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J. Phys. Chem. B 2001, 105, 1578-1586
Heterogeneous Electron Transfer of Cytochrome c on Coated Silver Electrodes. Electric Field Effects on Structure and Redox Potential Daniel H. Murgida and Peter Hildebrandt* Max-Planck-Institut fu¨ r Strahlenchemie, Stiftstrasse 34-36, D-45470 Mu¨ lheim, Germany ReceiVed: October 12, 2000; In Final Form: December 16, 2000
Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) of ω-carboxylalkanethiols that were covalently attached to Ag electrodes. Employing surface-enhanced resonance Raman (SERR) spectroscopy, the redox equilibria and the structural changes of the adsorbed Cyt-c were analyzed quantitatively for SAMs of different chain lengths ranging from 2-mercaptoacetic acid (C2-SAM) to 16mercaptohexadecanoic acid (C16-SAM). In the presence of Cyt-c in the bulk solution, the SERR spectra of the adsorbed Cyt-c display the characteristic vibrational band pattern of the native protein conformation denoted as state B1. The enhancement of the SERR signals decreases with increasing chain length, but even at distances as large as 24 Å (C16-SAM), SERR spectra of high quality could be obtained. Conversely, no SERR signals could be detected for SAMs including hydroxyl instead of carboxylate headgroups, implying that Cyt-c is adsorbed via electrostatic interactions. On the basis of potential-dependent SERR experiments, the redox equilibria of the adsorbed Cyt-c (B1) were analyzed, revealing ideal Nernstian behavior (n = 1). However, the redox potentials exhibit negative shifts compared to that of Cyt-c in solution, which increase with the chain length of the SAMs. In the absence of excess Cyt-c in solution (i.e., 0.2 µM), a new conformational state B2 of the adsorbed Cyt-c is observed. This state B2, which differs from the native state B1 by the heme pocket structure, includes three substates of different spin and coordination configurations. The distribution among these substates as well as the total contribution of state B2 varies with the chain length of the SAM such that the latter decreases from 73% at C2-SAM to 0% at C11- and C16-SAMs. These results imply that the formation of B2 is induced by the electric field at the binding site, generated by the potential drop across the electrode/SAM interface. When an electrostatic model for the interfacial potential distribution for the electrode/ SAM/protein device is employed, both the redox potential shifts and the electric-field-induced structural changes can be consistently explained. The impact of these findings for the processes of Cyt-c at biological interfaces is discussed.
Introduction The first step of many natural long-range interprotein electron transfer (ET) processes is the formation of a complex between the partner proteins, which, in general, results from electrostatic interactions via oppositely charged binding domains.1 Thus, ET must proceed through an array of charged residues and, hence, occurs under the influence of electric fields which may exhibit considerable strengths.2 If one of the reaction partners is embedded in a membrane, such electric fields may be further enhanced by a transmembrane potential gradient.3 A typical example is the complex of cytochrome c (Cyt-c) and the membrane-bound enzyme cytochrome c oxidase (CcO) for which the binding domains have been shown to exhibit several positively charged lysine and negatively charged glutamate (aspartate) residues, respectively.1a,b,4 Up to now, there are only a few studies dedicated to exploring the effect of electric fields on ET processes. For intramolecular ET processes in photosynthetic reaction centers, Boxer and coworkers found that ET rate constants varied by a factor of 4 upon changing the external electric field by ca. 1.2 × 106 V/cm on oriented samples.5 Sarti et al. studied CcO reconstituted in phospholipid vesicles and demonstrated that the generation of a transmembrane potential caused a ca. 10-fold reduction of * To whom correspondence should be addressed. Fax: +49 208 306 3951. E-mail:
[email protected].
the enzymatic activity that was attributed to an inhibition of the ET reactions.6 However, not only are the kinetics of the redox processes likely to be affected by electric fields. There are a variety of experimental studies which have shown that the formation of electrostatically stabilized complexes induces structural changes of the partner proteins.7 In the case of the Cyt-c/CcO complex, at least for Cyt-c,7a-d such changes can be considered to be primarily due to the action of electrostatic fields because for this protein qualitatively the same structural perturbations have been observed upon binding to phospholipid vesicles, polyanions, and electrodes.7d,8 Specifically, it was found that a portion of the bound Cyt-c is converted from the native form (denoted as state B1) to a conformational state B2 that lacks the axial Met-80 ligand.7d,8e Hence, electrostatic fields may substantially perturb the redox site structure, which, in turn, should have a pronounced effect on the ET properties of the heme protein.8a,e In the present work, we have investigated electric field effects on the redox process of Cyt-c electrostatically bound to electrodes that are covered with self-assembled monolayers (SAM) of carboxyl-terminated alkanethiols.9 Such coated electrodes represent more realistic models for charged biological interfaces than bare metal electrodes inasmuch as these monolayers exhibit similarities with phospholipid membranes with respect to the amphiphilic character of the constituents and the mode of self-organization. Furthermore, binding of Cyt-c to the
10.1021/jp003742n CCC: $20.00 © 2001 American Chemical Society Published on Web 02/06/2001
Heterogeneous Electron Transfer of Cytochrome c carboxylate headgroups of the SAM is likely to be controlled by electrostatic forces that are comparable to those upon binding to the carboxylate-rich interaction domain of CcO. A specific advantage of these coated electrodes is that the electric field strength at the interaction site can be altered in a systematic manner by varying the alkane chain length of the SAM and/or the electrode potential. In previous studies on Cyt-c bound to SAM-coated Au electrodes, electrochemical techniques have been employed albeit with conflicting results.10-13 Whereas Niki and co-workers reported redox potentials of the adsorbed Cyt-c identical to that in solution,10 negatively shifted values were reported by Bowden’s group.11 Furthermore, cyclic voltammetry (CV) and other electrochemical methods cannot identify the species involved in the redox process so that any conclusions concerning the structural integrity of the adsorbed Cyt-c that are exclusively based on the redox potential appear to be premature. These difficulties can be overcome when Ag instead of Au electrodes are used as supports for SAM coatings. These devices allow one to employ surface-enhanced resonance Raman (SERR) spectroscopy, which selectively probes the vibrational spectrum of the heme group solely of the adsorbed Cyt-c.14 Hence, stationary and time-resolved potential-dependent SERR measurements can provide information about the thermodynamics and kinetics of the interfacial redox process as well as of the redox site structures of the adsorbed species involved.8a,e,15 The present SERR spectroscopic study is dedicated to the analysis of the redox potential and the structural changes of Cyt-c adsorbed on SAM coatings of different chain lengths in order to gain more insight into the effect of electrostatic fields on the redox site structure and the ET properties. Materials and Methods Chemicals. Mercaptoacetic acid (C2), 3-mercaptopropionic acid (C3), 11-mercaptoundecanoic acid (C11), 16-mercaptohexadecanoic acid (C16), 2-mercaptoethanol, and 11-mercaptoundecanol were purchased from Sigma and used without further purification. 6-Mercaptohexanoic acid (C6) was synthesized according to published procedures16a and purified by highperformance liquid chromatography. Horse heart cytochrome c (Cyt-c; Sigma) was purified as previously published.8a SERR Spectroscopy. SERR spectra were measured with 413-nm cw excitation using a Kr ion laser (Coherent 302) with a power of ca. 60 mW at the sample. The scattered light (90°) was focused onto the entrance slit of a double monochromator (ISA U1000) working as a spectrograph and equipped with a liquid-nitrogen-cooled charge-coupled device camera. The spectral bandwidth was 4 cm-1 and the increment per data point 0.53 cm-1. The total accumulation time of the SERR spectra was between 5 and 30 s. After background subtraction, the SERR spectra were subjected to a component analysis in which complete spectra of the individual species are fitted to the measured spectra.17 Electrochemical Cell for SERR Experiments. For the SERR experiments, a home-built rotating electrode was used which differs in several aspects from the one described previously.15b Upon reduction of the length of the rotating shaft and the size and mass of the Delrin body housing the Ag ring, it was possible to lower the electrochemically active surface from 2.1 to 0.5 cm2 and the cell volume from 40 to 8 mL. The cell was machined from Delrin including quartz windows (Suprasil) for the incident laser beam and the scattered radiation. Platinum and Ag/AgCl electrodes were used as counter and reference electrodes, respectively. All potentials cited in this
J. Phys. Chem. B, Vol. 105, No. 8, 2001 1579 work refer to the saturated calomel electrode (SCE). The Ag ring is pressed against the stainless steel shaft by a Delrin cap and can easily be unscrewed for cleaning and roughening purposes. Electrical contact from the Ag working electrode to the potentiostat was achieved via a slip ring (Macon) attached to the rotating shaft. The rotational shaft is connected via an electrically insulating tube coupling to a dc motor equipped with a gear that is chosen according to the desired range of rotational frequencies. The present experiments were carried with rotational frequencies between 2 and 4 Hz. The advantages of this cell compared to the previously used setup are a considerable reduction of the amount of sample needed for the experiments as well as a higher mechanical stability of the rotational motion and a better optical throughput so that the efficiency of detecting the SERR scattered light is increased by a factor of ca. 10. Protocol for the SERR Experiments. SERR-active surfaces were prepared according to the previously published procedure.15b After activation, the Ag electrodes were immersed in a 2 mM ethanolic solution of the ω-carboxylalkanethiols for 24 h and then gently rinsed with ethanol and dried. Cyt-c was electrostatically adsorbed to the coated electrodes following two different procedures: (A) The modified electrode was immersed in a solution containing 2 µM Cyt-c in the supporting electrolyte for 45 min, then removed and rinsed with supporting electrolyte to eliminate unbound or loosely bound protein, and subsequently placed into the electrochemical cell containing a Cyt-c-free solution of the supporting electrolyte. (B) The modified electrode was directly placed into the electrochemical cell containing the supporting electrolyte and 0.2 µM Cyt-c, which was allowed to adsorb at open circuit for 45 min. The supporting electrolyte included a 12.5 mM potassium phosphate buffer (pH ) 7.0) and 12.5 mM K2SO4 in each case. All of the experiments were repeated several times to ensure reproducibility. Results Surface Enhancement on Coated Ag Electrodes. Cyt-c was electrostatically adsorbed on Ag electrodes coated with five ω-carboxylalkanethiols differing only by the chain length. For a given ω-carboxylalkanethiol, the SERR spectra of Cyt-c exhibit comparable signal-to-noise ratios and intensities regardless of the method of Cyt-c adsorption and the presence of Cyt-c in the bulk solution. These findings imply that the detected SERR signals exclusively originate from the adsorbed proteins and that Cyt-c is strongly adsorbed at an ionic strength of ca. 60 mM. The latter conclusion is in line with the slow temporal decay of the SERR intensity of less than 50% within 2 h. The SERR intensity of the adsorbed Cyt-c decreases with increasing chain length of the ω-carboxylalkanethiol (Figure 1). Nevertheless, high-quality spectra can be obtained even for C16. The distance dependence is in line with previous findings by Compagnini et al.18 Structural Changes of the Adsorbed Cytochrome c. It is well-known that adsorption of Cyt-c on a bare Ag electrode induces a conformational equilibrium between the native state, denoted as B1, in which the axial ligands of the heme iron His18 and Met-80 stabilize a six-coordinated low-spin (6cLS) configuration, and a new conformational state B2 that lacks the Met-80 axial ligand.7d,8e The B2 state consists of three substates in which the sixth coordination site of the heme either remains vacant, leading to five-coordinated high-spin (5cHS) configuration, or is occupied by a water molecule or by a new strongfield ligand, most likely His-33, to give a six-coordinated highspin (6cHS) or a six-coordinated low-spin (6cLS) configuration,
1580 J. Phys. Chem. B, Vol. 105, No. 8, 2001
Murgida and Hildebrandt
Figure 1. SERR spectra of Cyt-c electrostatically adsorbed on Ag electrodes coated with C2-, C11-, and C16-SAM. The spectra were measured under the same conditions.
Figure 3. SERR spectra of Cyt-c adsorbed on Ag electrodes coated with C2-SAM (top) and C3-SAM (bottom) measured at +0.03 V. The component spectra of the oxidized B1, the reduced B1, and the oxidized B2 species are given by the dotted, dashed, and dash-dotted lines, respectively.
Figure 2. SERR spectra of Cyt-c electrostatically adsorbed on Ag electrodes coated with C2-SAM (top) and C3-SAM (bottom). The spectra measured at -0.104, -0.194, and -0.404 V are shown by the solid, dashed, and dotted lines, respectively.
respectively. All of these species can be readily distinguished based on their characteristic SERR spectra, specifically, in the range between 1300 and 1700 cm-1.8e The frequencies of most of the bands in this region are well-established spectral indicators for the oxidation, spin, and coordination state of the heme iron.8a-e,19 Figure 2a shows SERR spectra of Cyt-c on a C2-coated Ag electrode, measured in the absence of Cyt-c in the bulk solution. The spectral range displayed includes the marker bands ν4 and ν3, which give rise to peaks with maxima at ca. 1360 and 1490 cm-1, respectively. These positions are characteristic of the reduced Cyt-c in the 6cLS configuration. In the spectrum measured at a potential of -0.104 V, both bands exhibit distinct shoulders at ca. 1373 and 1503 cm-1, respectively, that are
indicative of an oxidized 6cLS species. The intensity ratios of the conjugate bands remain essentially unchanged even if the potential is lowered to -0.194 V, i.e., to a potential much more negative than the redox potential of the native conformational state B1 (+0.01 V).20 Only when the potential is further lowered to -0.404 V is a complete reduction of the adsorbed Cyt-c achieved, as indicated by the disappearance of the 1373 and 1503 cm-1 band components. These findings imply that a portion of Cyt-c bound to C2-coated Ag electrodes exhibits a redox potential that is much more negative (
