- Email: [email protected]
The dependence of reversible potentials on the form of modification of edge plane pyrolytic graphite electrodes in voltammetric studies on rubredoxin and ferredoxin from Clostridium pasteurianum
www.elsevier.nl/locate/elecom Electrochemistry Communications 1 (1999) 309–314
The dependence of reversible potentials on the form of modification of edge plane pyrolytic graphite electrodes in voltammetric studies on rubredoxin and ferredoxin from Clostridium pasteurianum Zhiguang Xiao a, Megan J. Lavery a,1, Alan M. Bond b,*, Anthony G. Wedd a a
School of Chemistry, University of Melbourne, Parkville, Vic. 3052, Australia Department of Chemistry, Monash University, Clayton, Vic. 3168, Australia
b
Received 2 June 1999; accepted 7 June 1999
Abstract Reversible voltammetry of the anionic rubredoxin and ferredoxin proteins at a pyrolytic graphite edge plane electrode can be observed either by ex situ modification of the electrode with multivalent cations (poly(L-lysine), Cr(III) complexes) or by addition of these cations to the solution. However, poly(L-lysine) binds irreversibly to pyrolytic graphite and voltammograms obtained at an ex situ form of this modified electrode provide convenient reference points for estimation of the thermodynamic significance of solution phase interactions between the cationic modifiers and the proteins. Results obtained show that significant shifts in reversible potential are induced by the presence of free modifier in solution. Examination of the recombinant proteins and certain mutant forms suggests that association of the anionic proteins with cationic modifiers in solution is the primary cause of this shift. The effect of background ionic strength is minor in comparison. The conclusion is that the modifiers added to the solution phase are not thermodynamically innocent. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Square wave voltammetry; Rubredoxin; Ferredoxin; Reversible potentials; Electrode modification
1. Introduction
In principle, voltammetric techniques should provide ready access to the reversible potential of electron transfer metalloproteins. However, it has proven to be very difficult to observe well-defined voltammetric responses at bare electrodes, and chemically modified electrodes have usually been employed in voltammetric studies of these important biological molecules [1–4]. However, even when modified electrodes are used, acquisition of stable, reversible electrochemistry depends crucially upon the nature of the protein, the electrode and the specific redox-inactive surface modifier employed. For example, pyrolytic graphite and similar electrodes carry excess negative charge due to the presence of oxygenated surface groups [1,5]. Positively charged redox proteins such as cytochrome c give reversible voltammetry * Corresponding author. Fax: q613-9905-4597; e-mail: a.bond@ sci.monash.edu.au 1 Present address: Department of Biochemistry, G08, University of Sydney, Sydney, NSW 2006, Australia.
at such surfaces but negatively charged proteins usually require the introduction of a multivalent cationic modifier such as Mg2q, [Cr(NH3)6]3q, poly(L-lysine) or selected antibiotics [1,3,5–9] to observe any response in the region of the reversible potential. For many years it has been assumed that the electrode modification does not affect the thermodynamics of the electron transfer process. However, recent studies suggest that, at least in some cases, the modifier may influence the voltammetrically determined reversible potential. That is, the voltammetrically measured value need not be the same as that determined by classical potentiometric methods. For example, the recombinant cytochrome b5 of rat microsomal outer membrane (examined at a gold electrode modified with bmercaptopropionic acid) shows progressive positive shifts as the concentration of poly(L-lysine), Mg2q or [Cr(NH3)6]3q used to facilitate electron transfer at carbon electrodes is increased [7]. For poly(L-lysine), the observed half-wave potential levelled off at q8 mV versus SHE, a shift of at least 60 mV relative to values obtained at low modifier concentrations. At this point, the modifier:protein
1388-2481/99/$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII S 1 3 8 8 - 2 4 8 1 ( 9 9 ) 0 0 0 5 4 - 5
Monday Aug 09 10:28 AM
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
310
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
ratio was 2:1 and the authors concluded that a non-covalent 2:1 complex had formed. Similarly, the voltammetrically determined reversible potential for plastocyanins has been shown to depend on the concentration of Mg2q [10]. The iron–sulfur proteins rubredoxin (Rd, Fe(S-Cys)4; 6129 Da) and ferredoxin (Fd; 2=Fe4S4; 6203 Da) from Clostridium pasteurianum (Cp) exhibit stable, reversible (one-electron) cyclic voltammetry at an edge plane pyrolytic graphite (PGE) electrode in the presence of [Cr(NH3)6]3q [1,5], although conditions have been discovered for Fd and for the iron–sulfur enzyme aconitase where no multivalent cation is needed [11,12]. The present work reports the square wave voltammetry (SWV) of the recombinant and certain mutant forms of Rd and Fd at the PGE electrode in the presence of poly(L-lysine) and Cr(III) cationic electrode modifiers. The modifiers were adsorbed to the electrode surface prior to contact with the protein solution or were added to that solution and the reversible potentials calculated in order to determine the influence of the nature of the chemically modified electrode on the value determined. Commonly, cyclic voltammetric studies have been used to calculate the reversible potential. However, in this study, the far more sensitive square wave voltammetric method has been used to enhance the ability to extract the Faradaic response from the background when low levels of electrode modification are employed. Furthermore, the square wave response is not affected by the dc component of the experiment for a reversible process, so that reversible potentials are readily calculated even if only part of the electrode surface is modified, as will commonly be the case [4,13,14].
2. Materials and methods Recombinant (rRd, rFd) and mutant (G10A-Rd, G10VRd, D33,35,39N-Fd) forms of rubredoxin and ferredoxin from C. pasteurianum (Cp) were generated in E. coli as host and isolated as described elsewhere [15–17]. Unlike the native form, recombinant Rd is not formylated at the N terminus. The protein samples exhibited absorbance ratios of A490/A280s0.43 for rubredoxins and A390/A280s0.80 for ferredoxins. Poly(L-lysine) (degree of polymerisation, 20) was purchased from Sigma Chemicals. [Cr(en)3]Cl3 was prepared by a literature method [18]. [Cr(en)3]3q in water undergoes hydrolysis to form an equilibrium containing mononuclear and binuclear ions [19]. Voltammetric experiments employed a Cypress CS-1087 computer-controlled potentiostat. A standard three-electrode system was used. The working electrode was a 5 mm diameter disc of edge-cleaved pyrolytic graphite (Le Carbone-Lorraine) housed in a sheath of epoxy resin. The electrode was polished manually using a 0.3 mm alumina–water slurry followed by thorough rinsing with deionised water. The counter electrode was Pt wire. A AgNAgCl (saturated KCl) reference electrode was constructed and its potential (199 mV versus SHE at 258C) was verified against a saturated calomel elec-
Monday Aug 09 10:28 AM
trode (244 mV versus SHE at 258C). All potentials are reported relative to SHE. All experiments were carried out at 258C. The electrochemical data reported were acquired by SWV [5,20–23]. Reversible potentials were obtained directly from the value of maximum current of the square wave voltammogram. Solutions were typically 80 mM protein in 30 mM Tris– HClq0.1 M NaCl (pH 7.4). Dioxygen was removed by bubbling a slow stream of humidified, oxygen-free nitrogen through the solutions. The same gas passed across the solution surface during electrochemical measurements. Titration of protein solutions with modifiers involved the addition of aliquots of poly(L-lysine) (10 mM) or CrIII (160 mM) in buffer.
3. Results 3.1. Ex situ electrode modification by cations Smith and Feinberg [11,23] report a SWV peak current of about 11 mA at a PGE electrode (3 mm diameter) for a Fd solution (150 mM; Tris–HCl, 50 mM, pH 8) in the absence of added multivalent cations. Important aspects in obtaining this response appear to include: 1. anaerobic handling, which would minimise apo-protein contamination and consequent electrode blockage by irreversible adsorption of protein to the electrode, and 2. electrode pre-treatment by sonification of the electrode dipped in soap solution, which may lead to adsorption of detergent molecules and consequent masking of the negative charge on the electrode. The observations reported in [23] are confirmed in the present work. In addition, stable square wave voltammetric responses (;0.1 mA/mM) were observed when the PGE electrode employed in this work was modified by dipping into a 10 mM solution of poly(L-lysine) for 2 min followed by careful rinsing with distilled water, gentle removal of surface water by contact with absorbant tissue and placement in rFd or rRd solution (Fig. 1(a)). In contrast to the work of Smith and Feinberg, anaerobic handling of protein solutions prior to purging the cell of air for electrochemistry was not necessary. Table 1 contains the reversible potential determined from this kind of experiment. The observed peak potential obtained at this poly(L-lysine) modified electrode was independent of the square wave period t and plots of peak current Ip versus ty1/2 were linear (Fig. 1(b)), indicating the presence of reversible, diffusion-controlled electron transfer processes [20]. The peak widths at half-height of 127"1 mV (Es, 1 mV; Ew, 50 mV; ty1, 30 Hz) can be compared with the value of 126 mV theoretically expected for a reversible one-electron process under the conditions employed. When the electrode was modified with a hydrolysed [Cr(en)3]3q solution (160 mM: see Section 2, Materials and Methods), reversible responses were also observed but with lower peak currents which decayed upon repeated
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
311
weak and unstable voltammetric responses. Whilst the processes are reversible under all conditions of electrode modification, a key question to be addressed in this work is whether the measured reversible potentials are influenced by the modification of the electrode. Consequently, data obtained below consider results obtained by in situ forms of electrode modification and comparison with the data obtained by the ex situ method. 3.2. In situ addition of electrode modifier 3.2.1. Enhancement of peak currents as a function of solution phase modifier concentration When poly(L-lysine) (average charge, 20q) or [Cr(en)3]3q (fresh or hydrolysed) multivalent cations were added to the protein solutions, voltammetric responses were readily observed at the PGE electrode. No response was detected in the absence of modifier. The efficiency of the enhancement as estimated by the peak current magnitude under the same voltammetric conditions varied with the cation and with the protein. In rRd solutions, an optimum and stable response was reached at modifier:protein ratios of 0.1:1 for poly(L-lysine) and at 1:1 and 100:1 for hydrolysed and fresh ([Cr(en)3]3q) solutions, respectively.
Fig. 1. (a) Square wave voltammograms of rRd (80 mM; 30 mM Tris–HCl, pH 7.4; 0.1 M NaCl) at an ex situ poly(L-lysine)-modified PGE electrode: top trace, first scan; bottom trace, 20th scan (Ew, 50 mV, Ess1 mV, ty1, 30 Hz). (b) Plot of first scan peak current Ip vs. ty1/2.
Table 1 Reversible potentials a for proteins at poly(L-lysine) PGE electrodes determined in the presence and absence of poly(L-lysine) in solution b Protein
rRd G10A-Rd G10V-Rd rFd f
Reversible potential/mV vs. SHE Ex situ modified electrode c
Extrapolated value d
Limiting value e
y77 y104 y119 y400
y78 y104 y119 y400
y48 y74 y90 )y370
a Determined from the peak potential of square wave voltammograms ("4 mV). b 80 mM protein in Tris–HCl (30 mM; pH 7.4), NaCl (100 mM). c Modified by poly(L-lysine): see Experimental. d Estimated (e.g. Fig. 2) by extrapolation of solution phase modifier:protein concentration ratio to zero. e Estimated as potential where the reversible potential becomes independent of modifier concentration (when slope of e.g. Fig. 2(b), approaches zero). f Equivalent results obtain for E17Q, D39N and D33,35,39N mutant forms.
scanning of the potential. Equivalent experiments with Mg2q and fresh [Cr(en)3]3q solution produced reversible, but very
Monday Aug 09 10:28 AM
3.2.2. Effect of modifier concentration upon peak potential Reversible potentials determined from SWV as peak potentials were studied as a function of molar ratio of cationic modifiers to protein (Fig. 2; Table 1). In order to obtain a reproducible response at low solution modifier concentrations, the electrode used in these studies was modified ex situ with poly(L-lysine). Positive potential shifts were observed under these conditions with increasing concentrations of each modifier, the molar shifts being largest for poly(L-lysine) (average charge, 20q). The shifts were largest at low cation to protein ratios and approached limiting values at higher ratios. The effects of variable ionic strength do not account for the shifts in Ep at low modifier levels which are less than 50 mV/M for both rRd and rFd (as estimated by variation of NaCl concentration; see also Refs. [24,25]). However, the ionic strength term may be responsible for the minor variation of peak potential at high modifier:protein ratios (Fig. 2). As might be predicted, the peak potentials at zero modifier levels estimated by extrapolation (Table 1) were indistinguishable from those observed for the ex situ poly(L-lysine) modified electrodes discussed in Section 3.1. The voltammetric behaviour of rRd and its G10A and G10V mutant forms [16] at a PGE electrode modified ex situ by poly(L-lysine) in the absence and presence of poly(Llysine) in solution are compared in Fig. 3 and Table 1. The reversible peak potentials are different for the three proteins but their dependence on the solution phase modifier:protein concentration ratio is similar. On the other hand, for rFd and its E17Q, D39N and D33,35,39N mutant forms [17], both peak potentials and dependence on modifier:protein ratio are identical, within experimental error ("4 mV).
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
312
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
4. Discussion
Fig. 2. Dependence of the reversible potential (SWV Ep value) of rCpRd obtained at an ex situ poly(L-lysine)-modified PGE electrode on the solution phase molar concentration ratios [modifier]/[protein]: (a) square wave voltammograms at different solution phase poly(L-lysine) levels; (b) plots of Ep vs. [modifier]/[protein. Other conditions are as in Fig. 1.
Fig. 3. Dependence of reversible potentials (Ep values) obtained by SWV for r-CpRd, G10A-Rd and G10V-Rd at an ex situ poly(L-lysine)-modified PGE electrode on the solution phase molar concentration ratio [poly(Llysine)]:[Rd]. Other experimental conditions are as in Fig. 1.
Monday Aug 09 10:28 AM
Both poly(L-lysine) and Cr(III) appear to adsorb onto and hence modify the surface of a PGE electrode, so that reversible voltammetry of rubredoxin and ferredoxin may be observed. Stable, reversible SWV peaks were observed for both rRd and rFd at the ex situ modified poly(L-lysine) electrode: peak currents were unchanged after 20 scans (Fig. 1), indicating irreversible adsorption of the modifier to the electrode. Ex situ Cr(III)-modified electrodes, whilst enabling a reversible process to be observed, did not provide stable electrochemistry: peak currents decreased significantly with each successive scan. Apparently, these cations cannot prevent progressive protein denaturation from blocking the electrode or their affinities for the protein molecules exceed that for the electrode surface. Related cationic centres have been attached covalently to PGE electrodes by reduction of [Cr(NH3)6]3q in aqueous ammonia [26]. Well-behaved cyclic voltammetry was observed for the anionic protein plasticyanin at such electrodes. The reversible half-wave potentials for the recombinant cytochrome b5 of rat microsomal outer membrane showed a pronounced dependence upon the solution phase concentration of poly(L-lysine), Mg2q and [Cr(NH3)6]3q [7]. Consequently, it was concluded that the reduction potential of this protein may be modulated in vivo by Ca2q or Mg2q ions or by cationic electron transfer protein partners. For plastocyanins, Ep is a function of Mg2q concentration [10] and the present results suggest that a modulation or related mechanism is available to rubredoxin and ferredoxin systems. Addition of modifiers to rRd or rFd solutions caused reversible potentials to shift positively with increasing modifier:protein ratios (Fig. 2). Thus, these reagents are not innocent in a thermodynamic sense. Ion association of the negatively charged metalloprotein and positively charged modifier is the likely cause, leading to coulombic stabilisation of the reduced form of the protein relative to the oxidised form or to a conformational change which perturbs the protein active site. The details of such interactions have been examined in a number of systems [7,25,27], and in rRd, in particular [28]. The potential shift effect levels off with increasing modifier concentration as cation binding sites on the protein surface become saturated (Fig. 2). Extrapolation of reversible SWV peak potential Ep versus relative solution phase concentration plots (Fig. 2) to zero modifier concentration provides Ep values which are indistinguishable from those estimated using the ex situ poly(L-lysine)-modified electrodes (Table 1). These values are also independent of the modifier used and agree, within experimental error ("4 mV), with values estimated previously by SWV for Rd (y74 mV) and Fd (y400 mV) [5,11,23]. The potential of y55 mV reported for rRd at a PGE electrode in a solution containing MgCl2 (150 mM) is consistent with a positive shift induced by Mg2q [28].
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
The behaviour of the mutant proteins provides some further insight into the role of poly(L-lysine). Mutation of residue Gly 10 to Ala or Val (side chain H to CH3 or CH(CH3)2) perturbs the active site of Rd, thus altering the reversible potential [16]. However, this mutation does not alter the molecular charge and so the similar dependence upon modifier:protein ratio observed (Fig. 3, Table 1) is expected. Mutation of surface carboxylate residues (D, E) of rFd to the sterically equivalent carboxamides (N, Q) changes the molecular charge of 11y on the oxidised form [17], but change to the stereochemistry of the protein surface is expected to be minimal. Since the voltammetric properties of E17Q, D39N (charge 10y) and D33,35,39N (charge 8y) mutant forms are indistinguishable from that of rFd, it can be assumed that the high positive charge of poly(L-lysine) (average, 20q) dominates the solution phase properties of the complex. In summary, reversible electrochemistry of Rd and Fd at a PGE electrode can be facilitated by either ex situ modification of the electrode surface with cationic species or in situ addition of multivalent cations to the solution. Use of an ex situ poly(L-lysine)-modified PGE electrode provides a convenient method of estimation of the reversible potentials of Rd and Fd which are independent of the nature of the modifier when measured potentials are extrapolated to zero solution phase modifier concentration. Association of anionic proteins with cationic modifiers in solution is believed to be the primary cause of the positive shifts in reversible potential induced by increasing solution phase modifier:protein concentration ratios. The effect of background ionic strength to reversible potential shifts is minor. Thus, solution forms of these multivalent modifiers are not thermodynamically innocent. The influence on the thermodynamics of surface bound forms of the modifiers is not so clear cut. If the role of surfaceattached materials is solely to prevent surface blockage by denatured protein so that an uncomplicated diffusion-controlled process is observed, then considerations akin to those described recently by Honeychurch and Rechnitz [29] may need to be considered. Thus, while this specific issue has not been addressed herein, the ex situ form of modification may also not be fully innocent in a thermodynamic sense.
5. Abbreviations
A Cp D d E E. Es Ep Ew Fd
alanine Clostridium pasteurianum aspartate electrode diameter glutamate Escherichia step height peak potential square wave amplitude ferredoxin
Monday Aug 09 10:28 AM
G Ip N PGE Q Rd r SHE SWV t V
313
glycine square wave peak current asparagine edge plane pyrolytic graphite electrode glutamine rubredoxin recombinant standard hydrogen electrode square wave voltammetry square wave period valine
Acknowledgements A.G.W. thanks the Australian Research Council for financial support under Grant A29330611.
References [1] F.A. Armstrong, H.A.O. Hill, N.J. Walton, Acc. Chem. Res. 21 (1988) 407. [2] F.A. Armstrong, A.M. Bond, H.A.O. Hill, B.N. Oliver, I.S.M. Psalti, J. Am. Chem. Soc. 111 (1989) 9185. [3] F.A. Armstrong, Adv. Inorg. Chem. 38 (1992) 17. [4] A.M. Bond, Anal. Proc. 30 (1993) 218. [5] F.A. Armstrong, H.A.O. Hill, B.N. Oliver, N.J. Walton, J. Am. Chem. Soc. 106 (1984) 921. [6] F.A. Armstrong, P.A. Cox, H.A.O. Hill, V.J. Lowe, B.N. Oliver, J. Electroanal. Chem. 217 (1987) 331. [7] M. Rivera, M.A. Wells, F.A. Walker, Biochemistry 33 (1994) 2161. [8] H.A. Heering, Y.B.M. Bulsink, W.R. Hagen, T.E. Meyer, Biochemistry 33 (1995) 14675. [9] F.A. Armstrong, S.J. George, A.J. Thomson, M.G. Yates, FEBS Lett. 234 (1988) 107. [10] D.D. Niles McLeod, H.C. Freeman, I. Harvey, P.A. Lay, A.M. Bond, Inorg. Chem. 35 (1996) 7156. [11] E.T. Smith, D.W. Bennett, B.A. Feinberg, Anal. Chim. Acta 251 (1991) 27. [12] J. Tong, B.A. Feinberg, J. Biol. Chem. 269 (1994) 24920. [13] A.M. Bond, Anal. Proc. 29 (1992) 132. [14] A.M. Bond, Inorg. Chim. Acta 226 (1994) 293. [15] S.D.B. Scrofani, P.S. Brereton, A.M. Hamer, M.J. Lavery, S.J. McDowall, G.A. Vincent, R.T.C. Brownlee, N.J. Hoogenraad, M. Sadek, A.G. Wedd, Biochemistry 33 (1994) 14486. [16] M. Ayhan, Z. Xiao, M.J. Lavery, A.M. Hamer, K.W. Nugent, M. Guss, S.D.B. Scrofani, A.G. Wedd, Inorg. Chem. 35 (1996) 5902. [17] P.S. Brereton, M.J. Lavery, P.A. Tregloan, A.G. Wedd, Biochim. Biophys. Acta 1429 (1999) 307. [18] R.D. Gillard, P.R. Mitchell, Inorg. Synth. 13 (1972) 184. [19] H.L. Schlafer, O.Z. Kling, Anorg. Allg. Chem. 287 (1956) 296. [20] J.H. Christie, J.A. Turner, R.A. Osteryoung, Anal. Chem. 49 (1977) 1899. [21] J.A. Turner, J.H. Christie, M. Vukovic, R.A. Osteryoung, Anal. Chem. 49 (1977) 1904. [22] J.J. O’Dea, J. Osteryoung, R.A. Osteryoung, Anal. Chem. 49 (1977) 695. [23] E.T. Smith, B.A. Feinberg, J. Biol. Chem. 265 (1990) 14371.
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
314
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
[24] R.C. Prince, M.W.W. Adams, J. Biol. Chem. 262 (1987) 5125. [25] G. Battistuzzi, M. Borsari, D. Dallari, S. Ferretti, M. Sola, Eur. J. Biochem. 233 (1995) 335. [26] F.A. Armstrong, P.A. Cox, H.A.O. Hill, B.N. Oliver, A.A. Williams, J. Chem. Soc., Chem. Commun. (1985) 1236.
Monday Aug 09 10:28 AM
[27] G. Cheddar, G. Tollin, Arch. Biochem. Biophys. 278 (1990) 265. [28] Q. Zeng, E.T. Smith, D.M. Kurtz Jr, R.A. Scott, Inorg. Chim. Acta 242 (1996) 245. [29] M. Honeychurch, G.A. Rechnitz, J. Phys. Chem. 101 (1997) 7472 and Refs. therein.
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
The dependence of reversible potentials on the form of modification of edge plane pyrolytic graphite electrodes in voltammetric studies on rubredoxin and ferredoxin from Clostridium pasteurianum Zhiguang Xiao a, Megan J. Lavery a,1, Alan M. Bond b,*, Anthony G. Wedd a a
School of Chemistry, University of Melbourne, Parkville, Vic. 3052, Australia Department of Chemistry, Monash University, Clayton, Vic. 3168, Australia
b
Received 2 June 1999; accepted 7 June 1999
Abstract Reversible voltammetry of the anionic rubredoxin and ferredoxin proteins at a pyrolytic graphite edge plane electrode can be observed either by ex situ modification of the electrode with multivalent cations (poly(L-lysine), Cr(III) complexes) or by addition of these cations to the solution. However, poly(L-lysine) binds irreversibly to pyrolytic graphite and voltammograms obtained at an ex situ form of this modified electrode provide convenient reference points for estimation of the thermodynamic significance of solution phase interactions between the cationic modifiers and the proteins. Results obtained show that significant shifts in reversible potential are induced by the presence of free modifier in solution. Examination of the recombinant proteins and certain mutant forms suggests that association of the anionic proteins with cationic modifiers in solution is the primary cause of this shift. The effect of background ionic strength is minor in comparison. The conclusion is that the modifiers added to the solution phase are not thermodynamically innocent. q 1999 Elsevier Science S.A. All rights reserved. Keywords: Square wave voltammetry; Rubredoxin; Ferredoxin; Reversible potentials; Electrode modification
1. Introduction
In principle, voltammetric techniques should provide ready access to the reversible potential of electron transfer metalloproteins. However, it has proven to be very difficult to observe well-defined voltammetric responses at bare electrodes, and chemically modified electrodes have usually been employed in voltammetric studies of these important biological molecules [1–4]. However, even when modified electrodes are used, acquisition of stable, reversible electrochemistry depends crucially upon the nature of the protein, the electrode and the specific redox-inactive surface modifier employed. For example, pyrolytic graphite and similar electrodes carry excess negative charge due to the presence of oxygenated surface groups [1,5]. Positively charged redox proteins such as cytochrome c give reversible voltammetry * Corresponding author. Fax: q613-9905-4597; e-mail: a.bond@ sci.monash.edu.au 1 Present address: Department of Biochemistry, G08, University of Sydney, Sydney, NSW 2006, Australia.
at such surfaces but negatively charged proteins usually require the introduction of a multivalent cationic modifier such as Mg2q, [Cr(NH3)6]3q, poly(L-lysine) or selected antibiotics [1,3,5–9] to observe any response in the region of the reversible potential. For many years it has been assumed that the electrode modification does not affect the thermodynamics of the electron transfer process. However, recent studies suggest that, at least in some cases, the modifier may influence the voltammetrically determined reversible potential. That is, the voltammetrically measured value need not be the same as that determined by classical potentiometric methods. For example, the recombinant cytochrome b5 of rat microsomal outer membrane (examined at a gold electrode modified with bmercaptopropionic acid) shows progressive positive shifts as the concentration of poly(L-lysine), Mg2q or [Cr(NH3)6]3q used to facilitate electron transfer at carbon electrodes is increased [7]. For poly(L-lysine), the observed half-wave potential levelled off at q8 mV versus SHE, a shift of at least 60 mV relative to values obtained at low modifier concentrations. At this point, the modifier:protein
1388-2481/99/$ - see front matter q 1999 Elsevier Science S.A. All rights reserved. PII S 1 3 8 8 - 2 4 8 1 ( 9 9 ) 0 0 0 5 4 - 5
Monday Aug 09 10:28 AM
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
310
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
ratio was 2:1 and the authors concluded that a non-covalent 2:1 complex had formed. Similarly, the voltammetrically determined reversible potential for plastocyanins has been shown to depend on the concentration of Mg2q [10]. The iron–sulfur proteins rubredoxin (Rd, Fe(S-Cys)4; 6129 Da) and ferredoxin (Fd; 2=Fe4S4; 6203 Da) from Clostridium pasteurianum (Cp) exhibit stable, reversible (one-electron) cyclic voltammetry at an edge plane pyrolytic graphite (PGE) electrode in the presence of [Cr(NH3)6]3q [1,5], although conditions have been discovered for Fd and for the iron–sulfur enzyme aconitase where no multivalent cation is needed [11,12]. The present work reports the square wave voltammetry (SWV) of the recombinant and certain mutant forms of Rd and Fd at the PGE electrode in the presence of poly(L-lysine) and Cr(III) cationic electrode modifiers. The modifiers were adsorbed to the electrode surface prior to contact with the protein solution or were added to that solution and the reversible potentials calculated in order to determine the influence of the nature of the chemically modified electrode on the value determined. Commonly, cyclic voltammetric studies have been used to calculate the reversible potential. However, in this study, the far more sensitive square wave voltammetric method has been used to enhance the ability to extract the Faradaic response from the background when low levels of electrode modification are employed. Furthermore, the square wave response is not affected by the dc component of the experiment for a reversible process, so that reversible potentials are readily calculated even if only part of the electrode surface is modified, as will commonly be the case [4,13,14].
2. Materials and methods Recombinant (rRd, rFd) and mutant (G10A-Rd, G10VRd, D33,35,39N-Fd) forms of rubredoxin and ferredoxin from C. pasteurianum (Cp) were generated in E. coli as host and isolated as described elsewhere [15–17]. Unlike the native form, recombinant Rd is not formylated at the N terminus. The protein samples exhibited absorbance ratios of A490/A280s0.43 for rubredoxins and A390/A280s0.80 for ferredoxins. Poly(L-lysine) (degree of polymerisation, 20) was purchased from Sigma Chemicals. [Cr(en)3]Cl3 was prepared by a literature method [18]. [Cr(en)3]3q in water undergoes hydrolysis to form an equilibrium containing mononuclear and binuclear ions [19]. Voltammetric experiments employed a Cypress CS-1087 computer-controlled potentiostat. A standard three-electrode system was used. The working electrode was a 5 mm diameter disc of edge-cleaved pyrolytic graphite (Le Carbone-Lorraine) housed in a sheath of epoxy resin. The electrode was polished manually using a 0.3 mm alumina–water slurry followed by thorough rinsing with deionised water. The counter electrode was Pt wire. A AgNAgCl (saturated KCl) reference electrode was constructed and its potential (199 mV versus SHE at 258C) was verified against a saturated calomel elec-
Monday Aug 09 10:28 AM
trode (244 mV versus SHE at 258C). All potentials are reported relative to SHE. All experiments were carried out at 258C. The electrochemical data reported were acquired by SWV [5,20–23]. Reversible potentials were obtained directly from the value of maximum current of the square wave voltammogram. Solutions were typically 80 mM protein in 30 mM Tris– HClq0.1 M NaCl (pH 7.4). Dioxygen was removed by bubbling a slow stream of humidified, oxygen-free nitrogen through the solutions. The same gas passed across the solution surface during electrochemical measurements. Titration of protein solutions with modifiers involved the addition of aliquots of poly(L-lysine) (10 mM) or CrIII (160 mM) in buffer.
3. Results 3.1. Ex situ electrode modification by cations Smith and Feinberg [11,23] report a SWV peak current of about 11 mA at a PGE electrode (3 mm diameter) for a Fd solution (150 mM; Tris–HCl, 50 mM, pH 8) in the absence of added multivalent cations. Important aspects in obtaining this response appear to include: 1. anaerobic handling, which would minimise apo-protein contamination and consequent electrode blockage by irreversible adsorption of protein to the electrode, and 2. electrode pre-treatment by sonification of the electrode dipped in soap solution, which may lead to adsorption of detergent molecules and consequent masking of the negative charge on the electrode. The observations reported in [23] are confirmed in the present work. In addition, stable square wave voltammetric responses (;0.1 mA/mM) were observed when the PGE electrode employed in this work was modified by dipping into a 10 mM solution of poly(L-lysine) for 2 min followed by careful rinsing with distilled water, gentle removal of surface water by contact with absorbant tissue and placement in rFd or rRd solution (Fig. 1(a)). In contrast to the work of Smith and Feinberg, anaerobic handling of protein solutions prior to purging the cell of air for electrochemistry was not necessary. Table 1 contains the reversible potential determined from this kind of experiment. The observed peak potential obtained at this poly(L-lysine) modified electrode was independent of the square wave period t and plots of peak current Ip versus ty1/2 were linear (Fig. 1(b)), indicating the presence of reversible, diffusion-controlled electron transfer processes [20]. The peak widths at half-height of 127"1 mV (Es, 1 mV; Ew, 50 mV; ty1, 30 Hz) can be compared with the value of 126 mV theoretically expected for a reversible one-electron process under the conditions employed. When the electrode was modified with a hydrolysed [Cr(en)3]3q solution (160 mM: see Section 2, Materials and Methods), reversible responses were also observed but with lower peak currents which decayed upon repeated
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
311
weak and unstable voltammetric responses. Whilst the processes are reversible under all conditions of electrode modification, a key question to be addressed in this work is whether the measured reversible potentials are influenced by the modification of the electrode. Consequently, data obtained below consider results obtained by in situ forms of electrode modification and comparison with the data obtained by the ex situ method. 3.2. In situ addition of electrode modifier 3.2.1. Enhancement of peak currents as a function of solution phase modifier concentration When poly(L-lysine) (average charge, 20q) or [Cr(en)3]3q (fresh or hydrolysed) multivalent cations were added to the protein solutions, voltammetric responses were readily observed at the PGE electrode. No response was detected in the absence of modifier. The efficiency of the enhancement as estimated by the peak current magnitude under the same voltammetric conditions varied with the cation and with the protein. In rRd solutions, an optimum and stable response was reached at modifier:protein ratios of 0.1:1 for poly(L-lysine) and at 1:1 and 100:1 for hydrolysed and fresh ([Cr(en)3]3q) solutions, respectively.
Fig. 1. (a) Square wave voltammograms of rRd (80 mM; 30 mM Tris–HCl, pH 7.4; 0.1 M NaCl) at an ex situ poly(L-lysine)-modified PGE electrode: top trace, first scan; bottom trace, 20th scan (Ew, 50 mV, Ess1 mV, ty1, 30 Hz). (b) Plot of first scan peak current Ip vs. ty1/2.
Table 1 Reversible potentials a for proteins at poly(L-lysine) PGE electrodes determined in the presence and absence of poly(L-lysine) in solution b Protein
rRd G10A-Rd G10V-Rd rFd f
Reversible potential/mV vs. SHE Ex situ modified electrode c
Extrapolated value d
Limiting value e
y77 y104 y119 y400
y78 y104 y119 y400
y48 y74 y90 )y370
a Determined from the peak potential of square wave voltammograms ("4 mV). b 80 mM protein in Tris–HCl (30 mM; pH 7.4), NaCl (100 mM). c Modified by poly(L-lysine): see Experimental. d Estimated (e.g. Fig. 2) by extrapolation of solution phase modifier:protein concentration ratio to zero. e Estimated as potential where the reversible potential becomes independent of modifier concentration (when slope of e.g. Fig. 2(b), approaches zero). f Equivalent results obtain for E17Q, D39N and D33,35,39N mutant forms.
scanning of the potential. Equivalent experiments with Mg2q and fresh [Cr(en)3]3q solution produced reversible, but very
Monday Aug 09 10:28 AM
3.2.2. Effect of modifier concentration upon peak potential Reversible potentials determined from SWV as peak potentials were studied as a function of molar ratio of cationic modifiers to protein (Fig. 2; Table 1). In order to obtain a reproducible response at low solution modifier concentrations, the electrode used in these studies was modified ex situ with poly(L-lysine). Positive potential shifts were observed under these conditions with increasing concentrations of each modifier, the molar shifts being largest for poly(L-lysine) (average charge, 20q). The shifts were largest at low cation to protein ratios and approached limiting values at higher ratios. The effects of variable ionic strength do not account for the shifts in Ep at low modifier levels which are less than 50 mV/M for both rRd and rFd (as estimated by variation of NaCl concentration; see also Refs. [24,25]). However, the ionic strength term may be responsible for the minor variation of peak potential at high modifier:protein ratios (Fig. 2). As might be predicted, the peak potentials at zero modifier levels estimated by extrapolation (Table 1) were indistinguishable from those observed for the ex situ poly(L-lysine) modified electrodes discussed in Section 3.1. The voltammetric behaviour of rRd and its G10A and G10V mutant forms [16] at a PGE electrode modified ex situ by poly(L-lysine) in the absence and presence of poly(Llysine) in solution are compared in Fig. 3 and Table 1. The reversible peak potentials are different for the three proteins but their dependence on the solution phase modifier:protein concentration ratio is similar. On the other hand, for rFd and its E17Q, D39N and D33,35,39N mutant forms [17], both peak potentials and dependence on modifier:protein ratio are identical, within experimental error ("4 mV).
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
312
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
4. Discussion
Fig. 2. Dependence of the reversible potential (SWV Ep value) of rCpRd obtained at an ex situ poly(L-lysine)-modified PGE electrode on the solution phase molar concentration ratios [modifier]/[protein]: (a) square wave voltammograms at different solution phase poly(L-lysine) levels; (b) plots of Ep vs. [modifier]/[protein. Other conditions are as in Fig. 1.
Fig. 3. Dependence of reversible potentials (Ep values) obtained by SWV for r-CpRd, G10A-Rd and G10V-Rd at an ex situ poly(L-lysine)-modified PGE electrode on the solution phase molar concentration ratio [poly(Llysine)]:[Rd]. Other experimental conditions are as in Fig. 1.
Monday Aug 09 10:28 AM
Both poly(L-lysine) and Cr(III) appear to adsorb onto and hence modify the surface of a PGE electrode, so that reversible voltammetry of rubredoxin and ferredoxin may be observed. Stable, reversible SWV peaks were observed for both rRd and rFd at the ex situ modified poly(L-lysine) electrode: peak currents were unchanged after 20 scans (Fig. 1), indicating irreversible adsorption of the modifier to the electrode. Ex situ Cr(III)-modified electrodes, whilst enabling a reversible process to be observed, did not provide stable electrochemistry: peak currents decreased significantly with each successive scan. Apparently, these cations cannot prevent progressive protein denaturation from blocking the electrode or their affinities for the protein molecules exceed that for the electrode surface. Related cationic centres have been attached covalently to PGE electrodes by reduction of [Cr(NH3)6]3q in aqueous ammonia [26]. Well-behaved cyclic voltammetry was observed for the anionic protein plasticyanin at such electrodes. The reversible half-wave potentials for the recombinant cytochrome b5 of rat microsomal outer membrane showed a pronounced dependence upon the solution phase concentration of poly(L-lysine), Mg2q and [Cr(NH3)6]3q [7]. Consequently, it was concluded that the reduction potential of this protein may be modulated in vivo by Ca2q or Mg2q ions or by cationic electron transfer protein partners. For plastocyanins, Ep is a function of Mg2q concentration [10] and the present results suggest that a modulation or related mechanism is available to rubredoxin and ferredoxin systems. Addition of modifiers to rRd or rFd solutions caused reversible potentials to shift positively with increasing modifier:protein ratios (Fig. 2). Thus, these reagents are not innocent in a thermodynamic sense. Ion association of the negatively charged metalloprotein and positively charged modifier is the likely cause, leading to coulombic stabilisation of the reduced form of the protein relative to the oxidised form or to a conformational change which perturbs the protein active site. The details of such interactions have been examined in a number of systems [7,25,27], and in rRd, in particular [28]. The potential shift effect levels off with increasing modifier concentration as cation binding sites on the protein surface become saturated (Fig. 2). Extrapolation of reversible SWV peak potential Ep versus relative solution phase concentration plots (Fig. 2) to zero modifier concentration provides Ep values which are indistinguishable from those estimated using the ex situ poly(L-lysine)-modified electrodes (Table 1). These values are also independent of the modifier used and agree, within experimental error ("4 mV), with values estimated previously by SWV for Rd (y74 mV) and Fd (y400 mV) [5,11,23]. The potential of y55 mV reported for rRd at a PGE electrode in a solution containing MgCl2 (150 mM) is consistent with a positive shift induced by Mg2q [28].
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
The behaviour of the mutant proteins provides some further insight into the role of poly(L-lysine). Mutation of residue Gly 10 to Ala or Val (side chain H to CH3 or CH(CH3)2) perturbs the active site of Rd, thus altering the reversible potential [16]. However, this mutation does not alter the molecular charge and so the similar dependence upon modifier:protein ratio observed (Fig. 3, Table 1) is expected. Mutation of surface carboxylate residues (D, E) of rFd to the sterically equivalent carboxamides (N, Q) changes the molecular charge of 11y on the oxidised form [17], but change to the stereochemistry of the protein surface is expected to be minimal. Since the voltammetric properties of E17Q, D39N (charge 10y) and D33,35,39N (charge 8y) mutant forms are indistinguishable from that of rFd, it can be assumed that the high positive charge of poly(L-lysine) (average, 20q) dominates the solution phase properties of the complex. In summary, reversible electrochemistry of Rd and Fd at a PGE electrode can be facilitated by either ex situ modification of the electrode surface with cationic species or in situ addition of multivalent cations to the solution. Use of an ex situ poly(L-lysine)-modified PGE electrode provides a convenient method of estimation of the reversible potentials of Rd and Fd which are independent of the nature of the modifier when measured potentials are extrapolated to zero solution phase modifier concentration. Association of anionic proteins with cationic modifiers in solution is believed to be the primary cause of the positive shifts in reversible potential induced by increasing solution phase modifier:protein concentration ratios. The effect of background ionic strength to reversible potential shifts is minor. Thus, solution forms of these multivalent modifiers are not thermodynamically innocent. The influence on the thermodynamics of surface bound forms of the modifiers is not so clear cut. If the role of surfaceattached materials is solely to prevent surface blockage by denatured protein so that an uncomplicated diffusion-controlled process is observed, then considerations akin to those described recently by Honeychurch and Rechnitz [29] may need to be considered. Thus, while this specific issue has not been addressed herein, the ex situ form of modification may also not be fully innocent in a thermodynamic sense.
5. Abbreviations
A Cp D d E E. Es Ep Ew Fd
alanine Clostridium pasteurianum aspartate electrode diameter glutamate Escherichia step height peak potential square wave amplitude ferredoxin
Monday Aug 09 10:28 AM
G Ip N PGE Q Rd r SHE SWV t V
313
glycine square wave peak current asparagine edge plane pyrolytic graphite electrode glutamine rubredoxin recombinant standard hydrogen electrode square wave voltammetry square wave period valine
Acknowledgements A.G.W. thanks the Australian Research Council for financial support under Grant A29330611.
References [1] F.A. Armstrong, H.A.O. Hill, N.J. Walton, Acc. Chem. Res. 21 (1988) 407. [2] F.A. Armstrong, A.M. Bond, H.A.O. Hill, B.N. Oliver, I.S.M. Psalti, J. Am. Chem. Soc. 111 (1989) 9185. [3] F.A. Armstrong, Adv. Inorg. Chem. 38 (1992) 17. [4] A.M. Bond, Anal. Proc. 30 (1993) 218. [5] F.A. Armstrong, H.A.O. Hill, B.N. Oliver, N.J. Walton, J. Am. Chem. Soc. 106 (1984) 921. [6] F.A. Armstrong, P.A. Cox, H.A.O. Hill, V.J. Lowe, B.N. Oliver, J. Electroanal. Chem. 217 (1987) 331. [7] M. Rivera, M.A. Wells, F.A. Walker, Biochemistry 33 (1994) 2161. [8] H.A. Heering, Y.B.M. Bulsink, W.R. Hagen, T.E. Meyer, Biochemistry 33 (1995) 14675. [9] F.A. Armstrong, S.J. George, A.J. Thomson, M.G. Yates, FEBS Lett. 234 (1988) 107. [10] D.D. Niles McLeod, H.C. Freeman, I. Harvey, P.A. Lay, A.M. Bond, Inorg. Chem. 35 (1996) 7156. [11] E.T. Smith, D.W. Bennett, B.A. Feinberg, Anal. Chim. Acta 251 (1991) 27. [12] J. Tong, B.A. Feinberg, J. Biol. Chem. 269 (1994) 24920. [13] A.M. Bond, Anal. Proc. 29 (1992) 132. [14] A.M. Bond, Inorg. Chim. Acta 226 (1994) 293. [15] S.D.B. Scrofani, P.S. Brereton, A.M. Hamer, M.J. Lavery, S.J. McDowall, G.A. Vincent, R.T.C. Brownlee, N.J. Hoogenraad, M. Sadek, A.G. Wedd, Biochemistry 33 (1994) 14486. [16] M. Ayhan, Z. Xiao, M.J. Lavery, A.M. Hamer, K.W. Nugent, M. Guss, S.D.B. Scrofani, A.G. Wedd, Inorg. Chem. 35 (1996) 5902. [17] P.S. Brereton, M.J. Lavery, P.A. Tregloan, A.G. Wedd, Biochim. Biophys. Acta 1429 (1999) 307. [18] R.D. Gillard, P.R. Mitchell, Inorg. Synth. 13 (1972) 184. [19] H.L. Schlafer, O.Z. Kling, Anorg. Allg. Chem. 287 (1956) 296. [20] J.H. Christie, J.A. Turner, R.A. Osteryoung, Anal. Chem. 49 (1977) 1899. [21] J.A. Turner, J.H. Christie, M. Vukovic, R.A. Osteryoung, Anal. Chem. 49 (1977) 1904. [22] J.J. O’Dea, J. Osteryoung, R.A. Osteryoung, Anal. Chem. 49 (1977) 695. [23] E.T. Smith, B.A. Feinberg, J. Biol. Chem. 265 (1990) 14371.
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64
314
Z. Xiao et al. / Electrochemistry Communications 1 (1999) 309–314
[24] R.C. Prince, M.W.W. Adams, J. Biol. Chem. 262 (1987) 5125. [25] G. Battistuzzi, M. Borsari, D. Dallari, S. Ferretti, M. Sola, Eur. J. Biochem. 233 (1995) 335. [26] F.A. Armstrong, P.A. Cox, H.A.O. Hill, B.N. Oliver, A.A. Williams, J. Chem. Soc., Chem. Commun. (1985) 1236.
Monday Aug 09 10:28 AM
[27] G. Cheddar, G. Tollin, Arch. Biochem. Biophys. 278 (1990) 265. [28] Q. Zeng, E.T. Smith, D.M. Kurtz Jr, R.A. Scott, Inorg. Chim. Acta 242 (1996) 245. [29] M. Honeychurch, G.A. Rechnitz, J. Phys. Chem. 101 (1997) 7472 and Refs. therein.
StyleTag -- Journal: ELECOM (Electrochemistry Communications)
Article: 64