between proteins, is being ex-ploited increasingly for obtaining electrochemicalinformation on metalloproteins [11,12]. Some investiga-tors have used direct electrochemistry at a graphiteelectrode and a modified gold electrode to study thebehavior of a mixture of cytochrome c and cytochrome* Corresponding author.

0022-0728/98/$19.00 © 1998 Elsevier Science S.A. All rights reserved.PII S0022-0728(98)00043-6

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199188

b5 [13,14]. Although they observed that cytochrome cor cytochrome b5 can promote mutual electrochemistryunder certain conditions, the true mechanism of theinteraction between the two proteins, or the interactionbetween a binary protein–protein complex and elec-trode remains to be clarified. It has been suggested thatthe electrochemical behaviour of metalloproteins is de-pendent on several factors, such as the nature ofproteins, the solution environments, the sensitivity ofelectrodes, etc. In this paper, we report a cyclic voltam-metry study on cytochrome c and cytochrome b5 usinga gold disc electrode modified with cysteine in enhanc-ing the electron transfer. The cysteine, which containsboth acidic (�COOH) and basic (�NH2) functionalgroups, should be suitable for the electrochemistry ofboth cytochrome c (positively charged protein, ca. +7at pH 7.0) and cytochrome b5 (negatively chargedprotein ca. −8 at pH 7.0) [15,16]. Therefore, both theelectrochemistry of cytochrome c and cytochrome b5

can be monitored simultaneously. The difference ofelectrochemical behavior between proteins alone andprotein–protein complexes suggested that a stronglyelectrostatic interaction existed. On the basis of thedifferent structures of the two proteins we proposeddifferent protein–electrolyte–electrode interactionmodels in illustrating the individual electrochemicalbehaviours of cytochrome c and cytochrome b5.

2. Experimental

2.1. Materials

Horse heart cytochrome c, type VI, was purchasedfrom Sigma and purified by ion-exchange chromatogra-phy using carboxymethyl-cellulose resin (CM-52, What-man Biochemicals) [16,17]. Bovine liver microsomalcytochrome b5 was expressed in Escherichia coli andpurified as described previously [18]. Hepes (4-(2-hy-droxyethyl)-1-piperazineethane-sulfonic acid) was pur-chased from Farco Chemical Supplies. The surfacemodified, L-cysteine, was obtained from B.D.H. as thehydrochloride salt. All other reagents were of analyticalquality. Protein solutions of known concentration wereprepared in Hepes buffer (1 mM, pH 7.0). The ionicstrength was adjusted by adding small amounts ofconcentrated KCl solution.

2.2. Measurements

Electrochemical experiments were performed at 2591°C using a PAR M273 potentiostat galvanostat (PARPrinceton, NJ) which was interfaced to an IBM com-puter and controlled by PAR M270 software. The cellconsists of a conventional three-electrode system with asmall volume sample (V�0.5 ml). The working elec-

trode was a 2 mm diameter gold disc electrode, withplatinum wire-coil as counter electrode and Ag�AgCl�3M KCl as reference electrode. Before each measure-ment, the gold electrode was polished with 0.075 mmalumina and washed with doubly distilled water, soni-cated to remove adhesive alumina, then thoroughlywashed again. Then the electrode was cycled between+1.2 V and −0.6 V versus a Ag�AgCl electroderepeatedly in 1 mM, pH 7.0 Hepes buffer solutioncontaining 10 mM KCl, until the typical pattern of aclean gold surface was obtained. The modification withcysteine was completed by dipping the working elec-trode in 10 mM cysteine for 15 min and rinsing withtwice distilled water. Then the washed electrode wasimmersed immediately into the protein solution. Cyclicvoltammetric experiments were carried out underanaerobic conditions by bubbling anaerobic nitrogengas into the solution for 20 min. During the measure-ments, a stream of humidified nitrogen flowed over thesurface of the solution. Sweep rates of 20–200 mV s−1

were used. All the experiments were repeated severaltimes and the values of half wave potentials, E1/2, whichwere related to the formal reduction potentials andcalculated by the formula (Epa+Epc)/2, (where Epa andEpc are anodic and cathodic peak potentials, respec-tively) were found to be reproducible within 95 mV.All the potentials reported in this paper are referencedto SHE unless otherwise stated. The apparent surfaceexcess of protein was determined by potential-stepchronocoulometry according to the literature [19,20].

3. Results and discussion

3.1. Cyclic 6oltammetry of cytochrome c andcytochrome b5 at the modified electrode

Fig. 1A shows a series of typical cyclic voltam-mograms of cytochrome c at a cysteine-modified goldelectrode at various scan rates. One pair of redox peakswas observed. The half wave potential, E1/2, was mea-sured as +282 mV at 50 mV s−1, suggesting that thevoltammetric response was due to the electron transferof cytochrome c at the electrode [21–23]. We noted thatthe half wave potential changed from +290 to +272mV with a change of the scan rate from 20 to 200 mVs−1, and the values of the peak separation, DEp, whichwere dependent on the scan rate, changed from 105 to158 mV. (Table 1) These results implied that there wasa strong electrostatic interaction between positivelycharged cytochrome c and the electrode at low ionicstrength [24]. However, the peak-shaped voltammetricresponses at all scan rate ranges demonstrated thatmass transport with linear diffusion should be takeninto account. No significant electrochemical response ofcytochrome b5 was observed under the same conditions

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199 189

(see curve a in Fig. 2). This suggests that the electro-chemical reaction of cytochrome b5 cannot occur on thegold electrode modified with cysteine.

3.2. The effect of Mg2+ion on the cyclic 6oltammetryof cytochrome c and cytochrome b5

To understand the electrochemistry further, we stud-ied the Mg2+ion dependence of the cyclic voltammetricbehavior of the two proteins. When adding a smallamount of Mg2+ion into cytochrome c solution, the

electrochemical behavior was improved; its cyclicvoltammogram was distinguished from that in the ab-sence of Mg2+ion just as shown in Fig. 1B. We ob-served well-defined peak-shaped curves even at a scanrate of 200 mV s−1. The values of E1/2 remainedapproximately constant, and the peak separation wassmaller than that in the absence of Mg2+ion, andchanged from 80 to 120 mV at scan rates of 20 to 200mV s−1. (see Table 1) The peak current, Ip, was higherthan that in the absence of Mg2+ion at the same scanrate, and was proportional to the root of the scan rate.These results indicated that the presence of smallamounts of Mg2+ion unlocks a partially locked surface(probably by strong adsorption of cytochrome c on theelectrode), rendering the entire surface electroactiveshowing a quasi-reversible electrochemical process [25].The mass transport to the electrode was dominated bya linear diffusion process. The half wave potential forcytochrome c at a scan rate of 50 mV s−1 was +280mV, which was in good agreement with that reportedpreviously in the absence of Mg2+ion. Hence, theMg2+ion does not influence the values of E1/2 of cy-tochrome c in this range of scan rates. The well-definedpeak-shaped redox curves remained unchanged untilthe Mg2+ion concentration was above 4 mM. AtMg2+ion concentrations above 5.5 mM, the peak cur-rent decreased, the peak separation increased markedly,and resulted in sigmoidal-like curves. (see Table 2 andcurve b, c in Fig. 3A) However, from Table 2 it can beseen that despite the dramatic effect of Mg2+ion on thewave shape, the half wave potentials are essentiallyindependent of Mg2+ion concentration. This impliesthat at higher concentrations, the Mg2+ion competeswith the electrode surface and at the positively chargedcytochrome c, the mass transport occurs by radialdiffusion to the residual electroactive sites. The resultsdemonstrate that the Mg2+cations affect the masstransport rather than the thermodynamics of the elec-tron transfer process.

A steady well-defined peak-shaped response appearedwhen 0.2 mM cytochrome b5, was titrated with MgCl2.The minimum concentration of divalent cation neces-sary to produce a near-reversible voltammogram wasabout 4 mM. The typical voltammogram of cy-tochrome b5 containing 4 mM Mg2+ion is shown ascurve b in Fig. 2. The half wave potential, whichvaried slightly with scan rate, was +8 mV at 50 mVs−1, and was positively shifted compared with thevalues reported previously [26,27]. In the presence of 4mM Mg2+ion, the peak separation was about 74–84mV, and did not vary markedly with different scanrates. (Table 1) The peak currents were proportional toboth protein concentrations and the square root of scanrate, 61/2. Obviously, the best Mg2+ion concentration,for a comparative study of cytochrome c and cy-tochrome b5, was 4 mM. We also noted that at higher

Fig. 1. Cyclic voltammograms of 0.2 mM cytochrome c at goldelectrode modified with cysteine. A: in the absence of Mg2+ion andB: in the presence of 4 mM Mg2+ion. Scan rate: (a) 20 (b) 50 (c) 100and (d) 200 mV s−1. Experimental conditions: pH 7.0, 1 mM Hepes,2591°C.

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199190

Table 1Cyclic voltammetric results of cytochrome c and cytochrome b5 alone at different scan rate

Scan rate/mV s−1 Cyt b5Cyt c

In 4 mM Mg2+No Mg2+ In 4 mM Mg2+

DEp/mV E1/2/mV DEp/mV E1/2/mV DEp/mVE1/2/mV

105 +28420 80+291 +9 7650 +282 120 +281 84 +8 7480 +274 133 +283 93 +8 82

151 +280 109+272 +9100 83158 +281200 120+276 +10 84

DEp obtained from cyclic voltammogram. Protein concentration 0.2 mM, pH 7.0, 1 mM Hepes, 2591°C.

Mg2+ion concentrations (above 20 mM), in spite of thecurrent decreasing slightly, the contribution from thelinear diffusion voltammetric responses still dominatedthe electron transfer process. However, the reversibleformal potential shifted to more positive values. (seeFig. 3B and Table 2) These experimental results demon-strated that the Mg2+ion not only promoted the elec-trochemical behaviour of negatively chargedcytochrome b5, but also shifted the equilibrium redoxpotential.

In summary, from the results shown above, bothcytochrome c and cytochrome b5 undergo a lineardiffusion-controlled electrochemical process in the pres-ence of a small amount of Mg2+ion.

3.3. The effects of KCl on the 6oltammetric beha6ioursof cytochrome c and cytochrome b5 alone

In order to probe the effect of the elec-trode�electrolye-solution interfacial properties on thethermodynamics of the electron transfer process, weperformed the same experiments by adding the sameamount of KCl to the protein solution instead ofdivalent electrolyte MgCl2. No marked promotion ofthe electrochemical response of the two proteins wasobserved even at higher KCl concentration. This indi-cates that a monovalent cation could not promote thedirect electrochemistry of both cytochrome c and cy-tochrome b5. On the contrary, for cytochrome c, higherKCl concentrations (about 50 mM) resulted in a nota-ble inhibition of the linear diffusion heterogeneouselectron transfer reaction between cytochrome c andthe electrode. (Fig. 4A) However, the peak-shapedvoltammetric response with linear diffusion mass trans-port still dominated the electron transfer process. Thevalues of the peak separation, DEp, at different concen-trations of KCl presented in Table 3 demonstrated thatthe peak separation of cytochrome c became biggerwith increasing concentration of KCl, indicating theincrease of the radial diffusion component. On thecontrary, in the presence of 4 mM Mg2+ion, the values

of DEp, of cytochrome b5, remained approximatelyconstant with the increase of KCl concentration. (Fig.4B) This implies that a peak-shaped voltammetric re-sponse contributing by the linear diffusion mass trans-port of cytochrome b5 to the modified electrode was notinfluenced by higher ionic strength. It is noteworthythat the changes in the half wave potential of bothcytochrome c and cytochrome b5 with KCl concentra-tion varied from 5 mM to about 400 mM, were about19 mV for cytochrome c and 8 mV for cytochrome b5.It is suggested that there are different protein–elec-trolyte–electrode properties in these two systems.Therefore, it is necessary for us to propose a differentmechanism to interpret the protein–electrode interac-tion as follows.

Fig. 2. Cyclic voltammograms of 0.2 mM cytochrome b5 at goldelectrode modified with cysteine. a: in the absence of Mg2+ion. b: inthe presence of 4 mM Mg2+ion. c: the blank solution. Experimentalconditions: pH 7.0, 1 mM Hepes, 2591°C, scan rate: 50 mV s−1.

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199 191

Table 2The effect of Mg2+ion concentration on the peak separation ofcytochrome c and cytochrome b5

Cyt c[Mg2+]/mM Cyt b5

DEp/mVE1/2/mV E1/2/mV DEp/mV

120 —0 —28284 13278 744

2827 106 17 76110 1822 88276160 23280 8654

276108 194 39 88

E1/2 and DEp obtained from cyclic voltammogram. Protein concentra-tion 0.2 mM, pH 7.0, 1 mM Hepes, 2591°C, scan rate 50 mV s−1.

per part containing heme possesses −9 charges, thelower part of the molecule has +1 charge. Therefore,the negatively charged electrode surface is not fa-vourable for the binding of cytochrome b5, which has agreat number of carboxyl groups distributed on theheme exposed surface of the protein because of therepulsion between the negatively charged cytochromeb5 and the electrode. As a result, cytochrome b5 showedelectrochemical inactivity at the cysteine-modified elec-trode (depicted in Fig. 5A).

Fig. 3. Effects of Mg2+ion concentration on the cyclic voltam-mograms at gold electrode modified with cysteine. A: 0.2 mM cy-tochrome c. a. 4 mM Mg2+ion. b. 5.5 mM Mg2+ion. c. 20 mMMg2+ion. B: 0.2 mM cytochrome b5. a. 4 mM Mg2+ion. b. 20 mMMg2+ion. c. 50 mM Mg2+ion. Experimental conditions: pH 7.0, 1mM Hepes, 2591°C, scan rate: 50 mV s−1.

3.4. The different interaction models ofprotein–electrolyte–electrode for cytochrome c andcytochrome b5

In order to elucidate the different electrochemicalbehaviour of two proteins, we describe the arrangementof modifier on the surface of the gold electrode asshown in Fig. 5. From the results mentioned above, itis reasonable to assume that at pH 7.0 an electrodedouble layer consisting of the –NH2 group and anegatively charged group of �COO− has formed on thesurface of the electrode. The �COO− group was ori-ented towards the solution, and approached close to thebulk solution. For cytochrome c, which has severallysine groups distributed on the surface of the protein,the modified electrode can provide a sufficient numberof carboxyl groups to interact with the lysine residuesof cytochrome c, leading to a mass transport with lineardiffusion. Our experimental results were consistent withthe hypothesis that the specific recognition betweencytochrome c and the electrode involved the surfacelysine groups of the protein and the functional groupsof the electrode surface through hydrogen bonding orthe salt bridge. Our experimental results also demon-strated that in the absence of electrolyte, electrostaticinteraction between the protein and the electrode wasso strong that the denaturation of cytochrome c oc-curred at the surface of the electrode resulting in apoorly reversible electron transfer process with largerpeak potential separation at high scan rate (see Table 1and Fig. 1A). This strong attraction at low ionicstrength led to cytochrome c being adsorbed on theelectrode with partial denaturation, the denaturedproteins blocked the reversible electron transfer be-tween the cytochrome c in solution and the electrode,producing a poorly reversible electrochemical response.On the contrary, cytochrome b5 is a negatively chargedprotein (net charge is −8 at pH 7.0) with unevencharge distribution on its surface. The hydrophilic up-

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199192

Fig. 4. Effects of ionic strength on the cyclic voltammograms at goldelectrode modfied with cysteine. A: 0.2 mM cytochrome c in thepresence of 4 mM Mg2+ion. a. no KCl b. 50 mM KCl. B: 0.1 mMcytochrome b5 in the presence of 4 mM Mg2+ion. a. no KCl b. 100mM KCl c. 200 mM KCl. Experimental conditions: pH 7.0, 1 mMHepes, 2591°C, scan rate: 50 mV s−1.

ion or KCl did not markedly change the half wavepotential of cytochrome c and, on the other hand, thelinear diffusion electrochemical behaviour of cy-tochrome c was inhibited by higher concentrations ofMg2+ion or KCl, it is possible to consider that theMg2+ion or K+ion did not coordinate specifically tothe carboxyl groups of cytochrome c. Indeed, becauseof the repulsion between positively charged cytochromec and the electrochemically inactive cation, the Mg2+orK+ion could not bind firmly to the surface of cy-tochrome c. It is more likely that the role of lowerconcentration Mg2+or K+ion seems merely to adjustthe ionic strength, and reduce the irreversible adsorp-tion of the denatured cytochrome c at the surface of theelectrode. There is no influence on the thermodynamicprocess. (Fig. 5B) Furthermore, in the presence of highconcentrations of Mg2+or K+ion the electroactive sitesat the negatively charged electrode surface were par-tially blocked by Mg2+or K+ion. Under this circum-stance the electron transfer reaction between positivelycharged cytochrome c and the electrode could occuronly at a few remaining electroactive sites, therefore,the mass transport process was dominated by the radialdiffusion process, resulting in a bigger peak–peak sepa-ration and lower peak current. (Fig. 5C) From theabove results, it is evident that for the positivelycharged cytochrome c both Mg2+and K+cations be-have similarly, affecting the electrochemical mass trans-port process of the protein. We propose that theinteraction of cytochrome c with the electrode involvedstrong electrostatic interaction; the role of smallamounts of Mg2+ion or K+ion is only in reducing thestrong interaction between cytochrome c and the elec-trode rather than influencing the thermodyanimicequilibrium.

We have noted that the voltammetric behaviour ofnegatively charged cytochrome b5 at the surface of themodified electrode was stimulated by addition of smallamounts of Mg2+ion rather than K+ion. Furthermore,the voltammetric behaviour retained the linear diffusionresponse not only at a lower concentration of Mg2+ionbut also at higher concentration. These experimentalresults suggested that the electrostatic interaction be-tween protein–electrolyte–electrode did not play a keyrole in the electron transfer reaction; alternatively, thespecific binding of Mg2+ion with cytochrome b5 mustbe considered. The fact that the Mg2+ion coordinatedwith heme propionate of cytochrome b5 has been con-firmed by NMR studies [26,27]. It increased the positivecharge density of the prosthetic group of proteins,which affects the oxidation state stability of the FeIII/FeII couple in heme, and meanwhile, reduced the elec-trostatic repulsion between cytochrome b5 and themodified electrode. Therefore, the inactive Mg2+ionnot only stimulates the electrochemical activity of cy-tochrome b5 at the modified electrode, but also influ-

From Tables 1 and 3, it is clear that at 50 mV s−1

scan rate, the DEp of cytochrome c in the presence of 4mM Mg2+ion or lower concentration KCl were lessthan that at high concentration of Mg2+ion or KCl.This implies that the denaturation of protein at thesurface of the modified electrode caused by the strongelectrostatic interaction between cytochrome c and theelectrode was reduced by the addition of a smallamount of electrolyte. Again, from the facts that Mg2+

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199 193

ences the thermodynamic redox equilibrium. It wasobserved that, for the system of cytochrome b5 alone,the peak-shaped redox curves changed only slightlyeven at higher Mg2+ion concentration (see Fig. 3B)and, the peak–peak separation remained constant ap-proximately. This implies the linear diffusion compo-nent of cytochrome b5 towards the electrode was notdecreased. The fact, that the half wave potentials wereshifted positively by addition of a large amount ofMg2+ion into the solution, demonstrated that theMg2+ion was simultaneously bound with heme propi-onate groups of cytochrome b5 and the carboxyl groupsdistributed on the surface of electrode, and confirmedfurther that the binding did influence the reduction andoxidation equilibrium and not only the mass transport.(Fig. 5B,C) In summary, the Mg2+ion has played a‘bridge’ role in binding between negatively chargedcytochrome b5 and the modified electrode. Further-more, the cyclic voltammetric response of cytochromeb5 in the presence of Mg2+ion was almost independentof KCl concentration. (Table 3) A peak-shaped redoxcurve remained even at higher KCl concentration, andthe peak–peak separation changed only slightly. Onceagain, these results indicated that the ‘bridge’ bindingaction between cytochrome b5 and the electrode was sostrong that it could not be broken down even at highionic strength. On the other hand, these results alsosuggested that the K+ions as an electrolyte could notaffect both the thermodynamics of the electron transferprocess and the mass transport of cytochrome b5.

Freeman et al. [28] had studied the effects of Mg2+

ion on the voltammetric response of the positivelycharged A. 6ariabilis plastocyanin at the graphite elec-trode. They observed that the linear diffusion compo-nent of the mass transport process was decreased andthe radial diffusion component was increased by addi-tion of Mg2+ion. However, in their paper they did notaddress the possibility that a small amount of Mg2+ioncould promote the linear diffusion electrochemical be-haviour as in the experimental results obtained by us.The difference might result from either the nature of

proteins studied or the different electrodes used. Ourstudies might be more significant for understanding theredox behaviour of metalloproteins at the modifiedelectrode surface, because the results can be related tothat of the redox proteins or their complexes associatedwith biological membranes. Our experimental resultsalso imply that the reduction potential of cytochrome cand cytochrome b5 may be modulated by physiologicalconcentration of Mg2+ions in vivo. In other words, asmall amount of Mg2+ion, existing in different com-partments in vivo, may be favourable for the redoxreaction between cytochrome c and cytochrome b5, orvariation of Mg2+ion caused by a number of factors invivo can be used to control its reactions with differentpartner proteins.

3.5. Cyclic 6oltammetric beha6iour of Cyt c/Cyt b5

complex in the absence of Mg2+ion

There is a fundamental reason why we study theelectrochemistry of cytochrome c and cytochrome b5

using a cysteine-modified electrode, because we couldobtain the cyclic voltammetric response of both cy-tochrome c and cytochrome b5 at the electrode simulta-neously. In addition, the multi-function groups ofcysteine have better electrochemical performance thanother binary functional promoters such as thioglycolicacid, 1,4-bipyridine, etc. [26,29]. This approach allowsus to obtain more detailed information concerning theinteraction between these two proteins. Based on theseadvantages, further experiments were performed tostudy the electrochemistry occurring either on the twoprotein surface or the protein–electrode interface. Asreported previously in this paper, cytochrome c pre-sented a peak-shaped-like electrochemical behaviour inthe absence of Mg2+ion at the modified electrode.When equal molar amounts of cytochrome b5 wereadded into the cytochrome c solution, only one pair ofredox peaks attributed to the redox of cytochrome cwas obtained; no electrochemical response of b5 wasobserved (curve a in Fig. 6). This implied that we did

Table 3The influence of ionic strength on the behaviour of cyclic voltammetry of cytochrome c and cytochrome b5 alone in the presence of 4 mMMg2+ion

Cyt c Cyt b5KCl/mM

DEp/mV Ipa/mADEp/mVE1/2/mVE1/2/mV Ipa/mA

282 92 0.316 10 82 0.2965282 92 0.31615 9 86 0.291

124263 0.31645 84110.3220.2848491 11261 0.303130

263 134 0.292 20 81 0.304213263 173 0.296 18 84 0.300391

E1/2, DEp and Ipa obtained from cyclic voltammogram. Protein concentration 0.2 mM, pH 7.0, 1 mM Hepes, 2591°C, scan rate 50 mV s−1.

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199194

Fig. 5. Schematic representation of the interaction between themodified electrode and the proteins. A: cytochrome c and cytochromeb5 alone in the absence of Mg2+ion. Cytochrome c suffered strongelectrostatic interaction at the surface of the modified electrode andresulted in a conformational change. Cytochrome b5 could not ap-proach to the surface of the modified electrode due to repulsion. B:cytochrome c and cytochrome b5 alone in the presence of a smallamount of Mg2+ion. Both proteins were in good configurationappropriate for facile electron transfer. C: cytochrome c and cy-tochrome b5 alone in the presence of a larger amount of Mg2+ion.With the addition of Mg2+ion into the solution, the electrode surfacebecame positively charged and was partially blocked, being unfa-vourable for the linear diffusion electron transfer process betweencytochrome c and the electrode. For cytochrome b5, the largeramounts of Mg2+ion were bound with the carboxyl groups dis-tributed on the surface of cytochrome b5, and forming a highlypositively charged protein. Although it demonstrated little effect onthe peak current, (Fig. 3B) it was still available for a linear masstransport to the negatively charged electrode. D: The 1:1 Cyt c/Cyt b5

complex in the absence of Mg2+ion. The complex migrated to thesurface of the electrode by means of the electrostatic attractionbetween cytochrome c and the electrode (details see text). E: The 1:1Cyt c/Cyt b5 complex in the presence of 4 mM Mg2+ion. Bothcytochrome c and cytochrome b5 were electrochemically active, butthe Mg2+ion affected the cyclic voltammetric behaviour of cy-tochrome c and cytochrome b5 in different ways (details see text). Thecomplex migrated to the surface of the electrode in a manner parallelto the surface of the electrode. The mutual interaction between twoproteins facilitated heterogeneous electron transfer reaction betweencomplex and the electrode.

+269 mV at a 1:1 molar ratio of proteins, which wasnegatively shifted compared with that of either cy-tochrome c alone in the absence of Mg2+ion (E1/2+282 mV) or in the presence of 4 mM Mg2+ion(E1/2+280 mV). It is noteworthy that, for cytochromec, with the addition of cytochrome b5, both the ca-thodic and anodic peak currents increased (from 0.22 to0.26 mA), but the latter is less than the value (0.32 mA)of cytochrome c mediated with Mg2+ion. So, eventhough cytochrome c cannot promote the electrochemi-cal activity of cytochrome b5 at the electrode, on thecontrary, the cytochrome b5 did stimulate the electro-chemistry of cytochrome c in a different way from theMg2+ion. Obviously, cytochrome b5 not only bound tocytochrome c, moving together with cytochrome c to-wards the electrode, but also modified the redox prop-erty of cytochrome c by shifting the thermodynamicequilibrium of the oxidation state. Furthermore, wevaried the ratio of Cyt c to Cyt b5 in a large range(from 10:1 to 1:10), but still failed to observe thepromoting action of cytochrome c on the electrochem-istry of cytochrome b5. However, it is also interesting tonote that the potential separation of cytochrome creached its minimum value in the equamolar proteinmixture, and the peak current was up to its maximumvalue, compared with those of either the protein-proteinmixtures with other molar ratios or cytochrome c alone.These experimental results can be interpreted in termsof the formation of 1:1 Cyt c/Cyt b5 complex [30]. Weassume that the protein complex, perhaps, approachesthe surface of the electrode by means of the electro-static attraction between the cytochrome c side of the

Fig. 6. Cyclic voltammograms of the protein–protein complex at 1:1molar ratio.a: in the absence of Mg2+ion. b: in the presence of 4 mMMg2+ion. Other experimental conditions are the same as Fig. 1.

not observe the electrochemical promotion of cy-tochrome c to cytochrome b5. At 50 mV s−1 scan rate,the calculated half wave potential of cytochrome c was

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Table 4Summary of cyclic voltammetric results of Cyt c/Cyt b5 mixtures in the presence of 4 mM Mg2+ion at various molar ratios

Cyt c (in 1:1 complex)Ratio Cyt c/Cyt b5 Cyt b5 (in 1:1 complex)

DEp/mV Ipa/mA E1/2/mVE1/2/mV DEp/mV Ipa/mA

2:1 284 110 0.263 15 84 0.252108 0.261 12 831.7:1 0.250287103 0.263 17282 861.4:1 0.252

2741.2:1 99 0.272 17 83 0.25574 0.316 181:1 64270 0.291

108 0.250 17274 761:1.2 0.2722711:1.4 113 0.250 12 82 0.248

113 0.240 171:2 83279 0.248

E1/2, DEp and Ipa obtained from cyclic voltammogram. pH 7.0, 1 mM Hepes, 2591°C, scan rate 50 mV s−1.

1:1 protein complex and the electrode as described inFig. 5D. Because of the repulsion between cytochromeb5 and the electrode, no direct contact accounts for thelack of cytochrome b5 voltammetric response observed.This is just the same as in the case of cytochrome b5

alone described in the previous section. The decreasingpotential separation brought about is possibly due tothe decrease of the electrostatic interaction betweencytochrome c and the electrode. In other words, itimplies that the protein interaction during the forma-tion of the protein–protein complex enhances the elec-tron transfer between cytochrome c and the electrode.Our experiments demonstrate that both cytochrome b5

and the Mg2+ion promote the electrochemistry of cy-tochrome c but in different ways.

The peak separation of cytochrome c in this equimo-lar protein complex became markedly larger with theaddition of KCl up to 0.05 M. Under such conditions,the value of DEp was equal almost to that of cy-tochrome c alone containing 0.05 M KCl. This isbecause the electrostatic interaction between cy-tochrome c and cytochrome b5 has been reduced and itindicated that the complex could not exist in a stableform at higher KCl concentration. Our experimentalresults also demonstrate that the protein–protein com-plex, formed by the salt ‘bridge’, promotes the electro-chemical activity, and that it is sensitive to theconcentration of electrolyte.

3.6. Cyclic 6oltammetric beha6iour of Cyt c/Cyt b5

complex in the presence of 4 mM Mg2+ion

The Cyt c/Cyt b5 mixture was further investigated inthe presence of the promoter Mg2+ion by titratingcytochrome c solution with cytochrome b5. Because ofthe promoting effect of Mg2+ion on the electrochem-istry of the two proteins, we observed not only one pairof well-defined redox peaks of cytochrome c, but alsothe other pair of redox peaks attributed to the redox ofcytochrome b5. (curve b in Fig. 6) It can be seen from

Table 4 that both peak currents of the two proteinswere growing gradually with the addition of cy-tochrome b5 into the cytochrome c solution andreached their maximum values at the protein molarratio of 1:1. And also the peak separations of the twoproteins retains their minimum values, 74 mV (cy-tochrome c) and 64 mV (cytochrome b5). Interestingly,the data in Table 4 indicate that any slight excess ofeither cytochrome c or cytochrome b5 decreases theelectrochemical reversibility. The half wave potentialsof cytochrome c and cytochrome b5 in 1:1 proteincomplex, were +270 mV and +18 mV, respectively.The value of +270 mV was negatively shifted about 10mV with respect to the half wave potential (+280 mV)of cytochrome c alone in the presence of Mg2+ion, butit was consistent with the value of +269 mV obtainedin 1:1 protein complex in the absence of Mg2+ion.Meanwhile, for cytochrome b5, the half wave potential,+18 mV, was positively shifted with respect to that ofcytochrome b5 alone under the same conditions. Weconsidered this result as that the interaction betweenpositively charged cytochrome c and negatively chargedcytochrome b5 altered the charge distribution on thesurface of both proteins, and influenced the stability ofboth heme groups in different directions. These resultsprovide unambiguous evidence showing that the 1:1protein complex was formed under such conditions andmutually stimulates the electrochemical activity of thesetwo proteins on the modified electrode. These resultsare also in good agreement with Hill’s observation ontitration experiments at an edge-plane graphite elec-trode, where the peak separations at a given scan ratedecrease as the cytochrome c to cytochrome b5 ratioincreases until the value is 1:1 [30]. As they clearlyindicate, cytochrome c promotes the electrochemistry ofcytochrome b5, rather than acts as a mediator of elec-trons to it.

To understand the protein–protein interaction andthe electrochemistry of the 1:1 complex further, theionic strength dependence of their cyclic voltammetry

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199196

Table 5The influence of ionic strength on the behaviour of cyclic voltammetry of cytochrome c and cytochrome b5 in 1:1 complex in the presence of 4mM Mg2+ion

Cyt b5 (in 1:1 complex)KCl/mM Cyt c (in 1:1 complex)

E1/2/mV DEp/mV Ipa/mA E1/2/mV DEp/mV Ipa/mA

71 0.311 17 665 0.30328179 0.312 12277 7115 0.294

27745 85 0.295 10 71 0.28284 0.282 1591 83277 0.29594 0.273 27265 81213 0.296

257391 125 0.275 32 86 0.273

E1/2, DEp and Ipa obtained from cyclic voltammogram. pH 7.0, 1 mM Hepes, 2591°C, scan rate 50 mV s−1.

was also studied. Considering cytochrome c first, whenKCl was added to the equimolar protein solution, theDEp gradually increased, the value being 125 mV at 0.4M KCl which is almost identical to the DEp of cy-tochrome c alone at 0.04 M KCl (124 mV). Relatively,the electrolyte, KCl, exerted a stronger effect on thesimple cytochrome c system than the Cyt c/Cyt b5

system. Obviously, the formation of the protein com-plex shielded disturbance from the electrolyte. (Table 5)However, it should be pointed out that for cytochromeb5 in the 1:1 protein–protein system there are markedDEp-ionic strength and E1/2-ionic strength dependencesthan in the protein system alone. This is due to both theelectrostatic interaction and the coordination occurringsimultaneously when the electron transferred betweenthe 1:1 protein–protein complex and the modified elec-trode. The binding of cytochrome b5 in the complexwith Mg2+ion was affected by interacting with posi-tively charged cytochrome c. So, the electrochemicalbehaviour of cytochrome b5 in the complex presentedmarked ionic strength dependence. The electrochemicalbehaviour of cytochrome c in the complex indicatedthat the electrostatic interaction between cytochrome cand cytochrome b5 in the presence of 4 mM Mg2+ion,can been weakened and even broken down by highionic strength. In other words, increasing the ionicstrength to approximately 0.4 M decreased the stabilityof the 1:1 protein complex, and also resulted in thedecrease of the reversibility of the electron transferreaction between cytochrome c and the electrode.Therefore, we can assume that the Mg2+ion and theformation of the 1:1 protein–protein complex are ap-propriate for stimulating both proteins’ electrochemicalactivity on the modified electrode. This conclusion wassupported by the best electrochemical performance(lowest DEp and highest Ip, listed in Table 6) of thissystem in four different systems. Meanwhile, the forma-tion of the protein complex does not alter the channelof heterogeneous electron transfer of cytochrome c andcytochrome b5 at the electrode compared with that ofthe two proteins alone.

The different electrochemical behaviour of 1:1 Cytc/Cyt b5 complex with or without Mg2+ion may reflecttheir different ways of interacting with the electrode.Although it clearly showed that the formation of theCyt c/Cyt b5 complex improved the electrochemistry ofcytochrome c in the absence of Mg2+ion, however,only the cytochrome c side of the complex made con-tact with the electrode, allowing the electron transferreaction. (Fig. 5D) In the presence of Mg2+ion, theinteraction pattern was altered. The 1:1 protein–proteincomplex approached the surface of the electrode in amanner parallel to the surface of the electrode; (Fig.5E); both cytochrome c and cytochrome b5 performedelectron transfer dynamically and individually betweenthe protein and the electrode according to the interac-tion model proposed in previous sections.

3.7. The surface excess of proteins on the modifiedelectrode

In the previous section, we described the cyclicvoltammetry of cytochrome c and cytochrome b5 at agold electrode modified with cysteine. In the presence of4 mM Mg2+ion, the promotion of Mg2+ion to theheterogeneous electron transfer reaction betweenproteins and the electrode has been investigated. To

Table 6Summary of electrochemical parameters of four different proteinsystems

E1/2/mVSpecies Ipa/mADEp/mV

+282Cyt c 120 0.221Cyt b5 —— —

+280Cyt c+Mg2+ 84 0.316Cyt b5 +Mg2+ +8 74 0.321Cyt c (in Cyt c/Cyt b5) 0.26479+269

—Cyt b5 (in Cyt c/Cyt b5) ——Cyt c (in Cyt c/Cyt b5+Mg2+) +270 74 0.310Cyt b5 (in Cyt c/Cyt b5+Mg2+) +18 64 0.312

Experimental conditions: [Mg2+]=4 mM, pH 7.0, 1 mM Hepes,2591°C, scan rate 50 mV s−1.

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199 197

Fig. 7. Qf− t curves obtained from single potential-step chronocoulometry. (a) 0.2 mM cytochrome c in the presence of 4 mM Mg2+ion. (b) 0.2mM cytochrome b5 in the presence of 4 mM Mg2+ion. (c) 0.2 mM cytochrome c in the absence of Mg2+ion. (d) the 1:1 protein–protein complexin the absence of Mg2+ion. (e) the 1:1 protein–protein complex in the presence of 4 mM Mg2+ion. (f) blank for potential step from −400 mVto +400 mV vs. (Ag�AgCl), pH 7.0, 1 mM Hepes, 2591°C.

confirm the mode of interaction and the different be-haviour of cytochrome c and cytochrome b5, we mea-sured the surface excesses, Go, of two proteins alone aswell as the 1:1 protein complex on the surface of theelectrode, respectively, by potential step chronocou-lometry. Fig. 7 shows the charge-time (Qf� t) be-haviour, which was obtained as the potential changesfrom −400 mV to +400 mV vs Ag�AgCl. The surfaceexcess of proteins at the electrode calculated by anintercept of the plot of Qf� t1/2 according to the fol-lowing equation: [19,20]

Qf=2nFAc0(D0t/p)1/2+nFAG0+Qdl

where n is the number of electrons transferred; A is theaverage area of the electrode, and the c0 and D0 are theprotein concentration and diffusion coefficient, respec-tively. In order to obtain exact values of G0, the capac-itive charge, Qdl, was corrected by double potential stepchronocoulometry. We determined the values of G0 ofsix protein systems, and calculated the average ofmolecular area of these proteins, interacting with themodified electrode at the surface of electrode. Theseexperimental results are listed in Table 7. In the pres-ence of Mg2+ion, the surface excesses of cytochrome cand cytochrome b5 are 1.16×10−11 mol cm−2 and1.09×10−11 mol cm−2, respectively, corresponding tothe molecular area, 1432986 A2 and 1470994 A2.The experimental values agreed well with the valuereported previously, 1107�2372 A2, of adsorbed cy-tochrome c at a tin oxide working electrode at differentionic strength and different pH, reported by Bowden et

al. [31,32]. These values also suggested a single molecu-lar layer interaction between the protein and themodified electrode. It should be emphasized that themolecule area of the 1:1 protein complex, 29549370A2, is almost equal to the combination of individualcytochrome c and cytochrome b5, and demonstratedthat both cytochrome c and cytochrome b5 have inter-acted with the electrode simultaneously under experi-mental conditions. This result supports the mechanismpresented before in this paper.

However, in the absence of Mg2+ion, we obtained asmaller surface excess, 0.76×10−11 mol cm−2 of the1:1 protein complex and a bigger molecular area,21729200 A2 (compared with that of cytochrome c orcytochrome b5 alone). A reasonable explanation is be-cause of the repulsion between cytochrome b5 and theelectrode, the complex could not fully contact with theelectrode in a parallel manner, just as described Fig.5D. The value, 18659147 A2, of cytochrome c alone inthe absence of Mg2+ion, is slightly bigger than that of1432986 A2, of cytochrome c alone in the presence ofMg2+ion. This result is appropriate for a change in anative configuration and physiological conformation ofcytochrome c [32] in the absence of Mg2+ion at thesurface of the electrode.

These experiments are in good agreement with themechanism of the heterogeneous electron transfer reac-tion of protein alone and the protein–protein complexwith the modified electrode. It confirms that Mg2+ionis important for the recognition of the protein and theelectrode and supported the mechanism of the hetero-

W. Qian et al. / Journal of Electroanalytical Chemistry 447 (1998) 187–199198

Table 7The surface excess and the molecular area of cytochrome c and cytochrome b5 alone and the Cyt c/Cyt b5 complex at a modified electrodedetermined by potential-step chronocoulometry

Cyt b5 Cyt c/Cyt b5Cyt c

4 mM Mg2+ No Mg2+No Mg2+ 4 mM Mg2+ No Mg2+ 4 mM Mg2+

1.161011 G/mol cm−2 —0.89 1.09 0.76 0.561432 — 14701865 2172A/A2 2965

geneous electron transfer reaction between proteins andthe electrode modified with cysteine. Meanwhile, weassume that there is no direct interaction between thetwo heme groups of cytochrome c and cytochrome b5

just as described in Fig. 5D,E. Possibly, the 1:1 proteincomplex formed by binding carboxyl groups with lysinegroups adjacent to the heme groups of two proteins,distributed on the side of the two proteins.

4. Conclusion

In order to obtain a rapid and near-reversible elec-tron transfer, we investigated the cyclic voltammetry ofcytochrome c, cytochrome b5 and the Cyt c/Cyt b5

mixtures using a gold electrode modified with cysteine.The electrode gave rise to a sigmoidal-like cyclicvoltammetric behaviour of cytochrome c at high scanrate. No electrochemical response was observed oncytochrome b5. Titrations of cytochrome c with Mg2+

ion or cytochrome b5 demonstrate that the addition ofsmall amounts of Mg2+ion or equimolar cytochromeb5 improves the electrochemical behaviour of cy-tochrome c on the electrode in different ways, butcytochrome c does not promote the electrochemicalactivity of cytochrome b5. The peak-shaped voltam-mogram of cytochrome c is slightly improved by asmall amount of Mg2+ion, but the increasing presenceof Mg2+ion caused a deterioration in its electrochemi-cal performance. The fact that the half wave potentialschanged slightly with addition of Mg2+ion implies thatthe cation affects the mass transport rather than theredox process of cytochrome c. The electrochemicalprocess of cytochrome b5 is stimulated by addition ofdivalent cation, Mg2+ion, and the half wave potentialsshifts positively with the increase of Mg2+ion. Theseresults suggest that the Mg2+ion is bound to cy-tochrome b5, and modified its electrochemical be-haviour by shifting the redox equilibrium. Therefore,we proposed different interaction models involvingprotein–electrolyte–electrode to interpret differentvoltammetric behaviour of cytochrome c and cy-tochrome b5 at the modified electrode.

The best electrochemical behaviour of cytochrome cand cytochrome b5 is achieved in the 1:1 Cyt c/Cyt b5

system with the presence of 4 mM Mg2+ion. This isclearly shown by the electrochemical parameters DEp

and Ip, as crucial criteria. (Table 6) It is apparent fromour experiments that the electron transfer betweenproteins and the electrode, and reduction potential ofthe proteins are affected by the nature of the proteins,the pretreatment of electrode, the ionic strength and theconcentration of promoter. The formation of anequamolar protein complex and a possible interactionmode between proteins and the electrode are proposedto explain the results observed. This explanation, sup-ported by the cyclic voltammetry study, is also verifiedby the influence of the electrolyte KCl on the electro-chemical properties, and further confirmed by the re-sults of the potential-step chronocoulometry.

Finally, it should be stressed that the promotioneffect of small amounts of Mg2+ion on the heteroge-neous electron transfer reaction between either cy-tochrome c or cytochrome b5 and the electrode and onthe formation of the 1:1 protein–protein complex evenat higher ionic strength could be significant in furtherunderstanding the effect of Mg2+on the reduction ofproteins in biological systems. Recently, several investi-gators suggested that the Mg2+ion plays an importantrole in the conformational change of proteins [33]. Theexact role of Mg2+ion on the protein structure andfunction remains to be clarified.

Acknowledgements

This work is supported by the National ScienceFoundation of China and the State Key Laboratory ofGenetics of Fudan University.

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