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Effect of electrode surface microstructure on electron transfer induced conformation changes in cytochrome c monitored by in situ UV and CD spectroelectrochemistry
Spectrochimica Acta Part A 61 (2005) 943–951
Effect of electrode surface microstructure on electron transfer induced conformation changes in cytochrome c monitored by in situ UV and CD spectroelectrochemistry Xiue Jiang, Zheling Zhang, Hanying Bai, Xiaohu Qu, Junguang Jiang, Erkang Wang, Shaojun Dong∗ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Received 15 January 2004; accepted 18 May 2004
Abstract Electrochemical redox processes of bovine heart cytochrome c were investigated by in situ UV–vis and CD spectroelectrochemistry at bare glassy carbon electrode (GCE) and single-wall carbon nanotubes (SWNTs) modified glassy carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. The spectra obtained at GCE and SWNTs/GCE reflect electrode surface microstructure-dependent changes in protein conformation during redox transition. Potential-dependent conformational distribution curves of cytochrome c obtained by analysis of in situ circular dichroism (CD) spectra using singular value decomposition least square (SVDLS) method show that SWNTs can retain conformation of cytochrome c. Some parameters of the electrochemical reduction process, i.e. the product of electron transfer coefficient and number of electrons (αn = 0.3), apparent formal potential (E0 = 0.04 V), were obtained by double logarithmic analysis and standard heterogeneous electron transfer rate constant k0 was obtained by electrochemistry and double logarithmic analysis, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Electrode surface microstructure; Spectroelectrochemistry; SWNTs modified electrode; Conformational transitions of cytochrome c
1. Introduction Direct electron transfer reactions between electrodes and redox active groups in heme proteins have been focused in recent years because such studies may contribute to understand electron transport mechanisms in biological systems. Cytochrome c (cyt c) is one of the most extensively studied heme proteins so far in this field. Cyt c, whose function is to act as electron carriers in the electron transport chain, plays an important role in the biological respiratory chain. This is a much complex process involving intermolecular and intramolecular electron transfer [1,2], and its mechanism has been disputed for a long time [3-12]. The hypothesis of the conformational rearrangement of cytochrome c during ∗
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1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2004.05.036
electron transfer process has been supposed [5-12]. But, none of the experiment can clearly verify the conformational changes inherent to redox transition up to now. The voltammetric technique is useful for characterizing the redox behavior of metalloproteins. Unfortunately, the voltammetric response of cyt c is poor at bare electrode since cyt c adsorbs strongly on the electrode surface and results in denaturation [13,14]. So, it is necessary to provide a stable and biocompatible electrode surface to keep protein structure while allowing fast electron transfer [15]. One of the examples is using single-wall or multiwall carbon nanotubes (SWNTs or MWNTs) modified electrodes to get the direct electrochemistry of cyt c [16,17]. All the results indicate that the biocompatible electrode surface can accelerate the electron transfer between electrode and cyt c, and probably retain the conformation of cyt c. Hildebrandt groups have reported a series of important results about the effect of biocompatible
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on the structure and redox properties of cyt c with in situ Raman spectroscopy [18–21]. But the Raman spectroscopy can only identify the structural changes of cyt c that include the inner coordination sphere of the heme iron and not get the more information about the secondary structure changes. So a complementary study method should be further performed. Circular dichroism (CD) is a powerful tool for studying the conformation of the samples [22]. Studies combining CD spectrometry with thin layer electrochemistry were performed here. When the electrolytic time is long enough in a long optical path thin layer cell, the diffusion layer is approximate to the thin layer of the cell [23]. So CD thin layer spectroelectrochemistry can be used to investigate the conformational transitions of proteins in electrochemical redox process [24]. In this paper, in situ UV–vis and CD spectroelectrochemistry with a LOPTLC are used to investigate the redox reactions of cyt c at GCE and SWNTs/GCE. The main contributions of our study are to give a more direct insight about (i) how the conformations of cyt c are changed under the redox transition, (ii) why the reversible electrochemistry of cyt c can be obtained at SWNTs modified electrode. We describe the results of experiments aimed at gaining further molecular information to insight into the mechanism of electron transfer for cyt c and how the biocompatible electrode surface microstructure affect the conformational changes of protein during electrochemical reduction process.
treated SWNTs modified glassy carbon (8 mm × 8 mm) electrode prepared according to literature [16] was used as working electrode, a twisted platinum wire as auxiliary electrode, and a Ag/AgCl (saturated KCl) as reference electrode. Before modification, the GCE was mechanically polished with 1.0, 0.3, and 0.05 m ␣-Al2 O3 slurry, successively, and then washed ultrasonically in water followed by ethanol for a few minutes at each step. The LOPTLC is of 10.0 mm optical path length and 0.2 mm thickness of thin layer. The incident light passed through the thin layer being parallel to the working electrode. Before each measurement of in situ CD or UV–visible spectrum, the electrolysis potential of LOPTLC was applied for 5 min to keep all the cytochrome c molecules interact with electrode surface completely.
2.3. Data analysis 2.3.1. Singular value decomposition least squares analysis (SVDLS) To ascertain the different forms of cyt c presented over a range of potentials, the CD spectral data were evaluated by SVDLS analysis. In this analysis, several CD spectra were measured with the same m (m = total wavelengths) wavelengths. The spectra were grouped into series taken under identical conditions except for the changes of a single variable (as the potentials). The SVDLS mathematical and computational details have been reported previously [27,28].
2. Experimental 2.1. Reagents Bovine heart cyt c, purchased from Sigma, was prepared in 0.1 M phosphate buffer solution (PBS). The concentration of solution was determined spectrophotometrically using a molar absorptivity of 1.06 × 105 mol−1 cm−1 at 410 nm for cyt c [25]. Buffer solutions used in all experiments were prepared by Na2 HPO4 ·12H2 O and NaH2 PO4 ·2H2 O (analytical grade), doubly purified water was from Milli-Q system. SWNTs were bought from Shenzhen Nanotech Port Co. Ltd. and were purified and functionalized through a well-established way with slight modification [26]. After the purification, the SWNTs contain abundant oxygen-contained functionalities. All the other chemicals were of reagent grade and used as received. 2.2. Instruments and methods Spectroelectrochemical experiments were carried out in a home-made long optical path thin layer cell (LOPTLC), AVIV 62A DS circular dichroism spectrometer (AVIV Co., USA) for CD spectrum measurement, Cary 500 UV–vis–NIR spectrometer (Varian Co., USA) for UV–visible spectrum measurement and a CHI 630 electrochemical instrument (CHI Co., USA) for electrochemical operation. The pre-
2.3.2. Double logarithmic analysis [29] In this paper, some parameters of the electrochemical reduction process of cyt c at SWNTs/GCE with applied potentials can be obtained based on the following double logarithmic analysis equation ln ln
Ain − Af Ai − A f
=
−αnFEi + ln RT +
αnFE0 RT
∆t d
k0 (1 − θ)
(1)
where Ai is the absorption of reactant on applied potential, Af and Ain the finial and initial absorption of reactant, ␣n the product of electron transfer coefficient and number of electrons, t the electrolytic time for each potential, d the thickness of the thin layer, k0 the standard heterogeneous electron transfer rate constant, θ the percentage of the surface coverage, Ei and E0 are the applied potential and apparent formal potential, respectively. Other symbols have their common meanings. The concrete deduction can be seen in ref. [29]. According to Eq. (1), E0 can be obtained from the peak position of differential curve of A’(E)∼ E with respect to E, and αn from the slope of the double logarithmic line. k0 can be calculated by normal non-linear regression method.
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cyt c shows no obvious electrochemical activity on the bare GCE. While, SWNTs/GCE leads to a pair of well-defined redox peaks with the formal potential (E0 = (Ep,a + Ep,c )/2) of 0.05 mV, which is almost agreement with the reported formal potential of cyt c in solution [16,30,31]. The inset of Fig. 1 shows that the oxidation peak current of cyt c is linearly dependent on the square root of scan rates in the range from 5 to 100 mV/s, indicating a diffusion-controlled electrode process. Cathodic and anodic peak currents are almost equal throughout our study scan rate range. Randles-Sevick expression is used here [32]. 1/2
ipo = 269n3/2 ADo ν1/2 c
(2)
where n is the number of electron transfer, A the area of the electrode, v the scan rate (V/s), Do the charge transport diffusion coefficient (cm2 /s), c the concentration (mol/l) of cyt c. The value of Do can be calculated according to the slope of ipo ∼ v1/2 , Do = 3.9 × 10−7 cm2 /s. Based on Nicholson theory [33] the apparent heterogeneous electrode reaction rate constant (ks ) can be estimated. The average value of ks is 6.0 × 10−3 cm/s. It demonstrates the electrode process is quasi-reversible within the investigated scan rates range. Fig. 1. Cyclic voltammograms of 0.05 mM cyt.c at bare GCE (dashed line) and the SWNTs/GCE (solid line) in 0.1 M PBS (pH 7.2). Scan rate: 20 mv/s.
3. Results 3.1. Direct electrochemistry of cyt c at SWNTs/GCE Fig. 1 shows the cyclic voltammograms of 0.05 mM cyt c at bare GCE (dashed line) and the SWNTs/GCE (solid line) in 0.1 M PBS (pH 7.2) at 20 mV/s. It can be seen from Fig. 1 that
3.2. In situ UV–vis spectroelectrochemistry study of cyt c The wavelength and intensity of characteristic absorption spectra of cyt c are sensitive to the conformational state of the protein, especially for Soret band [34]. Fig. 2 shows in situ UV–vis spectra of cyt c at GCE in 0.1 M PBS (pH 7.2). It can be seen that the oxidized state of cyt c (ferricytochrome c) exhibits two major bands, the Soret band at 410 nm and  band at 530 nm, respectively (Fig. 2(a), curve (1)). While, the reduced state of cyt c (ferrocytochrome c) exhibits three major bands, the Soret band at 414 nm, the ␣ band at 550 nm,
Fig. 2. In situ UV–vis spectra of 3.2 M cyt c in 0.1 M PBS (pH 7.2) solution at GCE in a LOPTLC. Time interval: 5 min; applied potentials: (a) (1–9) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.0, −0.05 V; (b) (10–12) −0.10, −0.20, −0.30 V.
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Fig. 3. In situ UV–vis spectra of 3.5 M cyt c in 0.1 M PBS (pH 7.2) solution at SWNTs/GCE. Time interval: 5 min; applied potentials: (a–m) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.0, −0.10, −0.20, −0.30, −0.40, −0.50 V.
and the  band at 520 nm (Fig. 2(a), curve (9)). The results obtained are identical with those reported [34]. Upon the applied potential shifting from 0.6 to −0.05 V, a transition of characteristic absorption spectrum from the ferricytochrome c to ferrocytochrome c occurs. This can be seen in Fig. 2(a) (curves (1–9)). The absorption peak of Soret band shows redshifts from 410 to 414 nm, the  band shows blue-shifts from 530 to 520 nm, and ␣ band appears gradually. All of the Soret, ␣ and  bands increase in intensity accompanying with potential negatively shifting. But in the range from −0.10 to −0.30 V, all the three bands decrease in intensity accompanying with the blue-shift of the Soret band. (Fig. 2(b), curves (10–12)). This indicates a seriously adsorption on the GCE occurs under the negative electric field. Because the wavelength and intensity of Soret band are sensitive to the conformational state of the protein, all the variations may be a reflection for the conformational changes of the adsorbed ferrocytochrome c on GCE. In situ UV–vis spectra of cyt c at SWNTs/GCE in 0.1 M PBS (pH 7.2) are shown in Fig. 3. As can be seen that the absorption bands of ferricytochrome c and ferrocytochrome c are located near 409, 530, and 411, 520, 550 nm, respectively (Fig. 3a and m). Compared with the spectra of Fig. 2, the absorption peak of Soret band red-shifts only from 409 to 411 nm with the applied potential shifting from 0.60 to −0.50 V (Fig. 3a–m). But the intensity of all absorption peaks does not decrease even the cyt c enriched with positive charge adsorbed on the SWNTs/GCE under the negative electric field. This is obviously different from the phenomena shown in Fig. 2, indicating that the ferrocytochrome c conformation remains almost unchanged in this case. Maybe, the smaller red-shift of Soret band also implies that SWNTs can keep the conformational state of cyt c. The plot of absorbance at 409 nm against applied potentials shows a sigmoidal curve (figure not shown), and the corresponding differential curve gives a one-peak curve (Fig. 4B). Based on the peak position, the apparent formal po-
Fig. 4. (A) Double logarithmic curve; (B) differential curve. Experimental conditions are the same as those in Fig. 3.
tential E0 is estimated to be 0.04 V, which is almost in agreement with the formal potential value obtained with cyclic voltammetry. The double logarithmic plot of the spectroelectrochemical data is an oblique line (Fig. 4A). This type of double logarithmic plot suggests that the redox process of cyt c at SWNTs/GCE is a simple electrochemical reaction [29]. From the slope of the oblique line, ␣n is estimated to be 0.30. Using E0 , ␣n and experimental conditions in normal non-linear regression program, k0 is calculated to be (5.3 ± 0.16) × 10−3 cm/s, which is almost equal to the ks value (6.0 × 10−3 cm/s) obtained by electrochemistry. This further proves that electrode process of cyt c at SWNTs/GCE is quasi-reversible. 3.3. Structure changes of cyt c in electrochemical reduction reaction 3.3.1. The effect of electrode surface on conformational changes of cyt c Cyt c (3.2 M) in 0.1 M PBS (pH 7.2) in a LOPTLC at GCE was used for CD spectroelectrochemical measurement in far-UV region. The CD spectra in far-UV region provide information about the conformation of the polypeptide backbone [35]. Fig. 5 shows the in situ CD spectra of cyt c at GCE. As can be seen from Fig. 5, the negative Cotton peak decreases gradually with the applied potential shifting from 0.60 to −0.30 V. Fig. 6 shows the spectra of five different conformations of cyt c obtained after treated the spectra of Fig. 5 by the SVDLS analysis [34]. According to Gauss-Markoff mode of Protein Secondary structure CD Spectra, curves a, b, c, d, and e are attributed to ␣-helix, parallel -sheet, random coil, antiparallel -sheet and -turn, respectively [37]. Fig. 7 shows the corresponding potential-dependent plot of fractional distribution of the five conformations. The five conformations, i.e., ␣-helix, parallel -sheet, random coil, an-
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Fig. 5. In situ CD spectra of 3.2 M cyt c in far-UV region in 0.1 M PBS (pH 7.2) at GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–k) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.00, −0.10, −0.20, −0.30 V.
tiparallel -sheet and -turn, coexist in the solution at 0.60 V with the fractions of 0.65, 0.003, 0.003, 0.003 and 0.34, respectively. When applied potential shifts to 0.20 V, the fractions of ␣-helix and -turn decrease rapidly to 0.11 and 0.23, respectively, whereas the parallel -sheet and antiparallel sheet fractions increase to 0.28 and 0.34, respectively. This implies that the conformational transitions are mainly from ␣-helix, -turn to -sheet. In the potential range from 0.60 to 0.20 V, the cyt c reduction does not occur, so the conformational transitions may be driven by the positive electric field. When the applied potential continually shifts from 0.20 to 0.0 V, the reduction of cyt c occurs, resulting in more complex conformational transitions. Take ␣-helix as example, there is dramatic increase during the redox transition. This cor-
Fig. 6. Five conformations CD spectra of cyt c obtained by SVDLS analysis, (a) ␣-helix (solid line), (b) parallel -sheet (dashed line), (c) random coil (dotted line), (d) antiparallel -sheet (dash dotted line), (e) -turn (dash dot dotted line).
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Fig. 7. Potential-dependent conformations distribution curves of cyt c obtained by SVDLS analysis, (a–e) ␣-helix, parallel -sheet, random coil, antiparallel -sheet, -turn.
responds to a much compact protein conformation. So, this may be the main reason that there is no apparent electrochemical signal at bare GCE. In potential range from 0.0 to −0.30 V, the conformational transitions may be driven by the heavy absorption of cyt c on GCE surface. This is because cyt c is positively charged at neutral pH and might adsorb on the electrode surface when the potential moves to negative values. The random coil structure becomes the main conformation in solution and ␣-helix decrease sharply. These results clearly indicate that electrochemical reduction of cyt c is accompanied with the complex conformational transitions. Fig. 8 shows the in situ CD spectra of cyt c in far-UV region at SWNTs/GCE in 0.1 M PBS (pH 7.2). Unlike CD spectra change of cyt c at GCE surface, the negative Cotton peak increases with applied potential range from 0.60 to −0.30 V at SWNTs/GCE. This implies that conformational
Fig. 8. In situ CD spectra of 3.5 M cyt c in far-UV region in 0.1 M PBS (pH 7.2) at SWNTs/GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–k) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.00, −0.10, −0.20, −0.30 V.
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transition process shown in Fig. 7. A dramatic decrease of ␣-helix fraction was induced during this process, which indicates a more opening protein conformation. This may be the main reason why direct electrochemistry can be obtained at biocompatible electrode. Under the effect of negative electric field, ␣-helix fraction is still the main conformation although the adsorption of cyt c also occurs at SWNTs/GCE. All the results indicate that SWNTs can retain the cyt c conformation.
Fig. 9. Five conformations CD spectra of cyt c obtained by SVDLS analysis, (a) ␣-helix (solid line), (b) parallel -sheet (dashed line), (c) random coil (dotted line), (d) antiparallel -sheet (dash dotted line), (e) -turn (dash dot dotted line).
changes of cyt c are influenced strongly by electrode surfaces microstructure. Five principal conformations and their CD spectra are extracted from the experimental CD spectra by SVDLS analysis (Fig. 9). The plot of potential-dependent fractional distribution of the five conformations is shown in Fig. 10. Comparing with Fig. 7, when applied potential shifts to 0.20 V, the fractions of all kinds of conformations change slightly. Especially in the case of ␣-helix, there is dramatic decrease at GCE whereas retain almost unchanged at SWNTs/GCE. This sufficiently proves that SWNTs do hold the conformation of cyt c even under the effect of positive electric field. This result is similar to that obtained by Hildebrandt group [20]. When the applied potential continually shifts from 0.20 to 0.0 V, the conformational changes inherent to cyt c reduction transition are much different from the
Fig. 10. Potential-dependent conformations distribution curves of cyt c obtained by SVDLS analysis, (a–e) ␣-helix, parallel -sheet, random coil, antiparallel -sheet, -turn.
3.3.2. The effect of electrode surface on tertiary structure changes in electrochemical reduction of cyt c The CD spectra in the Soret band can provide further insight into the environment of the heme [6]. The in situ CD spectrum of cyt c obtained at GCE in 0.1 M PBS (pH 7.2) shows a positive Cotton peak at 398 nm and a negative cotton peak at 420 nm at 0.60 V (Fig. 11a). Lowering the potential to −0.30 V, the negative Cotton peak at ca. 420 nm is replaced by two positive peaks at around 393 and 428 nm (Fig. 11f). This indicates that the structural microenvironment of the cyt c heme is changed accompanied with cyt c reduction when induced conformational transitions take place. Oellerich et al. [38] have reported the Soret CD spectrum of cyt c induced by adding dioleoylphosphatidylglycerol (DOPG). They found that the negative Cotton peak of cyt c at ca. 417 nm would been replaced by two positive peaks at around 393 and 417 nm when DOPG was added into cyt c solution, which parallels the formation of the 5cHS species. Similar consideration may be taken into account by our experiment. Maybe a kind of 5cHS specie would be formed during the redox transition of cyt c at GCE surface. Fig. 12 shows the in situ CD spectra of cyt c obtained at SWNTs/GCE in 0.1 M PBS (pH 7.2). The spectra changes
Fig. 11. In situ Soret CD spectra of 3.2 M cyt c in 0.1 M PBS (pH 7.2) at GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–f) 0.60, 0.25, 0.08, 0.00, −0.10, −0.30 V.
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Fig. 12. In situ Soret CD spectra of 3.5 M cyt c in 0.1 M PBS (pH 7.2) at SWNTs/GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–f) 0.60, 0.25, 0.08, 0.00, −0.10, −0.30 V.
of cyt c at SWNTs/GCE are different from those shown in Fig. 11. Lowering the potential induces a transition from positive Cotton peak to negative one together with a red-shift of the absorption peak to 410 nm. At the same time the negative cotton peak becomes positive accompanying a red-shift of the absorption peak to 425 nm (Fig. 12f). This indicates that the transition of potential-dependent heme configuration at SWNTS/GCE is different from that at GCE.
4. Discussion 4.1. The conformational transitions of cyt c coupled with electrochemical reduction The spectra obtained by in situ UV–vis and CD give a better insight into the conformational changes of cyt c induced by electrochemistry. Several intermediate states of cyt c will be produced when ferricytochrome is reduced to ferrocytochrome corresponding to the changes of wavelength and intensity of Soret band in in situ UV–vis spetra of cyt c (Fig. 2) [12]. The intense Soret band results from the additive effects of the transition dipole moments of the two orbital excitations a1u –eg and a2u –eg of the –∗ transitions of porphyrin ring of cyt c [39]. Therefore, the intensity of the Soret band will be affected by changes of the symmetry of the porphyrin ring resulting from protein conformational changes [39]. Because the intensity of the Soret band increases step by step during the electron transfer process of cyt c, there should be more than one conformational adjustment processes in this reaction. This phenomenon can be seen clearly from the potential-dependent conformational distribution curves of cyt c (Figs. 7 and 10). The change of state of heme axial coordination induced by the change of porphyrin ring symmetry results in the al-
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ternation of Fe atom microenvironment, which will induce a decrease of the positive ellipticity in Soret region CD spectra (Figs. 11 and 12). This is indicative of a progressive weakening of the axial heme bonds and flattening of the heme conformation [40]. Therefore, a major tertiary structure change has to be occurred, which probably also gives rise to the changes of the CD spectra in the far-UV region [40]. The negative band of CD spectrum at 420 nm in Soret region observed for cyt c derives from the direct interaction of the –∗ transition of the aromatic ring of Phe-82 with the –∗ transition of heme group [41]. The change from negative band to positive ellipticity at 420 nm indicates that this interaction has been changed, which may affect the –∗ electron transition of heme group. Because the transition electrons are mainly located in HOMOs, the energy levels and the shape of the HOMOs may be influenced by the change of interaction accompanied with electron transfer. This is identical with that reported [4]. The heme group in cyt c is covalently bound to the poplypeptide backbone by two thioether linkages from Cys14 and Cys-17, and histidine residue and methionine residue act as the fifth and the sixth ligands of heme group, respectively. So the adjustment of the heme structure corresponding to intramolecular and intermolecular electron transfer will result in the movement of poplypeptide backbone related to these residues [36]. These residues may include Asn-52, Tyr67, Thr-78, Phe-82 [8], and Trp-59 [10]; Cys-14 and Cys-17 are included in -turn, Tyr-67 is also in -turn connected two ␣-helices; Asn-52 is included in an ␣-helix; Met-80 and Phe82 are in a region of random coil [42]; and Trp-59 belongs to -sheet [10]. So the changes of cyt c secondary structure resulting from the movements of these residues will take place with redox transition (Figs. 7 and 10). 4.2. The effect of SWNTs on the conformational changes of cyt c Although previous report clearly indicates that the carbon nanotubes can retain the activity of proteins [16,17,43], it is the first time to give molecular level information about how the activity of protein is retained at SWNTs taking cyt c as an example. Comparing the absorption spectra collected in LOPTLC at GCE (Fig. 2) with the those at SWNTs/GCE (Fig. 3), a slight red-shifting in wavelength and no decrease in intensity are observed even when the applied potential attains to −0.50 V at SWNTs/GCE. The results indicate that the conformation of cyt c adsorbed on SWNTs/GCE can be retained. That is electrode surface microstructure do affect the electrode surface-dependent conformational changes of adsorbed cyt c. Hidebrandt group [18-21] have studied conformational state of cyt c adsorbed on Ag electrodes coated with selfassembled monolayer (SAM) of carboxyl-terminated alkanethiols by surface enhanced resonance Raman spectroscopy. Potential-dependent measurements indicate that there were two conformational states, the native state B1 (in which axial
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ligands of the heme iron His-18 and Met-80 stabilize a sixcoordinated low-spin) and state B2 (in which lacks the Met80 axial ligand), coexist on SAM-coated electrodes. They found that state B2 was not observed on SAM-coated electrodes when the electrodes were in contact with cyt c solution. But such a dependence of the B2 /B1 distribution on the cyt c concentration in solution was not found for cyt c adsorbed on bare electrodes [20]. They explained that intermolecular interactions between the adsorbed proteins on SAM-coated electrodes may cause a reorientation to a configuration on which the native conformational state B1 was the thermodynamically favored [20]. Antal`ık et al. [44] reported that the presence of a negatively charged field created by covalently binding of polyanion was necessary for cyt c to form a structure with more stable Met80-Fe bond. According to our experimental phenomena, SWNTs can retain the native configuration of adsorbed cyt c. This result is also inconsistent with that obtained by M¨antele group. They found that the interaction between the protein and the molecules of promoter covering the electrode surface plays an important role in the stabilization of the native structure of cyt c [36]. The SWNTs functionalized with abundant oxygencontained functionalities and the interaction between these groups and cyt c maybe provide a negatively charged field to stabilize the Met80-Fe bond. This can be further revealed by the Soret CD spectra of cyt c at GCE and SWNTs/GCE as described in Section 3.3.2, a kind of 5cHS specie may be formed during the redox transition of cyt c at GCE surface. The SWNTs coated on electrode surface can also decrease the effect of positive electric field on cyt c and retain its secondary structure. This can be investigated by distribution curves of the five conformations of cyt c with varied applied potential in far-UV region (Figs. 7 and 10). Fig. 7 shows that ␣-helix, -turn and -sheet are changed seriously before reduction taking place. But all kinds of conformations of cyt c are unchanged at SWNTs in the same condition (Fig. 10), which clearly proves that SWNTs can maintain the conformation of cyt c. Wackerbarth et al. [18] reported that the decreasing of chain length of the thiols of SAM, that is, increasing electric field strength would favor the formation of B2 conformational state. Liu’ group [10] suggested that anion could screen the lysine and arginine surface charges to reduce the lysine–lysine repulsions and suppress the protein fluctuations. Taking into account these results, SWNTs can decrease the disturbance of the electric field and maybe the interaction between COO− , OH− and cyt c provide a negatively charged field to screen the lysine and arginine surface charges, which makes the repulsions between lysine and lysine reduce. So the conformation of cyt c becomes more stable.
5. Conclusion As a conclusion, the electrochemical reduction process of cyt c and the effect of electrode surface microstruc-
ture on conformational changes were studied by CD and UV spectroelectrochemical methods followed by data analysis. The results showed the electrode surface-dependent conformational changes during the electrochemistry reduction. The main points of this study were summarized as follows: 1. The SWNTs/GCE could effectively decrease the effect of electric field on the conformational changes of cyt c and stabilize the Met80-Fe bond of adsorbed cyt c. 2. Electron transfer induced a more opening conformational change at SWNTs/GCE than that at GCE. 3. UV–vis spectroelectrochemical measurements at SWNTs/GCE and double logarithmic plot proved to be suitable tools for the extraction of thermodynamic and kinetic information of electrochemical reaction. It gave not only the information of mechanism but also the parameters of the redox process. 4. CD spectroelectrochemical measurement and SVDLS program were suitable for dealing with the conformational transition of proteins in an electrochemical process, which gives the number of components, CD spectrum of each component and its distribution fraction with applied potential.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 20211130506, 20275036).
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Effect of electrode surface microstructure on electron transfer induced conformation changes in cytochrome c monitored by in situ UV and CD spectroelectrochemistry Xiue Jiang, Zheling Zhang, Hanying Bai, Xiaohu Qu, Junguang Jiang, Erkang Wang, Shaojun Dong∗ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Received 15 January 2004; accepted 18 May 2004
Abstract Electrochemical redox processes of bovine heart cytochrome c were investigated by in situ UV–vis and CD spectroelectrochemistry at bare glassy carbon electrode (GCE) and single-wall carbon nanotubes (SWNTs) modified glassy carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. The spectra obtained at GCE and SWNTs/GCE reflect electrode surface microstructure-dependent changes in protein conformation during redox transition. Potential-dependent conformational distribution curves of cytochrome c obtained by analysis of in situ circular dichroism (CD) spectra using singular value decomposition least square (SVDLS) method show that SWNTs can retain conformation of cytochrome c. Some parameters of the electrochemical reduction process, i.e. the product of electron transfer coefficient and number of electrons (αn = 0.3), apparent formal potential (E0 = 0.04 V), were obtained by double logarithmic analysis and standard heterogeneous electron transfer rate constant k0 was obtained by electrochemistry and double logarithmic analysis, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Electrode surface microstructure; Spectroelectrochemistry; SWNTs modified electrode; Conformational transitions of cytochrome c
1. Introduction Direct electron transfer reactions between electrodes and redox active groups in heme proteins have been focused in recent years because such studies may contribute to understand electron transport mechanisms in biological systems. Cytochrome c (cyt c) is one of the most extensively studied heme proteins so far in this field. Cyt c, whose function is to act as electron carriers in the electron transport chain, plays an important role in the biological respiratory chain. This is a much complex process involving intermolecular and intramolecular electron transfer [1,2], and its mechanism has been disputed for a long time [3-12]. The hypothesis of the conformational rearrangement of cytochrome c during ∗
Corresponding author. Fax: +86 431 5689711. E-mail address: [email protected] (S. Dong).
1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2004.05.036
electron transfer process has been supposed [5-12]. But, none of the experiment can clearly verify the conformational changes inherent to redox transition up to now. The voltammetric technique is useful for characterizing the redox behavior of metalloproteins. Unfortunately, the voltammetric response of cyt c is poor at bare electrode since cyt c adsorbs strongly on the electrode surface and results in denaturation [13,14]. So, it is necessary to provide a stable and biocompatible electrode surface to keep protein structure while allowing fast electron transfer [15]. One of the examples is using single-wall or multiwall carbon nanotubes (SWNTs or MWNTs) modified electrodes to get the direct electrochemistry of cyt c [16,17]. All the results indicate that the biocompatible electrode surface can accelerate the electron transfer between electrode and cyt c, and probably retain the conformation of cyt c. Hildebrandt groups have reported a series of important results about the effect of biocompatible
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on the structure and redox properties of cyt c with in situ Raman spectroscopy [18–21]. But the Raman spectroscopy can only identify the structural changes of cyt c that include the inner coordination sphere of the heme iron and not get the more information about the secondary structure changes. So a complementary study method should be further performed. Circular dichroism (CD) is a powerful tool for studying the conformation of the samples [22]. Studies combining CD spectrometry with thin layer electrochemistry were performed here. When the electrolytic time is long enough in a long optical path thin layer cell, the diffusion layer is approximate to the thin layer of the cell [23]. So CD thin layer spectroelectrochemistry can be used to investigate the conformational transitions of proteins in electrochemical redox process [24]. In this paper, in situ UV–vis and CD spectroelectrochemistry with a LOPTLC are used to investigate the redox reactions of cyt c at GCE and SWNTs/GCE. The main contributions of our study are to give a more direct insight about (i) how the conformations of cyt c are changed under the redox transition, (ii) why the reversible electrochemistry of cyt c can be obtained at SWNTs modified electrode. We describe the results of experiments aimed at gaining further molecular information to insight into the mechanism of electron transfer for cyt c and how the biocompatible electrode surface microstructure affect the conformational changes of protein during electrochemical reduction process.
treated SWNTs modified glassy carbon (8 mm × 8 mm) electrode prepared according to literature [16] was used as working electrode, a twisted platinum wire as auxiliary electrode, and a Ag/AgCl (saturated KCl) as reference electrode. Before modification, the GCE was mechanically polished with 1.0, 0.3, and 0.05 m ␣-Al2 O3 slurry, successively, and then washed ultrasonically in water followed by ethanol for a few minutes at each step. The LOPTLC is of 10.0 mm optical path length and 0.2 mm thickness of thin layer. The incident light passed through the thin layer being parallel to the working electrode. Before each measurement of in situ CD or UV–visible spectrum, the electrolysis potential of LOPTLC was applied for 5 min to keep all the cytochrome c molecules interact with electrode surface completely.
2.3. Data analysis 2.3.1. Singular value decomposition least squares analysis (SVDLS) To ascertain the different forms of cyt c presented over a range of potentials, the CD spectral data were evaluated by SVDLS analysis. In this analysis, several CD spectra were measured with the same m (m = total wavelengths) wavelengths. The spectra were grouped into series taken under identical conditions except for the changes of a single variable (as the potentials). The SVDLS mathematical and computational details have been reported previously [27,28].
2. Experimental 2.1. Reagents Bovine heart cyt c, purchased from Sigma, was prepared in 0.1 M phosphate buffer solution (PBS). The concentration of solution was determined spectrophotometrically using a molar absorptivity of 1.06 × 105 mol−1 cm−1 at 410 nm for cyt c [25]. Buffer solutions used in all experiments were prepared by Na2 HPO4 ·12H2 O and NaH2 PO4 ·2H2 O (analytical grade), doubly purified water was from Milli-Q system. SWNTs were bought from Shenzhen Nanotech Port Co. Ltd. and were purified and functionalized through a well-established way with slight modification [26]. After the purification, the SWNTs contain abundant oxygen-contained functionalities. All the other chemicals were of reagent grade and used as received. 2.2. Instruments and methods Spectroelectrochemical experiments were carried out in a home-made long optical path thin layer cell (LOPTLC), AVIV 62A DS circular dichroism spectrometer (AVIV Co., USA) for CD spectrum measurement, Cary 500 UV–vis–NIR spectrometer (Varian Co., USA) for UV–visible spectrum measurement and a CHI 630 electrochemical instrument (CHI Co., USA) for electrochemical operation. The pre-
2.3.2. Double logarithmic analysis [29] In this paper, some parameters of the electrochemical reduction process of cyt c at SWNTs/GCE with applied potentials can be obtained based on the following double logarithmic analysis equation ln ln
Ain − Af Ai − A f
=
−αnFEi + ln RT +
αnFE0 RT
∆t d
k0 (1 − θ)
(1)
where Ai is the absorption of reactant on applied potential, Af and Ain the finial and initial absorption of reactant, ␣n the product of electron transfer coefficient and number of electrons, t the electrolytic time for each potential, d the thickness of the thin layer, k0 the standard heterogeneous electron transfer rate constant, θ the percentage of the surface coverage, Ei and E0 are the applied potential and apparent formal potential, respectively. Other symbols have their common meanings. The concrete deduction can be seen in ref. [29]. According to Eq. (1), E0 can be obtained from the peak position of differential curve of A’(E)∼ E with respect to E, and αn from the slope of the double logarithmic line. k0 can be calculated by normal non-linear regression method.
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cyt c shows no obvious electrochemical activity on the bare GCE. While, SWNTs/GCE leads to a pair of well-defined redox peaks with the formal potential (E0 = (Ep,a + Ep,c )/2) of 0.05 mV, which is almost agreement with the reported formal potential of cyt c in solution [16,30,31]. The inset of Fig. 1 shows that the oxidation peak current of cyt c is linearly dependent on the square root of scan rates in the range from 5 to 100 mV/s, indicating a diffusion-controlled electrode process. Cathodic and anodic peak currents are almost equal throughout our study scan rate range. Randles-Sevick expression is used here [32]. 1/2
ipo = 269n3/2 ADo ν1/2 c
(2)
where n is the number of electron transfer, A the area of the electrode, v the scan rate (V/s), Do the charge transport diffusion coefficient (cm2 /s), c the concentration (mol/l) of cyt c. The value of Do can be calculated according to the slope of ipo ∼ v1/2 , Do = 3.9 × 10−7 cm2 /s. Based on Nicholson theory [33] the apparent heterogeneous electrode reaction rate constant (ks ) can be estimated. The average value of ks is 6.0 × 10−3 cm/s. It demonstrates the electrode process is quasi-reversible within the investigated scan rates range. Fig. 1. Cyclic voltammograms of 0.05 mM cyt.c at bare GCE (dashed line) and the SWNTs/GCE (solid line) in 0.1 M PBS (pH 7.2). Scan rate: 20 mv/s.
3. Results 3.1. Direct electrochemistry of cyt c at SWNTs/GCE Fig. 1 shows the cyclic voltammograms of 0.05 mM cyt c at bare GCE (dashed line) and the SWNTs/GCE (solid line) in 0.1 M PBS (pH 7.2) at 20 mV/s. It can be seen from Fig. 1 that
3.2. In situ UV–vis spectroelectrochemistry study of cyt c The wavelength and intensity of characteristic absorption spectra of cyt c are sensitive to the conformational state of the protein, especially for Soret band [34]. Fig. 2 shows in situ UV–vis spectra of cyt c at GCE in 0.1 M PBS (pH 7.2). It can be seen that the oxidized state of cyt c (ferricytochrome c) exhibits two major bands, the Soret band at 410 nm and  band at 530 nm, respectively (Fig. 2(a), curve (1)). While, the reduced state of cyt c (ferrocytochrome c) exhibits three major bands, the Soret band at 414 nm, the ␣ band at 550 nm,
Fig. 2. In situ UV–vis spectra of 3.2 M cyt c in 0.1 M PBS (pH 7.2) solution at GCE in a LOPTLC. Time interval: 5 min; applied potentials: (a) (1–9) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.0, −0.05 V; (b) (10–12) −0.10, −0.20, −0.30 V.
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Fig. 3. In situ UV–vis spectra of 3.5 M cyt c in 0.1 M PBS (pH 7.2) solution at SWNTs/GCE. Time interval: 5 min; applied potentials: (a–m) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.0, −0.10, −0.20, −0.30, −0.40, −0.50 V.
and the  band at 520 nm (Fig. 2(a), curve (9)). The results obtained are identical with those reported [34]. Upon the applied potential shifting from 0.6 to −0.05 V, a transition of characteristic absorption spectrum from the ferricytochrome c to ferrocytochrome c occurs. This can be seen in Fig. 2(a) (curves (1–9)). The absorption peak of Soret band shows redshifts from 410 to 414 nm, the  band shows blue-shifts from 530 to 520 nm, and ␣ band appears gradually. All of the Soret, ␣ and  bands increase in intensity accompanying with potential negatively shifting. But in the range from −0.10 to −0.30 V, all the three bands decrease in intensity accompanying with the blue-shift of the Soret band. (Fig. 2(b), curves (10–12)). This indicates a seriously adsorption on the GCE occurs under the negative electric field. Because the wavelength and intensity of Soret band are sensitive to the conformational state of the protein, all the variations may be a reflection for the conformational changes of the adsorbed ferrocytochrome c on GCE. In situ UV–vis spectra of cyt c at SWNTs/GCE in 0.1 M PBS (pH 7.2) are shown in Fig. 3. As can be seen that the absorption bands of ferricytochrome c and ferrocytochrome c are located near 409, 530, and 411, 520, 550 nm, respectively (Fig. 3a and m). Compared with the spectra of Fig. 2, the absorption peak of Soret band red-shifts only from 409 to 411 nm with the applied potential shifting from 0.60 to −0.50 V (Fig. 3a–m). But the intensity of all absorption peaks does not decrease even the cyt c enriched with positive charge adsorbed on the SWNTs/GCE under the negative electric field. This is obviously different from the phenomena shown in Fig. 2, indicating that the ferrocytochrome c conformation remains almost unchanged in this case. Maybe, the smaller red-shift of Soret band also implies that SWNTs can keep the conformational state of cyt c. The plot of absorbance at 409 nm against applied potentials shows a sigmoidal curve (figure not shown), and the corresponding differential curve gives a one-peak curve (Fig. 4B). Based on the peak position, the apparent formal po-
Fig. 4. (A) Double logarithmic curve; (B) differential curve. Experimental conditions are the same as those in Fig. 3.
tential E0 is estimated to be 0.04 V, which is almost in agreement with the formal potential value obtained with cyclic voltammetry. The double logarithmic plot of the spectroelectrochemical data is an oblique line (Fig. 4A). This type of double logarithmic plot suggests that the redox process of cyt c at SWNTs/GCE is a simple electrochemical reaction [29]. From the slope of the oblique line, ␣n is estimated to be 0.30. Using E0 , ␣n and experimental conditions in normal non-linear regression program, k0 is calculated to be (5.3 ± 0.16) × 10−3 cm/s, which is almost equal to the ks value (6.0 × 10−3 cm/s) obtained by electrochemistry. This further proves that electrode process of cyt c at SWNTs/GCE is quasi-reversible. 3.3. Structure changes of cyt c in electrochemical reduction reaction 3.3.1. The effect of electrode surface on conformational changes of cyt c Cyt c (3.2 M) in 0.1 M PBS (pH 7.2) in a LOPTLC at GCE was used for CD spectroelectrochemical measurement in far-UV region. The CD spectra in far-UV region provide information about the conformation of the polypeptide backbone [35]. Fig. 5 shows the in situ CD spectra of cyt c at GCE. As can be seen from Fig. 5, the negative Cotton peak decreases gradually with the applied potential shifting from 0.60 to −0.30 V. Fig. 6 shows the spectra of five different conformations of cyt c obtained after treated the spectra of Fig. 5 by the SVDLS analysis [34]. According to Gauss-Markoff mode of Protein Secondary structure CD Spectra, curves a, b, c, d, and e are attributed to ␣-helix, parallel -sheet, random coil, antiparallel -sheet and -turn, respectively [37]. Fig. 7 shows the corresponding potential-dependent plot of fractional distribution of the five conformations. The five conformations, i.e., ␣-helix, parallel -sheet, random coil, an-
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Fig. 5. In situ CD spectra of 3.2 M cyt c in far-UV region in 0.1 M PBS (pH 7.2) at GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–k) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.00, −0.10, −0.20, −0.30 V.
tiparallel -sheet and -turn, coexist in the solution at 0.60 V with the fractions of 0.65, 0.003, 0.003, 0.003 and 0.34, respectively. When applied potential shifts to 0.20 V, the fractions of ␣-helix and -turn decrease rapidly to 0.11 and 0.23, respectively, whereas the parallel -sheet and antiparallel sheet fractions increase to 0.28 and 0.34, respectively. This implies that the conformational transitions are mainly from ␣-helix, -turn to -sheet. In the potential range from 0.60 to 0.20 V, the cyt c reduction does not occur, so the conformational transitions may be driven by the positive electric field. When the applied potential continually shifts from 0.20 to 0.0 V, the reduction of cyt c occurs, resulting in more complex conformational transitions. Take ␣-helix as example, there is dramatic increase during the redox transition. This cor-
Fig. 6. Five conformations CD spectra of cyt c obtained by SVDLS analysis, (a) ␣-helix (solid line), (b) parallel -sheet (dashed line), (c) random coil (dotted line), (d) antiparallel -sheet (dash dotted line), (e) -turn (dash dot dotted line).
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Fig. 7. Potential-dependent conformations distribution curves of cyt c obtained by SVDLS analysis, (a–e) ␣-helix, parallel -sheet, random coil, antiparallel -sheet, -turn.
responds to a much compact protein conformation. So, this may be the main reason that there is no apparent electrochemical signal at bare GCE. In potential range from 0.0 to −0.30 V, the conformational transitions may be driven by the heavy absorption of cyt c on GCE surface. This is because cyt c is positively charged at neutral pH and might adsorb on the electrode surface when the potential moves to negative values. The random coil structure becomes the main conformation in solution and ␣-helix decrease sharply. These results clearly indicate that electrochemical reduction of cyt c is accompanied with the complex conformational transitions. Fig. 8 shows the in situ CD spectra of cyt c in far-UV region at SWNTs/GCE in 0.1 M PBS (pH 7.2). Unlike CD spectra change of cyt c at GCE surface, the negative Cotton peak increases with applied potential range from 0.60 to −0.30 V at SWNTs/GCE. This implies that conformational
Fig. 8. In situ CD spectra of 3.5 M cyt c in far-UV region in 0.1 M PBS (pH 7.2) at SWNTs/GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–k) 0.60, 0.40, 0.25, 0.15, 0.12, 0.08, 0.04, 0.00, −0.10, −0.20, −0.30 V.
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transition process shown in Fig. 7. A dramatic decrease of ␣-helix fraction was induced during this process, which indicates a more opening protein conformation. This may be the main reason why direct electrochemistry can be obtained at biocompatible electrode. Under the effect of negative electric field, ␣-helix fraction is still the main conformation although the adsorption of cyt c also occurs at SWNTs/GCE. All the results indicate that SWNTs can retain the cyt c conformation.
Fig. 9. Five conformations CD spectra of cyt c obtained by SVDLS analysis, (a) ␣-helix (solid line), (b) parallel -sheet (dashed line), (c) random coil (dotted line), (d) antiparallel -sheet (dash dotted line), (e) -turn (dash dot dotted line).
changes of cyt c are influenced strongly by electrode surfaces microstructure. Five principal conformations and their CD spectra are extracted from the experimental CD spectra by SVDLS analysis (Fig. 9). The plot of potential-dependent fractional distribution of the five conformations is shown in Fig. 10. Comparing with Fig. 7, when applied potential shifts to 0.20 V, the fractions of all kinds of conformations change slightly. Especially in the case of ␣-helix, there is dramatic decrease at GCE whereas retain almost unchanged at SWNTs/GCE. This sufficiently proves that SWNTs do hold the conformation of cyt c even under the effect of positive electric field. This result is similar to that obtained by Hildebrandt group [20]. When the applied potential continually shifts from 0.20 to 0.0 V, the conformational changes inherent to cyt c reduction transition are much different from the
Fig. 10. Potential-dependent conformations distribution curves of cyt c obtained by SVDLS analysis, (a–e) ␣-helix, parallel -sheet, random coil, antiparallel -sheet, -turn.
3.3.2. The effect of electrode surface on tertiary structure changes in electrochemical reduction of cyt c The CD spectra in the Soret band can provide further insight into the environment of the heme [6]. The in situ CD spectrum of cyt c obtained at GCE in 0.1 M PBS (pH 7.2) shows a positive Cotton peak at 398 nm and a negative cotton peak at 420 nm at 0.60 V (Fig. 11a). Lowering the potential to −0.30 V, the negative Cotton peak at ca. 420 nm is replaced by two positive peaks at around 393 and 428 nm (Fig. 11f). This indicates that the structural microenvironment of the cyt c heme is changed accompanied with cyt c reduction when induced conformational transitions take place. Oellerich et al. [38] have reported the Soret CD spectrum of cyt c induced by adding dioleoylphosphatidylglycerol (DOPG). They found that the negative Cotton peak of cyt c at ca. 417 nm would been replaced by two positive peaks at around 393 and 417 nm when DOPG was added into cyt c solution, which parallels the formation of the 5cHS species. Similar consideration may be taken into account by our experiment. Maybe a kind of 5cHS specie would be formed during the redox transition of cyt c at GCE surface. Fig. 12 shows the in situ CD spectra of cyt c obtained at SWNTs/GCE in 0.1 M PBS (pH 7.2). The spectra changes
Fig. 11. In situ Soret CD spectra of 3.2 M cyt c in 0.1 M PBS (pH 7.2) at GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–f) 0.60, 0.25, 0.08, 0.00, −0.10, −0.30 V.
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Fig. 12. In situ Soret CD spectra of 3.5 M cyt c in 0.1 M PBS (pH 7.2) at SWNTs/GCE in a LOPTLC. Time interval: 5 min; applied potential: (a–f) 0.60, 0.25, 0.08, 0.00, −0.10, −0.30 V.
of cyt c at SWNTs/GCE are different from those shown in Fig. 11. Lowering the potential induces a transition from positive Cotton peak to negative one together with a red-shift of the absorption peak to 410 nm. At the same time the negative cotton peak becomes positive accompanying a red-shift of the absorption peak to 425 nm (Fig. 12f). This indicates that the transition of potential-dependent heme configuration at SWNTS/GCE is different from that at GCE.
4. Discussion 4.1. The conformational transitions of cyt c coupled with electrochemical reduction The spectra obtained by in situ UV–vis and CD give a better insight into the conformational changes of cyt c induced by electrochemistry. Several intermediate states of cyt c will be produced when ferricytochrome is reduced to ferrocytochrome corresponding to the changes of wavelength and intensity of Soret band in in situ UV–vis spetra of cyt c (Fig. 2) [12]. The intense Soret band results from the additive effects of the transition dipole moments of the two orbital excitations a1u –eg and a2u –eg of the –∗ transitions of porphyrin ring of cyt c [39]. Therefore, the intensity of the Soret band will be affected by changes of the symmetry of the porphyrin ring resulting from protein conformational changes [39]. Because the intensity of the Soret band increases step by step during the electron transfer process of cyt c, there should be more than one conformational adjustment processes in this reaction. This phenomenon can be seen clearly from the potential-dependent conformational distribution curves of cyt c (Figs. 7 and 10). The change of state of heme axial coordination induced by the change of porphyrin ring symmetry results in the al-
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ternation of Fe atom microenvironment, which will induce a decrease of the positive ellipticity in Soret region CD spectra (Figs. 11 and 12). This is indicative of a progressive weakening of the axial heme bonds and flattening of the heme conformation [40]. Therefore, a major tertiary structure change has to be occurred, which probably also gives rise to the changes of the CD spectra in the far-UV region [40]. The negative band of CD spectrum at 420 nm in Soret region observed for cyt c derives from the direct interaction of the –∗ transition of the aromatic ring of Phe-82 with the –∗ transition of heme group [41]. The change from negative band to positive ellipticity at 420 nm indicates that this interaction has been changed, which may affect the –∗ electron transition of heme group. Because the transition electrons are mainly located in HOMOs, the energy levels and the shape of the HOMOs may be influenced by the change of interaction accompanied with electron transfer. This is identical with that reported [4]. The heme group in cyt c is covalently bound to the poplypeptide backbone by two thioether linkages from Cys14 and Cys-17, and histidine residue and methionine residue act as the fifth and the sixth ligands of heme group, respectively. So the adjustment of the heme structure corresponding to intramolecular and intermolecular electron transfer will result in the movement of poplypeptide backbone related to these residues [36]. These residues may include Asn-52, Tyr67, Thr-78, Phe-82 [8], and Trp-59 [10]; Cys-14 and Cys-17 are included in -turn, Tyr-67 is also in -turn connected two ␣-helices; Asn-52 is included in an ␣-helix; Met-80 and Phe82 are in a region of random coil [42]; and Trp-59 belongs to -sheet [10]. So the changes of cyt c secondary structure resulting from the movements of these residues will take place with redox transition (Figs. 7 and 10). 4.2. The effect of SWNTs on the conformational changes of cyt c Although previous report clearly indicates that the carbon nanotubes can retain the activity of proteins [16,17,43], it is the first time to give molecular level information about how the activity of protein is retained at SWNTs taking cyt c as an example. Comparing the absorption spectra collected in LOPTLC at GCE (Fig. 2) with the those at SWNTs/GCE (Fig. 3), a slight red-shifting in wavelength and no decrease in intensity are observed even when the applied potential attains to −0.50 V at SWNTs/GCE. The results indicate that the conformation of cyt c adsorbed on SWNTs/GCE can be retained. That is electrode surface microstructure do affect the electrode surface-dependent conformational changes of adsorbed cyt c. Hidebrandt group [18-21] have studied conformational state of cyt c adsorbed on Ag electrodes coated with selfassembled monolayer (SAM) of carboxyl-terminated alkanethiols by surface enhanced resonance Raman spectroscopy. Potential-dependent measurements indicate that there were two conformational states, the native state B1 (in which axial
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ligands of the heme iron His-18 and Met-80 stabilize a sixcoordinated low-spin) and state B2 (in which lacks the Met80 axial ligand), coexist on SAM-coated electrodes. They found that state B2 was not observed on SAM-coated electrodes when the electrodes were in contact with cyt c solution. But such a dependence of the B2 /B1 distribution on the cyt c concentration in solution was not found for cyt c adsorbed on bare electrodes [20]. They explained that intermolecular interactions between the adsorbed proteins on SAM-coated electrodes may cause a reorientation to a configuration on which the native conformational state B1 was the thermodynamically favored [20]. Antal`ık et al. [44] reported that the presence of a negatively charged field created by covalently binding of polyanion was necessary for cyt c to form a structure with more stable Met80-Fe bond. According to our experimental phenomena, SWNTs can retain the native configuration of adsorbed cyt c. This result is also inconsistent with that obtained by M¨antele group. They found that the interaction between the protein and the molecules of promoter covering the electrode surface plays an important role in the stabilization of the native structure of cyt c [36]. The SWNTs functionalized with abundant oxygencontained functionalities and the interaction between these groups and cyt c maybe provide a negatively charged field to stabilize the Met80-Fe bond. This can be further revealed by the Soret CD spectra of cyt c at GCE and SWNTs/GCE as described in Section 3.3.2, a kind of 5cHS specie may be formed during the redox transition of cyt c at GCE surface. The SWNTs coated on electrode surface can also decrease the effect of positive electric field on cyt c and retain its secondary structure. This can be investigated by distribution curves of the five conformations of cyt c with varied applied potential in far-UV region (Figs. 7 and 10). Fig. 7 shows that ␣-helix, -turn and -sheet are changed seriously before reduction taking place. But all kinds of conformations of cyt c are unchanged at SWNTs in the same condition (Fig. 10), which clearly proves that SWNTs can maintain the conformation of cyt c. Wackerbarth et al. [18] reported that the decreasing of chain length of the thiols of SAM, that is, increasing electric field strength would favor the formation of B2 conformational state. Liu’ group [10] suggested that anion could screen the lysine and arginine surface charges to reduce the lysine–lysine repulsions and suppress the protein fluctuations. Taking into account these results, SWNTs can decrease the disturbance of the electric field and maybe the interaction between COO− , OH− and cyt c provide a negatively charged field to screen the lysine and arginine surface charges, which makes the repulsions between lysine and lysine reduce. So the conformation of cyt c becomes more stable.
5. Conclusion As a conclusion, the electrochemical reduction process of cyt c and the effect of electrode surface microstruc-
ture on conformational changes were studied by CD and UV spectroelectrochemical methods followed by data analysis. The results showed the electrode surface-dependent conformational changes during the electrochemistry reduction. The main points of this study were summarized as follows: 1. The SWNTs/GCE could effectively decrease the effect of electric field on the conformational changes of cyt c and stabilize the Met80-Fe bond of adsorbed cyt c. 2. Electron transfer induced a more opening conformational change at SWNTs/GCE than that at GCE. 3. UV–vis spectroelectrochemical measurements at SWNTs/GCE and double logarithmic plot proved to be suitable tools for the extraction of thermodynamic and kinetic information of electrochemical reaction. It gave not only the information of mechanism but also the parameters of the redox process. 4. CD spectroelectrochemical measurement and SVDLS program were suitable for dealing with the conformational transition of proteins in an electrochemical process, which gives the number of components, CD spectrum of each component and its distribution fraction with applied potential.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 20211130506, 20275036).
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