Electrochemical behavior of cytochrome c3 from desulfovibrio vulgaris hildenborough

241

J. Electroanal. Chem.. 249 (1988) 241-252 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

ELECI-ROCHEMICAL BEHAVIOR OF CYTOCHROME DESULFOVIBRIO VULGARIS HILDENBOROUGH PROMOTER

PIERRE

AClWlTV

AT THE PYROLYTIC

BIANCO, ABDELHAKIM

MANJAOUI

c3 FROM

GRAPHITE ELECTRODE

and JEAN HALADJIAN

L.aboratoire de Chimie et Electrochimie des Complexes, Laboratoire de Chimie Bact&ienne case 57, Universitt? a’e Provence, Place Victor Hugo, 13331 Marseille Cedex 3 (France ) MIREILLE

du C.N.R.S.,

BRUSCHI

Laboratoire de Chimie BactMenne

du C.N. R.S., B.P. 71, 13277 Marseille Cedex 9 (France)

(Received 23rd February 1988; in revised form 18th March 1988)

ABSTRACT The electron transfer reaction of Desulfovibrio vulgaris Hildenborough cytochrome cs at the mercury and the pyrolytic graphite electrode was investigated using differential pulse and cyclic voltammetry at various pH. Cytochrome cs molecules adsorb onto the pyrolytic graphite surface as shown by film-transfer measurements. The modified pyrolytic graphite electrode (PG/cytochrome es) can be used readily to achieve direct unmediated electron transfer to negatively charged redox proteins such as flavodoxin and ferredoxins.

INTRODUCTION

In general, rapid electron transfers between conventional electrodes and metalloproteins are difficult to achieve; direct rapid electron transfers are observed only in some cases, e.g. with polyheme c-type cytochromes. Fortunately, the modification of electrode surfaces by simple chemisorption has proved to be a simple yet effective method for the promotion of direct electron transfer to redox proteins. Direct electrochemistry at a gold electrode upon which 4,4’-bipyridine or 1,2-bis(4pyridyl)ethylene were adsorbed has been reported [l-5]. Taniguchi et al. have suggested bis-(4pyridyl) bisulfide as a strongly adsorbing and effective promoter for studying horse-heart cytochrome c [6,7]. It seems that electrostatic effects may play a prominent role in the promotion of electron transfer between proteins and electrode. In the case of the pyrolytic graphite (PG) electrode, the reduction of rubredoxin, ferredoxin, flavodoxin and plastocyanin, which are negatively charged at neutral pH, can be enhanced drasti0022-0728/88/$03.50

0 1988 Elsevier Sequoia S.A.

242

tally by adding cations such as Mg2+ [S] or positively charged polymers such as poly-L-lysine [9]. It was suggested by Armstrong et al. [lo] that the effect of cations may be attributed to the formation of transient electrostatic bridging between negatively charged zones on the protein and the electrode surface. Cytochrome cj from Desulfovibrio vzdgaris Hildenborough is a tetrahemic protein found in anaerobic sulfate-reducing bacteria. The four hemes are attached covalently to the polypeptide chain by thioether linkages provided by cysteinyl residues; two histidine residues are used as axial ligands for each heme. Its isoelectric point is 10.5 [ll], and consequently this cytochrome is positively charged at neutral pH. In the electron-carrier chain of D. uulgaris Hildenborough, it can exchange electrons with hydrogenase, flavodoxin or ferredoxin [12]. The redox potentials of the four hemes were determined from EPR spectroscopy measurements [13], and more recently by differential pulse polarography at -0.290, -0.335, -0.345 and -0.375 V (vs. the standard hydrogen electrode) 1141. In the present work, the electrochemical properties of D. vulgaris Hildenborough cytochrome cj at mercury and graphite electrodes, are studied and especially its adsorption onto the graphite electrode. Since this protein is positively charged at neutral pH, it was ~teres~g to test its ability to promote heterogeneous electron transfer between the graphite electrode and negatively charged proteins. EXPERIMENTAL

Cytochrome q from D. vulgaris Hildenborough was prepared and characterized as described previously [15]. Spinach ferredoxin was a kind gift from Dr. J. Nari (Centre de Biochimie et de Biologie Moleculaire du C.N.R.S., Marseille). It was purified according to the procedure described previously [16]. Ferredoxin I from De~~ovibrio de~u~furican~ Norway, flavodoxin from D. v~~go~s H~denborou~ and ferredoxin from Clostridium thermocelfum were prepared and purified as described previously [17-191. All the other chemicals were of reagent grade. Methods and apparatus The working electrode generally was a basal-plane pyrolytic graphite electrode (Le Carbone Lorraine, Paris) denoted further as PG. Additional experiments were performed using other solid (carbon or gold) or mercury electrodes. Prior to each experiment, solid electrodes were polished using ultra-fine emery paper. The auxiliary electrode was a platinum wire and the reference electrode was a Metrohm Ag/AgCl (saturated NaCl) electrode. Throughout this paper, all potentials are given vs. the Ag/AgCl reference electrode, unless otherwise specified; potentials vs. the standard hydrogen electrode (SHE) can be obtained by adding 0.210 V. The buffer solution, which also served as the supporting electrolyte, generally was 0.01 M Tris + HCl at pH 7.6, unless otherwise specified. When studying the effect of pH, buffer solutions were prepared using the (0.01 M: Tris + HCI + 0.01 M sodium acetate + 0.01 M sodium borate) mixed buffer.

243

Oxygen was purged from solutions by bubbling with U-grade nitrogen for 30 min before experiments. Temperature was maintained constant at 25 + 0.1” C. FMN and flavodoxin solutions were handled with a minimum exposure to light and the electrochemical measurements relative to these solutions were performed in a dark cell. For differential pulse polarography (DPP) or voltammetry (DPV) a PAR 174A Polarographic Analyzer equipped with an M 174/70 drop timer was used with a Sefram X-Y chart recorder. For cyclic voltammetry (CV), we used a PAR 173 Potentiostat. A Cary 14 spectrophotometer was employed for optical measurements. RESULTS

Effect of concentration

and pH

The effect of the cytochrome cj concentration on DPP and DPV curves has been studied in 0.01 M Tris + HCl buffer, pH 7.6, at the mercury and PG electrodes. Typical DP voltammograms are shown in Figs. la and b. When using the mercury electrode, two peaks and one shoulder at EP1 = - 0.18 V, EP2 - -0.45 V and EP3 = -0.53 V are detected, as observed in previous work [20]. Peak (1) is an adsorption pre-peak, shoulder (2) and peak (3) correspond to the reduction of hemes

Ip/nA

(a

60.

iI(a)

10

I

0

’

’

-0.2

’

*

-0.4

-

*

-0.6

*

15

20

25

*

-0.6

E/V

Fig. 1. DP polarogmm (a) and DP voltammogram at the PG electrode (b) of a 12 pM D. vulgaris cytochrome cg solution in 0.01 A4 Tris + HCl buffer, pH 7.6. Drop time: 1 s (a); pulse repetition rate: 2 s-1 (b). (. . . . . . ) Background solution. The dependence of the DPP and DPV peak heights on cytochrome cj concentration is given in (c) and (d), respectively.

244

I

fi

-

0.1

1

’

-0.3

c

’

-0.5

’

*

-0.7

1

E/V

Fig. 2. Effect of pH on the DP polarograms of a 15 /.tM D. udguris cytochrome cj solution. Drop time: 1 s.

which takes place in two distinct steps. When using the PG electrode, one very slight shoulder at Eri - - 0.45 V and one peak at Epz = - 0.52 V are detected. A linear dependence of peak heights on concentration is observed in Figs. lc and d in the range investigated. The height of peak (1) in Fig. lc first tends to a plateau, then decreases and attains a second lower plateau. This behavior confirms the existence of adsorption phenomena onto the mercury surface [20]. The effect of pH on the electrochemical behavior of cytochrome c3 has been studied in the pH range 3-11.7 using the mercury electrode. DPP curves were obtained for a drop time of 1 s. Typical polarograms for a 15 PM cytochrome c3 solution are shown in Fig. 2. When the pH is decreased, shoulder (2) and peak (3) begin to lower at pH - 6, then new slight peaks appear. When the pH is increased, shoulder (2) and peak (3) are lowered at pH - 10, then tend to disappear. It is interesting to note that peak (3) decreases more quickly than shoulder (2). Shoulder (2) and peak (3) are fully restored when the pH of the solution is raised back from 3 or lowered back from 11.7 to neutral values. Peak potentials remain virtually unchanged within the pH range 6-10. Cyclic voltammetry has shown that the electrochemical system is fast in the same pH range. Consequently, it can be concluded that the redox potentials of cytochrome cj are not modified significantly

245

in the pH range 6-10. New “acid” or “alkaline” forms appear outside this pH range, as observed previously in the case of cytochrome c3 from D. desulfuricans Norway [21]. Similarly, only minor changes in the UV-visible absorption spectra of oxidized cytochrome cJ from 300 to 600 nm are detected within the same pH range. The positions of the (Y, y and 6 bands remained virtually unchanged when the pH was varied. Only a marked increase of the height of the 6 band was noted above pH - 11. Consequently, it seems that the integrity of the native molecules of D. vulgaris cytochrome cj is preserved within the pH range 6-10. The modifications occurring at extreme pH are sufficiently significant to affect the electrochemical behavior of cytochrome c3, which becomes a slow system, as shown by CV experiments. However, a fast electrochemical response can be reobserved when coming back to neutrality. Ahorption

of cytochrome cj on the graphite electrode surface

The foregoing experiments tend to show that cytochrome c3 can adsorb on mercury. In the case of the graphite electrode, it is possible to study the electrochemistry of attached molecules by employing differential pulse or cyclic voltammetry. Direct and indirect ways of investigation can be proposed using the film-transfer technique (1) by transferring adsorbed cytochrome cg molecules to the supporting electrolyte solution, and (2) by transferring them to a solution containing electroactive substances. The effect of the transferred film on the voltammetric signal gives information about the strength and the extent of adsorption phenomena. Film transfer to the supporting electrolyte solution The adsorbed oxidized film was examined directly by equilibrating the PG electrode in cytochrome c3 solutions for increasing times of exposure and at various concentrations. The experimental procedure was as follows. A freshly polished electrode surface was exposed to a stirred cytochrome c3 solution during a specified time, then the electrode was removed, washed with distilled water and transferred to the 0.01 M Tris + HCl solution (pH 7.6), unless otherwise specified. Differential pulse voltammograms before and after the treatment of the PG electrode by cytochrome c3 solution are shown in Fig. 3A. In this case the treatment was applied for 1 min using a 5 PM cytochrome c3 solution. Two slight peaks (1) V and E,, - -0.53 and (2) at Epl - -0.33 V, respectively, are detected after treatment. The effect of the treatment time of the PG electrode on peak heights and peak potentials was studied at various cytochrome cg concentrations. From these experiments it followed that the peak heights increased when the treatment time was increased and tended to constant values i,, and i,,, which were attained all the more swiftly the higher the cytochrome cj concentration. It was also observed that the peak heights decreased noticeably when the PG electrode remained immersed in the supporting electrolyte for 5 min before recording the DP voltammogram.

246

i

I i

I

2

4

6

8

10

PH

Fig. 3. (A) DP voltage obtained after the transfer of a treated PG electrode ( -)ara WISGE f- - -) to 0.01 M Tris + HCl pR 7.5 buffer scdution. Eleet~odes wefe treatedfor 1 min with a obtained in the absence of 5 PM Lt. utt&wzs cytocbrome q solution.( - . . I. -) DP vo~t~o~~ treatment. (B) Dependence of the potentials of peaks (1) and (2) on pH.

In order to determine the origin of peak (I) in Fig. 3A, DP vuhammograms were recorded using a paraffin wax-~pre~t~ spectroscopic graphite electrode (WISGE) (Fig. 3A). A marked peak was observed at Ep = -0.32 V. As shown in previous work [22], it corresponds to the catalytic reduction of oxygen adsorbed on the graphite surface. DP voltammograms at the treated PG electrode were also recorded at different pH of the support~g electrolyte; in this case we used the (0.01 M Tris + HCl + 0.01 M sodium acetate + 0.01 N sodium borate) mixed buffer. Figure 33 shows the pH dependence of the DPV peak potentials. The potential of peak (2) does not vary with pH, but the shift of ea. 50 mV per pH unit detected for peak (1) resembles the behavior observed in the case of solutions c~nt~ning oxygen. Thus, peak (1) might correspond to the reduction of small amounts of oxygen adsorbed on the electrode surface, which are not eliminated by bubbling. Peak (2), at Epz = -0.53 V, is assigned to the reduction of cytochrome cj adsorbed on the PG electrode surface; peak potential Ep2 agrees well with the value observed for the reduction of cyt~~ome c, in solution (see above).

247

Electrochemical properties of the cytochrome c,-coated PG electrode (cytochrome cj / PG electrode) The foregoing experiments have shown that cytochrome cs molecules adsorb on the graphite surface. Thus a cytochrome c,/PG modified graphite electrode can be obtained simply by immersion into a cytochrome cj solution. The stability and the performances of this modified electrode can be estimated by studying the electrochemical behavior of other molecules. In the case of a graphite surface treated with D. uulgaris cytochrome cj solutions, two apparently opposite effects might therefore be expected to occur, depending on the type of molecules investigated: (a) Adsorbed cytochrome c3 molecules may inhibit the electrochemical response of other electroactive molecules by forming a more or less insulating film. (b) It is known that D. uufgaris cytochrome c3 molecules are positively charged at physiological pH. Since in previous work [8-lo] attention has been drawn to the promotion of direct reduction-reoxidation of negatively charged molecules such as ferredoxins and flavodoxins, it is possible to envisage a promotion of the electron transfer to these negative proteins, which are usually slow electrochemical systems. To obtain more ample information about the capabilities of the cytochrome c,/PG electrode, three types of compounds were investigated: hexacyanoferrate(III), a small electroactive anion which gives fast electron-exchange reactions at polished graphite electrodes; FMN, which adsorbs strongly onto the graphite surface [23]; and negatively charged proteins (flavodoxin and ferredoxins). Effect on hexacyanoferrate(III)

and FMN systems

The cytochrome c,/PG electrode was transferred to solutions containing either 500 PM potassium hexacyanoferrate(II1) or 50 PM FMN. Cyclic voltammograms at a scan rate of 50 mV s-l were recorded just after the transfer (Fig. 4). The cathodic and anodic peaks of the hexacyanoferrate(III)/(II) couple were lowered drastically and the peak separation was increased (the same effects were observed when using a FMN/PG electrode [23]). A lowering of the cathodic and anodic peaks of FMN was also detected when using the cytochrome c,/PG electrode. These experiments show that the film of adsorbed cytochrome c3 molecules which is formed on the graphite surface retards electron transfers in this case. Study of D. vulgaris flavodoxin In previous work [23] we have shown that the presence of a positively charged polypeptide such as poly-L-lysine promotes the electron transfer between the PG electrode and D. uulgaris Hildenborough flavodoxin molecules. The same effect is shown here for the cytochrome cJPG electrode. Cyclic voltammograms for a 76 pM D. uulgaris flavodoxin solution are given in Fig. 5A. When using a bare polished PG electrode, a typical curve with only couple (lc-la) was observed. This couple corresponds to the reduction-reoxidation reac-

248

L

I

’

0.3

0.5

-0.1

0.1

E/v

Fig. 4. Effect of treatment of the PG electrode with cytochrome es on the CV curve potassium hexacyanoferrate(II1) solution. (a) With the untreated polished PG electrode (. ). Scan rate: 50 mV s-l. the cytochrome c3/PG electrode (-

CiC)’

’

)

of a 500 PM

. . . . .), (b) with,

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1

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(‘C) ..* . ..-*- : *...... ** *.’

.

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3

electrode of (A) 76 CM D. vulgaris flavodoxin in thermocellum ferredoxin in 0.45 M Tris+ HCI, pH 7.6; (C) 188 pM D. desulfurican.s Norway ferredoxin I in 0.01 M Tris+HCl, pH 7.6; (D) 140 CM spinach ferredoxin in 0.01 M Tris+ HCl, pH 7.6. Scan rate: 5 mV s-t. (. . . . . .) Cyclic voltanunograms at the untreated PG electrode. Fig. 5. Cyclic voltammograms

at the cytochrome

c,/PG

0.01 M Tris+ HCl, pH 7.6; (B) 150 p M Ckxtridium

249

tion of free FMN. When using the cytochrome c,/PG electrode, an additional couple (2c-2a) was observed at Epzc - - 0.65 V and EpZa - -0.57 V. The peak separation, AEr, of about 0.080 V was consistent with a quasi-reversible one-electron process. As reported in previous work [23], this couple is assigned to the semiquinone/ hydroquinone redox couple of D. uulguris flavodoxin. Study of ferredoxins The direct electrochemistry of ferredoxins can be promoted in a similar manner by using the cytochrome c,/PG electrode. Cyclic voltammetry experiments were conducted with ferredoxin from Clostridium thermocellum, ferredoxin I from D. desulfuricuns Norway and with spinach ferredoxin (Figs. SB, C and D, respectively). At a bare polished PG electrode, no redox process is observed, but a well-shaped couple (lc-la) is detected with the modified PG electrode at Epl, - -0.66 V, E Pla - -0.53 V (B); EPIC- -0.66 V, Epla - -0.54 V (C); Epl, - -0.66 V, Epla = - 0.53 V (D). The peak separations AEP are approx. 0.12 V and the respective half-wave potentials E,,, = -0.59 V, -0.60 V and -0.59 V (i.e. -0.40 V, -0.39 V and -0.40 V vs. SHE), in accord with the values obtained previously [9,24] for the redox potentials, when a promoting agent such as poly-L-lysine is added to the protein solutions. Stability of the cytochrome c3 / PG electrode From the foregoing experiments, it follows that spinach ferredoxin may be chosen as a test system for studying the degree of coverage of the graphite surface with time by cytochrome c3 and the stability of the cytochrome c,/PG electrode. Cyclic voltammograms of a 247 PM spinach ferredoxin solution were recorded at increasing times following the treatment of a freshly polished PG electrode with cytochrome c3 solution at different concentrations. The time dependence of the height of the anodic peak (see Fig. 5) corresponding to the reoxidation of spinach ferredoxin is given in Fig. 6 for three D. uulguris cytochrome cj solutions (1, 3 and 5 @l, respectively). These experiments indicate that the degree of coverage depends on concentration and time, but a unique plateau is obtained for the three concentrations investigated. This plateau corresponds to the limiting coating of the electrode surface. For 5 PM cytochrome c3 solution, the limiting promoting coverage is achieved in less than 1 s. The time-dependent cyclic voltammetric response of spinach ferredoxin solution at the cytochrome c,/PG electrode shows that the peak heights were lowered to approximately one half of their original value upon repeated cycling for 1 h. On the other hand, signals were detected even with a cytochrome cJPG electrode exposed to the air for 17 h before recording. In summary, the cytochrome c,/PG electrode has been found to display efficient promoting properties for studying flavodoxin and ferredoxins. Limitation could result from the desorption of cytochrome c3 molecules, in particular in the presence of other strongly adsorbable molecules. Moreover, a lack in persistence of electro-

250

I,1W 0.4000-0-0~) I--l-d=~ 16 A/A0," A/' ' AA4

f

Fig. 6. Effect of the treatment time of the PG electrode by D. vulgaris cytochrome c3 on the anodic peak height i, observed on the cyclic voltammogram (u = 10 mV s-l) of a 247 pM spinach ferredoxin after film-transfer experiments. Concentrations of the cytcchrome c3 solutions used for the treatment: (0) 5; (0) 3; (A) 1 PM.

chemical response excluded.

due to protein

denaturation

at the electrode

surface is not

DISCUSSION

Cytochrome cs from D. vulgaris Hildenborough constitutes a fast diffusion-controlled electrochemical sytem at mercury and graphite electrodes. Since no pH dependence is observed for its electrochemial behavior within the pH range 6-10, it may be concluded that for this pH range, the redox potentials remain invariant. On the other hand, the shifts in the resonance of some methyl groups in the pH range 5-8 as observed from ‘H NMR spectra measurements have been assigned by Moura et al. [25] to variations in the heme midpoint redox potentials. In fact, it seems that these NMR shifts do not correspond to noticeable variations of the redox potentials. In acidic and in alkaline medium, D. vulguris cytochrome cg becomes electrochemically inactive. It is assumed that the conformation of the molecule is modified due to protonation-deprotonation of acid or basic side chains of some amino acid residues. Effectively, five glutamic acid (pK - 4.3) and twelve aspartic acid (pK - 3.8) residues have been identified in the peptidic chain of D. vulgaris cytochrome c3 [26]. Eight propionic acid (pK - 4.9) chains (2 for each heme) are also present in the cytochrome cj molecule. Twenty lysine and one arginine residue are also found in the peptidic chain. The deprotonation of -NH: groups occurs at alkaline pH (- 10). Thus, it is not surprising that the protonation-deprotonation reactions of these residues have a prominent effect on the conformation of the cytochrome c1 molecule. In previous work on cytochrome cg from D. desulfuricuns Norway [21], it was suggested that hydrogen bonding does play an essential role in the electron transfer process and in maintaining the conformation of the native protein structure. The same conclusions can be put forward in the case of D. uulguris cytochrome c3.

251

As in the case of other redox proteins, the reduction mechanism of cytochrome c3 at mercury or solid electrodes must be described by taking into account adsorption phenomena [27]. The adsorption of D. vulgaris cytochrome c3 molecules is sufficiently strong to lower considerably the rate of electron transfer between the electrode and fast sytems such as hexacyanoferrate(III)/(II). In previous work, it has been found that the relative charge of both the protein and the electrode surface has a profound influence on the electrochemical behavior. Thus if protein molecules carry an excess of negatively charged residues and if their redox potentials lie in a domain where the electrode surface bears a negative capacitive charge, the reduction-reoxidation can be promoted by using positively charged agents such as polycations or poly-L-lysine. Reversible electrochemistry of flavodoxin and ferredoxins was observed directly by using a PG electrode modified through the adsorption of D. vulgaris cytochrome c3 molecules (= cytochrome c,/PG electrode). This electrode was easily prepared by soaking a bare polished graphite surface in D. uulguris cytochrome c3 solution directly, then transferring it into the solution of flavodoxin or ferrcdoxin investigated. Relatively fast responses were obtained and the redox potentials of flavodoxin and several ferredoxins were remeasured. The foregoing experimental features highlight the importance of electrostatic interactions at the protein/electrode interface for explaining the promotion of electron transfer. For ferredoxins and flavodoxin at neutral pH, the apparent absence of any faradaic response is consistent with the unfavorable interaction between the negatively charged electrode and the negatively charged proteins. The modification of the electrode/ solution interface resulting from the adsorption of D. uulguris cytochrome cg allows a closer approach of protein molecules to the electrode surface. Effectively, D. uulguris cytochrome cj molecules are positively charged at neutral pH, since the isoelectric point of this protein is at 10.5. Our experimental results suggest that the positive charges concentrated at the electrode/solution interface can stabilize the repulsive interactions between the electrode and protein molecules. Nevertheless, it seems that this interpretation does not apply to all cases. Mitochondrial horse-heart cytochrome c, which, like D. uulguris cytochrome c3, bears an overall positive charge at neutral pH, is unable to display a similar promoting effect. Other factors such as dipolar moment and superficial charge distribution also have to be considered. On the other hand, since the promoting molecules are immobilized on the electrode surface, it is not excluded that a transient electrostatic complex is formed. A modified electrode such as the cytochrome c,/PG electrode seems to be suitable for studying directly the electrochemistry of proteins, since it does not need the addition of foreign substances.

ACKNOWLEDGEMENT

We thank Dr. F. Guerlesquin present work.

for the purification

of the proteins used in the

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