Electric-Field Control of the pH-Dependent Redox Process of

May 30, 2012 - Biomolecular, Spectroscopy/LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1083, NL-1081 HV Amsterdam, The ...
1 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/JPCC

Electric-Field Control of the pH-Dependent Redox Process of Cytochrome c Immobilized on a Gold Electrode Bo Jin,†,∥ Gui-Xia Wang,†,∥ Diego Millo,‡ Peter Hildebrandt,§ and Xing-Hua Xia*,† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China ‡ Biomolecular, Spectroscopy/LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands § Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, Sekr. PC14, D-10623-Berlin, Germany

ABSTRACT: The pH-dependent redox processes of cytochrome c (cyt c) immobilized on a gold electrode that was coated with a self-assembled monolayer (SAM) of mercaptounadecanoic acid (MUA) were studied by electrochemical methods combined with quartz crystal microbalance (QCM) and surface enhanced infrared absorption (SEIRA) spectroscopy. Variation of the solution pH in the range from 4.0 to 10.0 determines the surface charge of the SAM, for which an apparent pKa of 6.0 was determined, whereas the structure of the electrostatically bound cyt c remains largely unchanged. Thus, the pH-dependence of the interfacial redox process reflects the electric-field control of cyt c immobilization which in turn has a pronounced impact on the electron transfer process. In the pH range between 7.0 and 4.0, the electrostatic interactions with the cationic protein are weakened due to the protonation of the carboxyl headgroups of the SAM such that the immobilized protein remains highly mobile and can rapidly adopt the orientation which is most favorable for electron transfer. Thus, the rate constant for direct electron transfer remains unchanged in this pH range, but it decreases upon increasing the pH above 7.0. The dramatic slowdown of the interfacial electron transfer is attributed to the increased strength of electrostatic binding which traps the protein in an orientation that is unfavorable for electron exchange with the electrode. The present study demonstrates that the solution pH is an important parameter that allows for optimizing interfacial electron transfer processes of electrostatically bound proteins.

■

bilayer and the integral proteins can cause a high electric field strength at the binding domains of functioning proteins.7,8 The electric field strength can reach up to 109 V/m which may influence the kinetics of charge-transfer processes and the conformations of the proteins, and thus may result in reaction mechanisms different from that of proteins in solutions. Similar local electric field strengths exist at the electrode/solution interface which thus is considered to be an appropriate biomimetic system to explore the consequences of high electric fields on biological processes. In fact, we recently found that the electric field at an electrode/solution interface may perturb the orientation of immobilized cyt c and thus changed the distance

INTRODUCTION Protein immobilization on electrodes has been widely employed to investigate the structural changes and dynamics of intermolecular electron transfer in native cells. Cytochrome c (cyt c), a single-heme protein that mediates electron transport between the integral membrane protein complexes of the respiratory chain, has been employed as a model protein in several studies on electron transfer, with implications for fundamental and applied research.1−6 As the natural reaction site of many redox proteins, the cell membrane possesses various ion pumps and ion-transporting channels, dealing with the transport of fat-insoluble and fatsoluble ions. Concentration gradient of ions across the membrane is a universal phenomenon, which generates a potential drop across the membrane. This potential drop as well as the heterogeneous charge distribution within the lipid © 2012 American Chemical Society

Received: April 18, 2012 Revised: May 26, 2012 Published: May 30, 2012 13038

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

Article

V·s−1 until a stable voltammogram was obtained. A typical nanoporous Au electrode was prepared by applying a constant potential of 1.4 V on the gold electrode in 2 M HCl for 40 s. This porous gold electrode designated as p-Au was rinsed with DI water and dried before use. The prepared nanoporous Au film electrode was incubated in 2 mM MUA ethanolic solution at 20 °C for 20 h. Then, the modified electrode was thoroughly rinsed with ethanol and DI water to remove the physically adsorbed MUA molecules. For the adsorption of cyt c, the electrode was incubated in a 10 mM pH 7.0 PBS solution containing 1 mg·mL−1 of cyt c at 4 °C for 6 h. Subsequently, the cyt c/MUA/p-Au electrode was rinsed by PBS thoroughly and then stored in the refrigerator prior to use. Electrochemical Analysis. Cyclic voltammetric (CV) and amperometric measurements were performed using a CHI 400 electrochemical workstation (CH Instruments, U.S.A.) in a three-electrode system with an Ag/AgCl (3 M KCl) as the reference electrode and a Pt wire as the counter electrode. Solutions were first purged with high-purity nitrogen for 10 min, and a nitrogen environment was then maintained in the cell during the whole electrochemical measurements. All experiments were performed at ambient temperature (25 ± 2 °C). Electrochemical impedance spectroscopy (EIS) measurements were performed on an Autolab electrochemical analyzer (Eco Chemie, The Netherlands) to characterize surface charging state of MUA-SAM on a gold electrode. Electrochemical impedance spectra were collected at 0.21 V in a 1 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) mixture in 0.1 M KCl with different pH values (adjusted by adding dilute KOH or HCl solution), and an alternating current voltage of 5.0 mV was applied within the frequency ranging from 0.1 Hz to100 kHz. Charge transfer resistance was calculated from the semicircle of obtained Nyquist plots. Quartz Crystal Microbalance (QCM) Characterization. QCM measurements were carried out with a CHI 440A electrochemical analyzer (CH Instruments). A gold QCM resonator (geometric area 0.196 cm2, fundamental frequency 8 MHz) was pretreated with piranha solution for two minutes (piranha solution, 1:3 volume ratio of 30% H2O2 and 98% H2SO4. Caution: piranha solution should be handled with extreme care, and only small volumes should be prepared at any time). The electrode was then washed with water and dried using nitrogen gas. The MUA-SAM was assembled on Au surface with the same method as on p-Au electrode. Then, the QCM electrode was placed in a Teflon electrochemical cell and fixed by O-rings. Thus one side of the electrode can contact with the solution and the other was sealed in air. SEIRA Spectroscopy. Infrared spectra were measured with a Bruke Tensor27 Fourier transform spectrometer in attenuated total reflection mode, using a spectral resolution of 4 cm−1. The gold NPs film deposited on i-hemisphere was electrochemically cleaned in H2SO4 solution after it was assembled in optical path. P-polarized IR radiation was totally reflected at the gold nanoparticles film/solution interface with an incident angle of 70° and was detected with a liquidnitrogen-cooled mercury cadmium telluride (MCT) detector. After a layer of MUA-SAM formed on the Au NPs film, the prism was rinsed and covered by 10 mM PBS solution (pH 7.0). Then, a reference spectrum was recorded and sample spectra were recorded as a function of time as cyt c (1

between the heme center and the support.4 The electron transfer rate constant of cyt c is the largest at an electrode potential near to the potential of zero charge (PZC, ca. −0.1 V vs SCE) and decreases rapidly as the electrode potential deviates from the PZC due to the increase of interfacial electric field strength. A theoretical study of the influence of the electric field on the electron transfer of cyt c has revealed the dipole moment alignment of the adsorbed cyt c molecules, which strongly affected the overall electron transfer rate.2 To elucidate fundamentals of the biological processes, it is therefore essential to understand the influence of the electric field on the interfacial electron transfer and conformation of proteins at an interface. In this respect, appropriately chemically modified electrodes can be used as convenient model systems to mimic the charge distribution on membrane/solution interfaces. In the last two decades, proteins immobilized on these biomimetic interfaces have been intensely explored to understand the thermodynamics and kinetics of redox reactions of protein monolayers.9−11 Self-assembled monolayers (SAMs) of ω-substituted alkanethiols, including hydroxyl12 and carboxyl-terminated alkanethiols,13−15 have been widely used as functional modifiers to study the electron transfer (ET) reaction of redox proteins and bioactivity of enzymes. Previous work has proven that a carboxyl-terminated alkanethiols monolayer on a gold electrode can provide ideal surface matrices for immobilizing the cyt c molecule and also for promoting its direct electrochemistry.16−24 Phosphonate-terminated SAMs can also provide a biofunctional interface for immobilization and direct electrochemistry as shown for hemoglobin.11 In addition, it has been reported that the direct electron transfer process for H2 oxidation by nickel−iron hydrogenase can be modulated by controlling the terminal group and the chain length of SAMs.25 In this work, we employ electrochemical techniques in combination with quartz crystal microbalance (QCM) measurements and surface enhanced infrared absorption (SEIRA) spectroscopy to study the pH-dependent dynamics of charge-transfer processes of cyt c on porous gold (p-Au) electrodes modified with 11-mercapto-1-undecanoic acid (MUA) SAMs. The results show that the interfacial electric field plays a crucial role in the direct electrochemistry of immobilized proteins. The present work may help to understand the fundamentals of interfacial redox processes of immobilized proteins and, in a wider context, may contribute to improve the performance of biosensors, bioelectronics, and biofuel cells.

■

EXPERIMENTAL SECTION Materials. 11-Mercaptoundecanoic acid (MUA) and horse heart cytochrome c were purchased from Sigma-Aldrich and used without further purification. Phosphate buffer solutions (PBS, pH 7.0) were prepared with KH2PO4 and K2HPO4. In all experiments, deionized water (DI water, >18 MΩ cm) from a Milli-Q purification system (Purelab Classic Corp., USA) was used. All chemicals were of reagent grade. Preparation of MUA/Porous Au Electrode and Cyt c/ MUA on Au Electrode. An Au disk electrode (diameter 2 mm, 99.99%) was used in our work. Prior to use, the Au electrode was mechanically polished to a mirror-like surface with 1.0, 0.3, and 0.05 μm Al2O3 slurry, subsequently. After ultrasonication in alcohol and water, the well-polished electrode was electrochemically pretreated by cycling the potential between 0 and 1.5 V in 0.5 M H2SO4 at a scan rate of 0.1 13039

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

Article

mg·mL−1) contained PBS solution was added. Typically, 128 scans were averaged.

■

RESULTS AND DISCUSSION Determination of Surface pKa for the MUA Monolayer. The surface pKa of acid-terminated SAMs is one of the most important parameters controlling the surface properties of the monolayer.26−29 Among the strategies to measure the apparent surface pKa,30−33 electrochemical titration is a simple and appropriate method. In our experiments, the negatively charged ferricyanide ion has been used as electrochemical probe to characterize the charging states of the MUA/p-Au using the electrochemical impedance spectroscopic (EIS) technique. Figure 1 shows a typical charge transfer resistance

Figure 2. Cyclic voltammograms for cyt c/MUA/p-Au (a) and MUA/ p-Au electrodes (b) in 10 mM PBS (pH 7.0) at a scan rate of 0.1 V·s−1.

coverage (13 pmol·cm−2).38 In the present measurements, there are some sites of the MUA/p-Au electrode that cannot be accessed by cyt c (2.5 × 2.5 × 3.7 nm339,40) possibly due to steric hindrance. In addition, the current peak potential separation for the heme center of cyt c is only 13 mV, and the ratio of the anodic peak current to cathodic peak current is equal to 1.0, indicating an electrochemically reversible redox process. The direct electron-transfer rate constant ks can be calculated as described by Laviron.41 Assuming the charge-transfer coefficient α as 0.5 and the number of electrons n = 1, the calculated ks is 15 s−1 for cyt c adsorbed on the MUA/p-Au electrode at pH 7.0, which is in the range of values determined for similar devices.42 Influence of Solution pH Value on the Electrochemistry of cyt c/MUA/p-Au Electrode. The pH-dependent voltammetric response of cyt c immobilized on the MUASAM was studied in a pH range from 3.0 to 10.0. In these experiments, a cyt c/MUA/p-Au device was prepared by incubating a MUA/p-Au electrode in a solution of 10 mM PBS (pH 7.0) containing 1 mg·mL−1 of cyt c at 4 °C for 6 h. After the electrode was washed with 10 mM PBS (pH 7.0), it was immersed in 10 mM PBS of different pH values. As shown in Figure 3A, with the decrease of the solution pH from 7.0 to 3.0, the anodic and cathodic peak currents of cyt c continuously decrease and the formal potential (E0′) shifts positively, whereas the peak shape and the peak separation remain unchanged. However, in alkaline solutions (from pH 7.0 to 10.0), the shape of peaks broadens and the separation between oxidization and reduction peaks is enlarged (Figure 3B). In addition, the redox peak currents decrease. Figure 3C displays the relationship of the formal potentials (E0′) as a function of solution pH obtained from Figure 3, panels A and B. From pH 3.5 to 6.5, the data follow a straight line (solid line) well described by a linear fit of E (mV) = 162 − 25.75 pH (linear correlation coefficient of 0.999), corresponding to the coupling of proton translocation and electron transfer at a ratio of 0.5 protons per 1 electron. This effect may be related to the hydrogen-bonding rearrangement associated with the transition process between the oxidized and reduced states of cyt c.43 However, this linear relationship between the formal potential and solution pH is not observed in alkaline solutions. The formal potential slightly increases from 7.0 to 9.0, reaches a maximum value of 4.5 mV, and then decreases from pH 9.0 to 10.0. In addition, the peak separation (ΔEp) shows different tendencies in acidic and alkaline solutions (Figure 4). At pH 3.0 to 7.0, ΔEp remains almost constant. It varies only in a very small region between 24 mV and 9 mV. This indicates that the kinetics of the electron transfer process is not affected by the

Figure 1. Charge transfer resistance (Rct) of 1 mM Fe(CN)63−/4−at the MUA/p-Au electrode at various solution pH values.

titration curve (Rct) of 1 mM Fe(CN)63−/4− at the MUA/p-Au electrode at different pH of the solutions. Below pH 5.0, the charge transfer resistance increases slightly with the pH but increases exponentially from pH 5.0 to 7.5, until it finally levels off above pH 7.5. This pH-dependent titration curve is ascribed to the electrostatic interactions between the protonated (neutral) and deprotonated (anionic) head groups of the SAM and the negatively charged ferricyanide probe. Above pH 7.5, the SAM head groups are deprotonated, resulting in a high charge transfer resistance for the ferricyanide due to the electrostatic repulsion with the SAM surface. Conversely, below pH 5.0, the charge transfer resistance for ferricyanide ions is lower since the protonation of the carboxylate group promotes the electrostatic interaction between the SAM and the electroactive probe. From this titration curve, the surface pKa of MUA SAMs can be determined to be ca. 6. It has been reported the surface pKa of MUA-SAMs is located in the range from 6.0 to 7.1 depending on the chain length (mercaptoalkanoic acid with a longer chain gives a larger surface pKa), which is 1.5 to 2.5 pH units higher than that in the bulk solutions due to the interactions among the assembled molecules.34 Electrochemical Properties of cyt c Immobilized on the MUA/p-Au Electrode. Figure 2 shows the cyclic voltammograms of cyt c electrostatically adsorbed on the MUA/p-Au electrode. A pair of well-defined reduction− oxidation peaks are observed at ca. −0.09 V (vs Ag/AgCl), which reflects the oxidation and reduction of the heme Fe(III)/ Fe(II) redox couple in cyt c.35 Anodic and cathodic peak currents increase linearly with the scan rate ranging from 0.05 to 1.0 V·s−1. These results indicate that the direct electrochemical reaction of cyt c/MUA/p-Au electrode is a surfaceconfined process.36 Integration of the peak currents results in a surface coverage of electroactive cyt c of 9.1 pmol·cm−2,37 slightly smaller than the theoretical value for a full monolayer 13040

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

Article

Figure 5A shows the CVs of the cyt c/MUA/p-Au electrode measured subsequently in pH 7.0, 4.5, and 7.0 of 10 mM PBS.

Figure 3. Cyclic voltammograms of the cyt c/MUA/p-Au electrode in 10 mM PBS at different pH values: (A) from 3.0 to 7.0 and (B) from 7.0 to 10.0. (C) Plot of the formal potential vs the solution pH. Data were obtained from the CVs at a scan rate of 0.1 V·s−1.

Figure 5. (A) CVs of cyt c/MUA/p-Au measured subsequently in 10 mM PBS at (a) pH 7.0, (b) 4.5, and (c) 7.0. (B) CVs of cyt c/MUA/pAu measured subsequently in 10 mM PBS at (a) pH 7.0, (b) 10.0, and (c) 7.0. (C) CVs of cyt c/MUA/p-Au at pH 7.0 (10 mM PBS) after immersion at pH 10.0 (10 mM PBS) for 10, 20, and 30 min (from right to left). Scan rate was 0.1 V·s−1.

All of the CVs show the similar shape with almost the same current peak separation of 9 mV. This indicates that the electron transfer rate constants for the immobilized cyt c in pH 7.0 PBS and the remaining immobilized cyt c in pH 4.5 PBS are the same (vide supra). However, the formal potential shifts considerably from −11 to 56 mV from pH 7.0 to 4.5. This is probably due to the participation of the proton and the effect of pH on the protonation state of the SAM that is known to affect the cyt c-SAM interaction and therefore the formal potential.44 In addition, there is a considerable decrease in peak currents at pH 4.5, which cannot be recovered when the pH was changed back to pH 7.0. The charge for the anodic current peak (subtracted by the background signal of the MUA/p-Au electrode in the same solutions) is calculated to be 1.732 μC at pH 7.0 and 1.039 μC at pH 4.5. The difference in charge transfer demonstrates that 58% immobilized cyt c was desorbed and/or inactivated by changing solution pH from 7.0 to 4.5. In order to verify this hypothesis, the quartz crystal microbalance technique was used to monitor mass variation of the modified electrode. The results are shown in Figure 6. The resonant

Figure 4. Dependence of current peak separation (ΔEp) and logarithm of electron transfer constant (ks) on solution pH value. The ks was calculated from the average value of ΔEp obtained from at least five measurements in 10 mM PBS at a scan rate of 0.1 V·s−1.

pH variation in this range, possibly due to a constant orientation of cyt c respect to the electrode surface (vide infra). On the contrast, ΔEp shows a rapidly increase in alkaline solutions from 9 (pH 7.0) to 250 mV (pH 10.0). According to the Laviron’s equation, an electron transfer constant ks for cyt c in solutions with different pH value was calculated (Figure 4, empty cycles). The highest electron transfer constants are found in acidic solutions with values between 5.94 and 15.0 s−1 from solution pH 3.0 to 7.0. However, the electron transfer constant decreases rapidly with increasing the pH from 7.0 to 10.0. 13041

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

Article

requires about 30 min as indicated in Figure 5C. CVs measured at pH 7.0 after exposure at pH 10.0 display recovery in current increasing with the time at pH 7.0. These findings imply a rather slow pH-dependent transition of the immobilized cyt c between the native state (pH 7.0) and a state of perturbed electron transfer capability (pH 10.0). In solution, cyt c undergoes a conformational transition at ca. pH 9.5 that involves protein structural change including the replacement of the Met80 heme ligand by a Lys residue.46,47 This conformational transition is associated with large negative shift of the redox potential which is, however, not observed in the CV shown in Figure 5B. To elucidate the underlying molecular processes, we have employed SEIRA spectroscopy that selectively probes the IRactive modes of the immobilized protein.48 Figure 7A shows the

Figure 6. Resonant frequency shift of the MUA/Au electrode in different pH solution. Trace a to b indicates the addition of the cyt c solution into 10 mM PBS with pH 7.0. Trace b to c indicates the change of solution pH from 7.0 to 4.5.

frequency of a MUA/p-Au electrode in 10 mM PBS (pH 7.0) decreases rapidly upon addition of cyt c solution due to the adsorption of cyt c on the MUA-SAM (Figure 6, from trace a to trace b). After the resonant frequency reached a constant value (−467.3 Hz), the solution pH was changed to 4.5. As expected, the resonant frequency rapidly increases and finally reaches a constant value of −188.4 Hz (Figure 6, from trace b to trace c). The change of solution pH from 7.0 to 4.5 results in an increase of resonant frequency of 278.9 Hz, which demonstrates 59% mass weight losses from the cyt c modified electrode surface according to the Sauerbrey equation.45 This decreased mass is in full agreement with the charge decrease observed in cyclic voltammograms. Therefore, it can be concluded that the decrease in peak currents observed in CVs by changing solution pH in the acidic region is mainly due to desorption of cyt c from the MUA/p-Au electrode surface and not due to other processes that can impair the direct electron transfer (e.g., reorientation). This finding can be well understood if we consider that the surface charge properties of cyt c are largely controlled by the amino side chains of the large number of lysine residues. These groups are essentially protonated (positively charged) in the pH range from 7.0 to 4.5 such that protonation of the carboxylate groups of the SAM below pH 5.0 weakens the electrostatic binding and thus causes the partial desorption of the protein from the surface (Figure 3A). In a similar way, CVs of the cyt c/MUA/p-Au electrode, prepared at pH 7.0, were also recorded after the modified electrode was immersed in 10 mM PBS at pH 10.0. Now considerably different CVs were observed (Figure 5B). The redox peaks of the cyt c/MUA/p-Au electrode are broader and the current peak separation ΔEp increases to 256 mV (Figure 5B, curve b). The enlarged peak separation in the alkaline solution as compared to pH 7.0 (curve a) demonstrates that the electron transfer constant of the immobilized cyt c has decreased. Nevertheless, the immobilized cyt c molecules can be recovered to their initial states by changing the solution with pH from 10.0 to 7.0 as indicated by the recovery of the current peak separation to 9 mV (Figure 5 B, curve c), in agreement with previous results.17 However, the original peak current cannot be fully restored which may be attributed to the desorption of a fraction (ca. 20% as calculated from the change in currents) of cyt c molecules due to the weakened electrostatic binding of cyt c with the MUA-SAMs via (partial) deprotonation of lysine side chains.17 The full recovery of the original peak separation of 9 mV is a relatively slow process as it

Figure 7. (A) SEIRA spectra of cyt c adsorbed on MUA SAMs on Au at pH 7.0 (blue trace) and pH 9.0 (red trace) and (B) the difference spectrum “pH 9.0” minus “pH 7.0” from (A).

SEIRA spectra measured of cyt c adsorbed on the MUA-coated Au surface in contact with solutions of pH 7.0 and 9.0. The spectra are dominated by the amide I and amide II bands of the polypeptide backbone. The frequency of the amide I is characteristic of the specific secondary structure elements of the protein, among which the α-helices (40% in cyt c)49 provide by far the strongest contribution to peak centered at 1658 cm−1. Other structural elements (e.g., β-turns, random coil), albeit comprising 60% of the total protein structure, give rise to much weaker spectral contributions as can be more clearly seen in the second derivative of the spectrum (data not shown). In fact, the surface enhancement of the IR signals depends on both the distance and the orientation of the molecular oscillator with respect to the metal surface50,51 such that the SEIRA intensities are not directly proportional to portions of the individual secondary structure elements. Conversely, changes of the amide I band envelope in the SEIRA spectrum may reflect alterations of the secondary structure and/or orientational changes of the immobilized protein. A careful inspection of the SEIRA spectrum measured at pH 9.0 indicates a slight intensity increase of the amide I at 1658 13042

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

■

cm−1 by ca. 8% compared to the spectrum at pH 7.0, as it is more clearly shown in the difference spectrum “pH 9.0” minus “pH 7.0” (Figure 7B). The difference spectrum displays a positive peak at 1642 cm−1 with a shoulder at 1669 cm−1 which falls in the amide I frequency range of unordered, α-helical, and β-turn segments.52 However, there are no negative signals which are expected in the case of secondary structure changes. Thus, we conclude that the underlying pH-dependent transition corresponds to a reorientation of the immobilized protein on the MUA surface. In principle, one cannot rule out that only individual peptide segments undergo an orientational change. However, this interpretation is considered to be less likely in view of the lack of any other spectral changes in the SEIRA spectrum. Instead, the idea of a protein reorientation brought about changes of the charge distribution in the SAM/cyt c interface is in line with previous findings and, moreover, can readily account for the altered electron transfer properties.17,53 In a recent surface enhanced resonance Raman spectroscopic study on cyt c immobilized on Ag electrodes coated with MUA, it was found that at pH 9.0 cyt c is trapped in a state that exhibits the same redox site structure but with strongly redox activity.17 These results were interpreted in terms of a reorientational change of the protein at pH 9.0. This view is consistent with the two-step electron transfer mechanism of cyt c immobilized on negatively charged surfaces as it was proposed on the basis of experimental and theoretical analyses.46−49 First, electrostatic binding takes place via the preferred binding domain which is not the most favorable electron tunneling site. In the second step, the immobilized cyt c reorients on the surface until a configuration is obtained which is most favorable for electron transfer.53−55 At higher pH (7.0−10.0), complete deprotonation of the COOH groups of the SAMs will cause a very tight electrostatic binding of cyt c via the yet protonated amino groups of cyt c. This will cause a rigid fixation of the protein in the orientation that is not so favorable for electron tunneling and thus electron transfer becomes slower (Figure 4). Conversely, the interfacial electron transfer of cyt c remains almost unchanged at lower pH values (7.0−4.5). Due to the protonation of the carboxylate groups of the SAM, the electrostatic interactions with the immobilized cyt c are not so strong such that protein reorientation, a prerequisite for a fast electron transfer, is not blocked.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National 973 Basic Research Program (2012CB933804), the National Natural Science Foundation of China (21035002), the National Science Fund for Creative Research Groups (21121091), and the Natural Science Foundation of Jiangsu province (BK2010009). D.M. acknowledges The Netherlands Organizasion for Scientific Research (NWO), Veni grant 722.011.003.

■

REFERENCES

(1) Stevens, J. M. Metallomics 2011, 3, 319−322. (2) Alvarez-Paggi, D.; Martín, D. F.; DeBiase, P. M.; Hildebrandt, P.; Martí, M. A.; Murgida, D. H. J. Am. Chem. Soc. 2010, 132, 5769−5778. (3) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263−331. (4) Song, Y. Y.; Jia, W. Z.; Yang, C.; Xia, X. H. Adv. Funct. Mater. 2007, 17, 2377−2384. (5) Song, Y. Y.; Li, Y.; Yang, C.; Xia, X. H. Anal. Bioanal. Chem. 2008, 390, 333−341. (6) Zhou, J. H.; Lu, X. B.; Hu, J. Q.; Li, J. H. Chem.Eur. J. 2007, 13, 2847−2853. (7) Murgida, D. H.; Hildebrandt, P. Acc. Chem. Res. 2004, 37, 854− 861. (8) Clarke, R. J. Adv. Colloid Interface Sci. 2001, 89, 263−281. (9) Leger, C.; Bertrand, P. Chem. Rev. 2008, 108, 2379−2438. (10) Armstrong, F. A.; Hill, H. A. O.; Walton, N. J. Acc. Chem. Res. 1988, 21, 407−413. (11) Chen, Y.; Jin, B.; Guo, L. R.; Yang, X. J.; Chen, W.; Gu, G.; Zheng, L. M.; Xia, X. H. Chem.Eur. J. 2008, 14, 10727−10734. (12) Terrettaz, S.; Cheng, J.; Miller, C. J. J. Am. Chem. Soc. 1996, 118, 7857−7858. (13) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847− 1849. (14) Leopold, M. C.; Bowden, E. F. Langmuir 2002, 18, 2239−2245. (15) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559−565. (16) Grochol, J.; Dronov, R.; Lisdat, F.; Hildebrandt, P.; Murgida, D. H. Langmuir 2007, 23, 11289−11294. (17) Millo, D.; Bonifacio, A.; Ranieri, A.; Borsari, M.; Gooijer, C.; van der Zwan, G. Langmuir 2007, 23, 9898−9904. (18) Nakano, K.; Yoshitake, T.; Yamashita, Y.; Bowden, E. F. Langmuir 2007, 23, 6270−6275. (19) Beales, P. A.; Bergstrom, C. L.; Geerts, N.; Groves, J. T.; Vanderlick, T. K. Langmuir 2011, 27, 6107−6115. (20) Techane, S. D.; Gamble, L. J.; Castner, D. G. J. Phys. Chem. C 2011, 115, 9432−9441. (21) Layfield, J. P.; Troya, D. J. Phys. Chem. B 2011, 115, 4662−4670. (22) Kasmi, A. E.; Wallace, J. M.; Bowden, E. F.; Binet, S. M.; Linderman, R. J. J. Am. Chem. Soc. 1998, 120, 225−226. (23) Imabayashi, S. I.; Mita, T.; Kakiuchi, T. Langmuir 2005, 21, 1470−1474. (24) Li, Y.; Li, J.; Xia, X. H.; Liu, S. Q. Talanta 2010, 82, 1164−1169. (25) Ciaccafava, A.; Infossi, P.; Ilbert, M.; Guiral, M.; Lecomte, S.; Giudici-Orticoni, M. T.; Lojou, E. Angew. Chem., Int. Ed. 2012, 51, 953−956. (26) Raj, C. R.; Behera, S. J. Electroanal. Chem. 2005, 581, 61−69. (27) Tominaga, M.; Maetsu, S.; Kubo, A.; Taniguchi, I. J. Electroanal. Chem. 2007, 603, 203−211. (28) Abiman, P.; Crossley, A.; Wildgoose, G. G.; Jones, J. H.; Compton, R. G. Langmuir 2007, 23, 7847−7852.

■

CONCLUSION In summary, we have found that the solution pH significantly influences the direct electrochemistry of cyt c immobilized on a MUA/p-Au electrode surface as revealed by electrochemical, QCM, and SEIRA spectroscopic measurements. In acidic solutions, the reversible electron transfer process is observed although the peak currents decrease with the decrease of solution pH, due to partial desorption of the protein. In alkaline solutions, the electron transfer rate decreases with increasing pH due to a rigid fixation of cyt c in the orientation that is unfavorable for a rapid electron transfer. SEIRA spectra rule out pH-dependent structural changes and confirm the view of pHdependent orientational changes. Thus, the present work has shown that the pH-dependence of the interfacial redox process of cyt c reflects the electric-field control of the protein orientation which in turn is essential for a rapid electron transfer. 13043

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044

The Journal of Physical Chemistry C

Article

(29) Schweiss, R.; Welzel, P. B.; Werner, C.; Knoll, W. Langmuir 2001, 17, 4304−4311. (30) Gershevitz, O.; Sukenik, C. N. J. Am. Chem. Soc. 2004, 126, 482−483. (31) Yu, H. Z.; Xia, N.; Liu, Z. F. Anal. Chem. 1999, 71, 1354−1358. (32) Smalley, J. F. Langmuir 2003, 19, 9284−9289. (33) Sugihara, K.; Shimazu, K.; Uosaki, K. Langmuir 2000, 16, 7101− 7105. (34) Feng, Z. Q.; Imabayashi, S.; Kakiuchi, T.; Niki, K. J. Chem. Soc. Faraday Trans. 1997, 93, 1367−1371. (35) Rusling, J. F.; Nassar, A. E. F. J. Am. Chem. Soc. 1993, 115, 11891−11897. (36) Murray, R. W.; Bard, A. J. Electroanalytical Chemistry; Marcel Dekker: New York, 1984; pp 191−368. (37) Laviron, E. J. Electroanal. Chem. 1979, 100, 263−270. (38) Avila, A.; Gregory, B. W.; Niki, K.; Cotton, T. M. J. Phys. Chem. B 2000, 104, 2759−2766. (39) Margoliash, E.; Smith, E. L. Nature 1961, 192, 1121−1123. (40) Scott, R. A.; Mauk, A. G. Cytochrome c: A Multidisciplinary Approach; University Science Books: Sausalito, CA, 1995. (41) Laviron, E. J. Electroanal. Chem. 1979, 101, 19−28. (42) Wisitruangsakul, N.; Zebger, I.; Ly, K. H.; Murgida, D. H.; Egkasit, S.; Hildebrandt, P. Phys. Chem. Chem. Phys. 2008, 10, 5276− 5286. (43) Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062−4068. (44) Petrovic, J.; Clark, R. A.; Yue, H.; Waldeck, D. H.; Bowden, E. F. Langmuir 2005, 21, 6308−6316. (45) Sauerbrey, G. Z. Phys. 1959, 155, 206−222. (46) Battistuzzi, G.; Borsari, M.; Sola, M. Eur. J. Inorg. Chem. 2001, 2989−3004. (47) Assfalg, M.; Bertini, I.; Dolfi, A.; Turano, P.; Mauk, A G.; Rosell, F. I.; Gray, H. B. J. Am. Chem. Soc. 2003, 125, 2913−2922. (48) Ataka, K.; Heberle, J. Biopolymers 2006, 82, 415−419. (49) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 585−595. (50) Ataka, K.; Heberle, J. Anal. Bioanal. Chem. 2007, 388, 47−54. (51) Osawa, M. Top. Appl. Phys. 2001, 81, 163−187. (52) Goormaghtigh, E.; Cabiaux, V.; Ruysschaert, J. M. Eur. J. Biochem. 1990, 193, 409−420. (53) Kranich, A.; Ly, H. K.; Hildebrandt, P.; Murgida, D. H. J. Am. Chem. Soc. 2008, 130, 9844−9848. (54) Ly, K. H.; Wisitruangsakul, N.; Sezer, M.; Feng, J. J.; Kranich, A.; Weidinger, I.; Zebger, I.; Murgida, D. H.; Hildebrandt, P. J. Electroanal. Chem. 2011, 660, 367−376. (55) Paggi, D. A.; Martín, D. F.; Kranich, A.; Hildebrandt, P.; Martí, M.; Murgida, D. H. Electrochim. Acta 2009, 54, 4963−4970.

13044

dx.doi.org/10.1021/jp303740e | J. Phys. Chem. C 2012, 116, 13038−13044