Direct electrochemistry of parsley plastocyanin on pyrolytic graphite electrodes

Journal of Electroanalytical Chemistry, 364 (1994) 17-22

17

JEC 02909

Direct electrochemistry graphite electrodes

of parsley plastocyanin on pyrolytic

Lars S. Conrad a, H. Allen 0. Hill b!*, Nicholas I. Hunt b and Jens Ulstrup a ’ Chemistry Department A, The Technical University of Denmark, DK-2800 Lyngby (Denmark) b Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR (UK) (Received

25 August

1992; in revised

form 22 April

1993)

Abstract A steady quasi-reversible electrochemical response due to parsley plastocyanin is reported at an edge-plane graphite electrode without the requirement for so-called adjunct-promoters. This observation is unusual in that studies involving plastocyanins from other sources require the addition of polyvalent cations to the solution in order to promote the heterogeneous electron transfer. Here cationic adjunct-promoters are required only under conditions of low ionic strength, where sigmoidal responses are obtained, characteristic of the presence of a low number of electroactive sites on the electrode surface such that conditions of radial, as opposed to linear, diffusion apply.

1. Introduction

Plastocyanin (PC>is a small blue copper protein with a molar mass of approximately 10500, present in the photosynthetic electron transport chain of plants, algae and cyanobacteria, where it conveys [ll electrons from the cytochrome f/b6 complex to photosystem I. Owing to its availability and relative robustness, PC has been extensively studied since its discovery some 30 years ago [2] and is now one of the best characterized [3] metalloproteins. The crystal structures of PC from poplar [4], Enteromorpha prolifera [51 and Oleander nerium [61, and the 3D NMR structures in solution of PC from Scenedesmus obfiquus [71, French bean [81, spinach [93 and parsley [lo] are available. These plastocyanins (PCS) possess very similar structures. The copper atom is coordinated by four ligands, His;37, Cys-84, His-87 and Met-92, and is located close (6 A1 to the protein surface in an area dominated by hydrophobic residues. PC from all sources but the cyanobacterium Anabaena uariabilis bears an excess negative chOarge, localized mainly in an acidic “patch” about 15 A from the copper centre. The acidic patch comprises residues 42-45 and 59-61 surround-

* To

whom correspondence

0022-0728/94/$7.00 SSDI 0022-0728(93)02909-2

should

be addressed.

ing the solvent exposed Tyr-83. The overall PC charge is between - 7 and -9 at pH 7, depending on the source. The most pronounced difference between parsley PC and the other PCSis a deletion of two amino acid residues at positions 57 and 58 in the sequence. This eliminates a turn [lo] in the poplar PC structure. In addition, the charge in the 59-61 region is less negative in parsley PC than in other plant and algae PCS. Detailed kinetic studies [3] have suggested that plant and algal PCS exhibit dual-path electron transfer (ET): one path is direct ET through the ligand His-87, while the other path is a longer, through-protein route via Tyr-83 at the acidic patch. The former path (often referred to as the “north” reaction site) is probably common to all PC reaction partners while the latter (the “east” site) is exploited only by reaction partners with a positively charged reaction site (such as cytochrome f 1. The electrochemistry of several PCShas been investigated, and well behaved, near reversible responses with pyrolytic graphite [ill, glassy carbon [121 and gold [131 electrodes have been obtained. However, all previous reports have stressed the need for modified electrodes, the addition of polyvalent cations, or low pH or temperature. Here we report direct unpromoted electrochemistry of parsley PC at an unmodified, edge-plane pyrolytic graphite (EPG) electrode at pH 7. This result 0

1994 - Elsevier

Sequoia.

All rights reserved

18

L.S. Conrad et al. / Direct electrochemistry

is significant for two reasons: reversible electrochemical kinetics of highly negatively charged proteins at unmodified electrodes do not appear to have been reported before; perhaps more importantly, the data indicate that even small structural differences between PCS can lead to very different electrochemical behaviour. 2. Experimental Parsley PC (a kind gift from Dr. S. Bagby) was isolated by the method of PlesniEar and Bendall [14] and further purified on a Mono Q HR lO/lO anion-exchange column (Pharmacia). The column was equilibrated with 20 mM Tris-HCI, pH 7.3 and the protein eluted with a 0.1 M KC1 gradient. The final absorbance ratio A &A 598 was 1.45 in the oxidized state. Oxidized samples were obtained by the addition of [Fe(CN>,13-, followed by extensive ultrafiltration @MC, fitted with YM-5 membranes, Amicon). PC concentrations were determined spectrophotometrically using the 598 nm molar absorption coefficient, for the fully oxidized species, of 4900 M-’ cm-’ [151. PC concentrations around 100 Z.LMwere used in all cyclic voltammetric experiments. [Cr(NH,),lCl, * H,O was synthesized following literature methods [161. All chemicals were analytical grade and all solutions were prepared with Milli-Q water (Millipore). Direct current cyclic voltammetry was carried out using an Ursar Scientific Instruments potentiostat with the voltammograms recorded on a Bryans 26000 A3 chart recorder. The electrochemical cell (volume approximately 400 ~1) was a standard two-compartment glass cell with a conventional three-electrode configuration. The side-arm containing the reference electrode (K401 saturated calomel electrode, Radiometer) was connected to the working compartment via a Luggin capillary. The working electrodes were constructed from approximately 2 X 3 mm* plates of pyrolytic graphite (Le Carbone-Lorraine), cut perpendicular to the u-b plane and housed in Teflon sheaths. Reported voltammetric parameters refer to the apparent working electrode area of 6 mm*. The counter-electrode was a piece of platinum gauze. Dioxygen was removed from the system by passing humidified oxygen-free argon through the cell. Prior to each experiment, the graphite electrode was polished in an alumina-water slurry (alumina particle size 0.3 pm), sonicated and rinsed with copious amounts of water. AI1 experiments were carried out at ambient temperatures using MES buffers (2-[N-morpholinolethane sulphonate) at pH 7.0. The ionic strength was adjusted with KCl. AI1 potentials quoted are referred to the standard hydrogen electrode.

of parsley plastocyanin

3. Results Cyclic voltammograms (CVs) at an EPG electrode of parsley PC in 20 mM MES (0.1 M KC11 were obtained at variable scan rates v. The corresponding Randles-SevEik plot is shown in Fig. 1. The midpoint potential, determined as the average of the cathodic and anodic peak potentials, was 383 f 3 mV. At scan rates below 20 mV s-l, the CVs were peak shaped, and both the cathodic and anodic peak currents and the peak separations (60-65 mV) were stable with time (Fig. 2). From the gradient of the Randles-SevEik plot, using the geometric area of the electrode, a diffusion coefficient of 1.3 X 1O-6 cm* s-l is calculated. The peak separation in the range Y = l-50 mV s-l increased from 60 to 100 mV, and using the Nicholson approach [17], an apparent exchange rate constant for the heterogeneous electron-transfer process of 4 x lop3 cm s-l can be estimated. However, a study of the effect of a change in protein concentration (50 PM to 250 PM) at low ionic strength revealed that the peak separations at a given scan rate (20 mV s-l, 50 mV s-l) were dependent on the protein concentration. This suggests 1181that the linear diffusion model may not apply accurately to the electrochemical response of parsley PC at an edge-plane graphite electrode. For 50 PM parsley PC at 20 mV s-l, a plot of the applied potential against log[(Zd - Z)/Z], where Z, is the limiting sigmoidal current, is linear (Fig. 31, with a gradient of 2.303RT/F (kO.004 V). This is consistent with quasi-reversible electron-transfer occurring at an array of microscopically small electroactive sites on the electrode surface, with radial diffusion of the protein to these sites [181. This would also explain why the Randles-Sevcik plot at high ionic strength tails off at high

0

0.1

0.2 0.3 0.4 ,,l,(“s_‘)‘lZ Scanrate

Fig. 1. Randles-Sevzik plot for parsley mM MES, pH 7.0 (0.1 M KCI).

plastocyanin

0.5

(100 FM);

20

L.S. Conrad et al. / Direct electrochemistry

of parsley plastocyanin

19

1 O,025/~A

,

0

n ”

1 _^

c

iMgz1 L””

“VU

vu”

Potential / mV Fig. 2. Cyclic voltammograms of parsley plastocyanin (100 PM), first five scans; 20 mM MES, pH 7.0 (0.1 M KCI); scan rate 10 mV s-l.

scan rate, as the peak current here is independent of scan rate. The CV behaviour is thus described in terms of the EPG electrode being considered as an array of microscopic electroactive sites where protein molecules approach and leave the surface sites by overlapping radial diffusion. With such a view, exchange rate constants independent of PC concentration and greater than 0.1 cm s-l are obtained.

‘”

/ mM

Fig. 4. Peak currents for the fifth anodic scan normalized with respect to parsley plastocyanin concentration as a function of Mgzf concentration; 1 mM MES, pH 7.0 (1 mM KCI); scan rate 20 mV SC’.

The electrochemistry of parsley PC at low ionic strength was also investigated, with the use of a 1 mM MES + 1 mM KC1 electrolyte, in the absence and presence of the polyvalent cations Mg*+ and

2500

-

2000

-

500

t 01 0.0

340

360

380

400

420

E I mV Fig. 3. Plot of log[(l,, - I)/11 against the applied potential parsley plastocyanin (50 PM), first scan, scan rate 20 mV s-‘; (1 mM MES, 1 mM KCl).

440 E for pH 7.0

I

0.5

I

I

I

1.0

1.5

2.0

[C?‘]

/ mM

Fig. 5. Peak currents for the fifth anodic scan normalized with respect to parsley plastocyanin concentration as a function of [CrtNH,),13’ concentration; 1 mM MES, pH 7.0 (1 mM KCI); scan rate 20 mV s-l.

L.S. Conrad et al. / Direct electrochemistry of parsley plastocyanin

20

mined as the average of the cathodic and anodic peak potentials, was 377 -t 5 mV when either magnesium or chromium hexammine was added. 4. Discussion

I

I

LOO Potential

600

/ mV

Fig. 6. Cyclic voltammograms of parsley plastocyanin (100 PM), first five scans; 1 mM MES, pH 7.0 (1 mM KCI); [Mg’+ I = 0.5 mM; scan rate 20 mV s-l.

[Cr(NH,),l 3+. Figures 4 and 5 show plots of anodic peak currents, obtained at 20 mV s-l, for the fifth scan normalized with respect to the PC concentration against the concentration of Mg2+ and [Cr(NH3)J3+ respectively. No stable electrochemical response due to parsley PC was obtained at low ionic strength without addition of the cations. Figures 4 and 5 show that on addition of Mg2+ and [Cr(NH3),13+, the general trend that more highly charged cations are more effective in promoting electrochemical responses is followed, and that the concentrations required to obtain a stable response are similar to those reported for spinach PC n91. At Mg2+ concentrations below 4 mM, the cathodic wave gradually lost its peak-shaped characteristics (Fig. 6) and approached sigmoidal behaviour before achieving stability. For [Cr(NH3>,13’ both waves remained peak shaped at all chromium concentrations used. The peak currents were stable in time for the lowest concentration of cations used (0.1 mM), although compared with the high ionic strength CVs they appeared more sigmoidal in nature, and with the larger peak separation of 100-110 mV. This is indicative of a lower number of electroactive sites at the electrode surface. The midpoint potential at low ionic strength, deter-

We have investigated the electrochemistry of the highly negatively charged parsley plastocyanin at an EPG electrode in the absence of promoters at ambient temperature. As shown in Fig. 1, at a low scan rate the peak currents are proportional to u112. Together with the observed peak separations this indicates that the reaction is diffusion controlled and in the quasi-reversible regime 1201. The midpoint potential determined at high ionic strength is in excellent agreement with the value of 375 mV obtained from kinetic data [3], and the decrease in midpoint potential with a decrease in ionic strength is similar to that observed for spinach PC [21]. The electrochemical response is stable in time. Well behaved and persistent unpromoted electrochemistry is thus achieved with parsley PC at a bare EPG electrode. This is in contrast to earlier reported studies of the electrochemistry of other PCS at EPG electrodes, where the addition of polyvalent cations and low temperature has been a requisite. The quasi-reversible behaviour of parsley PC at an EPG electrode is conceivably associated with two features in particular. First, and common to all the PCS, the electroactive sites of the EPG electrode are likely to be centred on the large number of oxidized C-O surface functionalities [22], such as phenols, alcohols and carboxylic acids [231. A pK value of 5.6 has been determined for these functionalities [191, and these dominate the overall charge of the EPG electrode surface, which is negatively charged at the pH applied in these studies. The functions of the polyvalent cations are believed [19] to be induction of reversible adsorption of the negatively charged proteins and to shield them electrostatically from the negatively charged electrode surface in a configuration that allows ET. Second, the specific behaviour of parsley PC compared with the other PCS may be as a result of small but significant differences in structure and charge distribution for PCS from different sources. These properties are summarized in Table 1. Residues 42-45 and 59-61 represent the “east site” and “electrochemical response” refers to unpromoted, quasi-reversible electrochemistry at a bare EPG electrode reported in the present and previous [11,12,241 work. The following observations and implications emerge from the table. (1) The overall charges of all the PCSare very similar and the unique behaviour of parsley PC is not likely to be associated with this feature alone nor with the small structural differences.

L.S. Conrad et al. / Direct electrochemistry of parsley plastocyanin

TABLE 1. Total charge, east site charge distribution and electrochemical response for plastocyanins on pyrolytic edge-plane graphite electrodes Source

Parsley S. obliquus Spinach Cucumber b

Total

Charge on residue 42-45

59-61

Electra chemical response

Reference a

charge -8

-4

-1

-t

This work

-8

-3

-2

-

24

-9 -9

-4 -4

-3 -3

-

11 12

a References are to the electrochemical information; the amino acid sequences can be found in ref. 3. b Polished glassy carbon electrode.

(2) A much more conspicuous difference between parsley PC and the other PCS is the single negative charge in the 59-61 region of the former and the multiple charges in the same region for the latter. Charge modulation at the north site due to the 59-61 residues is, moreover, stronger than from the 4?-45 residues owing to the shorter distances (11-12 A vs. 15-20 A>. The electrostatic potential close to the copper site can therefore be expected to be notably more positive for parsley PC than for other PCS. This view is substantiated by calculations of the electrostatic potentials around the copper atom of spinach PC [251 and parsley PC [lo], using the Delphi program. The potential is positive in either case, owing to the charge at the copper centre, both modulated by the east site negative charges. The spatial range of positive potentials is, however, notably wider for parsley PC than for spinach PC, in line with the weaker negative charge modulation in the former. (31 As noted, a dual-path ET pattern for plant PCS in homogeneous ET reactions is well documented experimentally [3] aad theoretically 124,261. While the longer route (15 A + reaction partner extension) is electronically facile, it is, Ohowever, only competitive with the shorter route (6 A + reaction partner extension) if supported by favourable inter-reactant work terms. Long distance, through-protein ET for the PCS therefore only applies to positively charged reaction partners. Electrochemical ET at an EPG electrode is dominated by negatively charged surface groups. Electrochemical ET is therefore most likely to involve “directly” the copper atom at the north site. More facile ET of parsley PC than for the other PCS is therefore suitably associated with the notably broader spatial north site region where the electrostatic potential is positive. Two effects would emerge from this difference. One is the more favourable work terms, or “double layer effects” associated with the electrode-PC interaction. This would lead to faster ET at a given

21

overpotential. The other, and more important, effect is that the more favourable interaction would also ensure a broader range of geometric orientations at the electrode surface favourable for facile “direct” ET. This electronic effect is thus the most likely explanation for the qualitatively different behaviour of parsley PC compared with the other PCS. The hypothesis regarding the additional charge effects is supported by three observations. (1) The introduction of a +3 charge at the east site in S. obliquus PC by Ru(III)-modification of His-59 [27] changes the electrochemistry from non-stable to well behaved and quasi-reversible in the absence of promoters. (2) The promoting polyvalent cations are known from NMR line broadening experiments to bind specifically at the east site 1281. Although the promoters may interact with the electrode functionalities, the fact that higher protein concentrations require higher promoter concentrations [19] indicates that specific protein-promoter interactions must also take place. (3) Unpromoted electrochemistry of spinach PC at EPG electrodes can be observed, but the response is not stable, and the voltammograms appear sigmoidal in nature, and are characterized by large peak-to-peak separations. Although linear diffusion is unlikely to be the predominant mode of mass transfer of the protein species to the electrode, the exchange rate constants calculated under the assumption of linear diffusion for spinach PC (2 x lop3 cm s-l) [ll] and parsley PC (4 X 10e3 cm s-l) are similar. This suggests that heterogeneous ET may still follow the same mechanism in the two proteins. The stability and quasi-reversible nature of the electrochemical response of parsley PC compared with that of other PCS is also indicative of a different parsley PC stability at the EPG electrode surface. The non-stability for example of the spinach PC response is characteristic of a reduction in time of the number of electroactive sites at the electrode surface, i.e. blocking of the electrode to ET occurs, probably owing to degradative adsorption of the protein at the electrode surface. The structure of parsley PC must therefore lead to more reversible electrode-protein complex formation, facilitating heterogeneous ET. In conclusion, although the solution chemistry of PCSfrom different sources is similar, the present report shows that their electrochemistry differs substantially. This indicates that the protein-electrode interaction is much more sensitive to the protein structure than the corresponding protein-reagent interaction in solution. The report also demonstrates that changes at a protein surface remote from both the reaction- and active site can lead to a qualitatively different electrochemical response.

22

L.S. Conrad et al. / Direct electrochemistry of parsley plastocyanin

Acknowledgments

LSC would like to thank Otto Monsteds Fond, Thomas B. Thriges Fond and Knud Hojgaards Fond for financial support. NIH would like to thank the AFRC for financial support. References 1 W. Haehnel, Annu. Rev. Plant Physiol., 35 (1984) 659. 2 S. Katoh and A. Takamiya, Nature, 189 (19611665. 3 A.G. Sykes, Struct. Bonding, 75 (1990) 175. A.G. Sykes, Adv. Inorg. Chem., 36 (1991) 377. 4 J.M Guss and H.C. Freeman, J. Mol. Biol., 169 (1983) 521. J.M. Guss, P.R. Harrowell, M. Murata, V.A. Norris and H.C. Freeman, J. Mol. Biol., 192 (1986) 361. 5 C.A. Collyer, J.M. Guss, Y. Sugimura, F. Yoshizaki and H.C. Freeman, J. Mol. Biol., 211 (1990) 617. 6 J. Han, E.T. Adman, T. Beppu, R. Codd, H.C. Freeman, L. Huq, T.M. Loehr and J. Sanders-Loehr, Biochem., 30 (1991) 10904. 7 J.M. Moore, W.J. Chazin, R. Powls and P.E. Wright, Biochem., 27 (1988) 7806. 8 J.M. Moore, C.A. Lepre, G.P. Gippert, W.J. Chazin, D.A. Case and P.E. Wright, J. Mol. Biol., 221 (1991) 533. 9 P.C. Driscoll, H.A.O. Hill and C. Redfield, Eur. J. Biochem., 170 (19871283. 10 S. Bagby, D. Phil. Thesis, University of Oxford, 1991. 11 F.A. Armstrong, H.A.O. Hill, B.N. Oliver and D. Whitford, J. Am. Chem. Sot., 107 (1985) 1473.

12 T. Sakurai, 0. Ikeda and S. Suzuki, Inorg. Chem., 29 (199014715. 13 P.D. Barker, K. DiGleria, H.A.O. Hill and V.J. Lowe, Eur. J. Biochem., 190 (19901 171. 14 M. PlesniEar and P.S. Bendall, Biochim. Biophys. Acta, 216 (19701 192. 15 D.J. Davis and A. SanPietro, Anal. Biochem., 95 (1979) 254. 16 A.L. Oppegard and J.C. Bailar, Jr., Inorg. Synth., 3 (1950) 153. 17 R.S. Nicholson, Anal. Chem., 37 (1965) 1351. 18 F.A. Armstrong, A.M. Bond, H.A.O. Hill, I.S.M. Psalti and C.G. Zoski, J. Phys. Chem., 93 (1989) 6485. 19 F.A. Armstrong, P.A. Cox, H.A.O. Hill, V.J. Lowe and B.N. Oliver, J. Electroanal. Chem., 217 (1987) 331. 20 A.J. Bard and L.R. Faulkner, Electrochemical Methods, Wiley, New York, 1980, Chapter 6. 21 H.E.M. Christensen, L.S. Conrad and J. Ulstrup, submitted for publication. 22 F.A. Armstrong, A.M. Bond, H.A.O. Hill, B.N. Oliver and I.S.M. Psalti, J. Am. Chem. Sot., 111 (1989) 9185. 23 R.E. Panzer and P.J. Elving, Electrochim. Acta, 20 (19751635. 24 H.E.M. Christensen, L.S. Conrad, K.V. Mikkelsen, M.K. Nielsen and J. Ulstrup, Inorg. Chem., 29 (19901 2808. 25 S.R. Durell, J.K. Labanowski and E.L. Gross, Arch. Biochem. Biophys., 277 (19901 241. 26 H.E.M. Christensen, L.S. Conrad and J. Ulstrup, Acta Chem. Stand., 46 (1992) 508. 27 F.A. Armstrong, J.N. Butt, K. Govindaraju, J. McGinnis, R. Powls and A.G. Sykes, Inorg. Chem., 29 (1990) 4858. 28 D.J. Cookson, M.T. Hayes, P.E. Wright, Biochim. Biophys. Acta, 591 (19801 162.