Adsorption Orientation of Horse Heart Cytochrome c on a Bare Gold

Dec 4, 2011 - Dependence of the direct electron transfer activity and adsorption kinetics of cytochrome c on interfacial charge properties. Gui-Xia Wa...
2 downloads 6 Views 2MB Size
Download PDF
ARTICLE pubs.acs.org/JPCC

Adsorption Orientation of Horse Heart Cytochrome c on a Bare Gold Electrode Hampers Its Electron Transfer Shourui Lin,†,‡ Xiue Jiang,*,† Lixu Wang,† Guihua Li,† and Liping Guo‡ †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China ‡ Department of Chemistry, Northeast Normal University, Changchun 130024, China ABSTRACT: Direct electron transfer of horse heart cytochrome c (HHCC) adsorbed on a bare metal electrode has been shown to be hard to achieve. The reason for that has been discussed in terms of conformational changes, protein unfolding, and even denaturation of the protein. We explored the adsorption of HHCC on a bare Au electrode by surfaceenhanced infrared absorption spectroscopy on the molecular level. Our results revealed that the native secondary structure of adsorbed HHCC was kept even on the bare Au surface. The hampered electron transfer was mainly caused by its adsorption orientation. The adsorption of HHCC on the bare gold surface led the tyrosine 97 residue to be close to the surface. In this orientation, most of the α-helices of adsorbed HHCC run parallel to the bare Au surface with a flatter orientation of the heme relative to its orientation on an 11mercaptoundecanoic acid-modified Au surface.

’ INTRODUCTION The electron transfer capability of metalloproteins has attracted extensive attention since it can be exploited for applications in biosensors,1,2 bioelectronics,3 and biofuel cells.4,5Horse heart cytochrome c (HHCC) is a small metalloprotein that mediates single-electron transfer between the integral membrane protein complexes of the respiratory chain and is often considered a model metalloprotein to study electron transfer within and between proteins. Its redox center consists of heme iron that is covalently bound to the protein matrix through thioether linkages from two cysteine residues. Its natural redox property and small dimensions make it easy to integrate in biosensors or bioelectronics. Electrons are directly injected into and/or retracted from HHCC after its contact with the electrode has been constructed. Direct electrochemistry of HHCC adsorbed on a self-assembled monolayer (SAM) from carboxyl-terminated alkanethiols developed on a metal electrode has been attained.6 8 The negatively charged carboxyl headgroup of the SAM forms electrostatic interactions with the positively charged lysine-rich domain around a heme-exposed cleft.8 10 This interaction sets up a proper orientation to facilitate electron transfer from the heme iron to the metal electrode. The strength of the negative charge of the SAM plays an important role in manipulating electron transfer by controlling the immobilized nature.11 However, the direct electrochemistry of HHCC adsorbed on a bare metal electrode was for a long time hampered. With adsorption on a gold surface, HHCC no longer showed a well-defined electrochemical response. This has been disputably ascribed to the impurities of HHCC,12 formation of aggregates,13 surface blocking by progressive adsorption of inactive molecules,14 and protein denaturation.15 These empirical claims were based on the observation of the electron-transfer r 2011 American Chemical Society

redox current. Although understanding should come from the molecular information of adsorbed HHCC on the bare gold electrode, in situ detection of conformation and orientation changes occurring on the monolayer of adsorbed protein is technically challenging. Surface-enhanced resonance Raman spectroscopy (SERRS) experiments have successfully been applied to study adsorbed HHCC on a metal surface to observe the microenvironment changes of the heme.16 18 Adsorption resulted in a transition of the heme iron ligation from low spin to high spin and changes of the heme either in random or in flat orientation. However, SERRS suffers from the limitation that only the chromophore is observed due to the resonance condition. Surface-enhanced infrared absorption (SEIRA) spectroscopy has been proved to be a powerful technology to study the secondary structural changes of adsorbed protein on a monolayer level and provide a wealth of molecular information.11,19 21 Here we employed SEIRA spectroscopy to in situ monitor the adsorption of HHCC on a bare gold electrode and investigated the redox-induced conformational changes of the adsorbed HHCC by SEIRA difference spectroscopy. By comparing the SEIRA and the redox-induced SEIRA difference spectra of HHCC adsorbed on an 11-mercaptoundecanoic acid (MUA)-modified gold electrode, we found that the hindrance of electron transfer of adsorbed HHCC on the bare gold electrode was not from protein denaturation induced by the structural changes but was from the different orientations and suppressed rotation of the adsorbed protein. Received: July 6, 2011 Revised: November 29, 2011 Published: December 04, 2011 637

dx.doi.org/10.1021/jp2063782 | J. Phys. Chem. C 2012, 116, 637–642

The Journal of Physical Chemistry C

ARTICLE

’ EXPERIMENTAL METHODS Au Film Preparation. The experimental procedures for Au film preparation have been described elsewhere.19 Briefly, a thin gold film was prepared on the flat surface of a triangular silicon prism by chemical deposition. The surface of the Si substrate was polished with aluminum oxide powder 1 μm in size, followed by immersion in a 40 wt % aqueous solution of NH4F for 1 min. Subsequently, the flat surface of the Si prism was exposed to a 1:1:1 volume mixture of (1) 0.03 M NaAuCl4, (2) 0.3 M Na2SO4 + 0.1 M Na2S2O3 + 0.1 M NH4Cl, and (3) 2.5 vol % HF solution for 90 s. After electrochemical cleaning, the gold-coated prism was mounted into a poly(trifluorochloroethylene) cell. The IR beam from the interferometer of the FT-IR spectrometer (IFS 66 V/S, Bruker, Ettlingen, Germany) was coupled into the silicon prism at an incident angle of 60°, and the reflected beam intensity was recorded with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector. Kinetic SEIRA Spectroscopy To Monitor the Adsorption of HHCC. HHCC (Sigma) was used without further purification. Adsorption of HHCC on the bare Au surface was attained by exposing the bare Au film to a 2 μM HHCC solution dissolved in 10 mM phosphate buffer solution (phosphate-buffered saline (PBS), pH 7.2). Before sample spectra were acquired, a reference spectrum of 10 mM phosphate buffer solution, pH 7.2, was recorded. Concomitant with the addition of HHCC, IR spectra were recorded at a spectral resolution of 4 cm 1. A series of 15 spectra with intervals of 10, 60, and 300 s were recorded. Alternatively, before adsorption of HHCC, the bare Au surface was modified with MUA (Sigma) by immersing the Au film in 0.1 mM MUA (in 500 μL of ethanol) for 90 min, and then the MUA-modified Au film was incubated with 2 μM HHCC solution dissolved in PBS solution (pH 7.2). A reference spectrum of the 10 mM PBS solution (pH 7.2) was recorded in the absence of HHCC. Sample spectra were recorded with the addition of HHCC. Electrochemical Properties and Potential-Induced FT-IR Difference Spectra of the Adsorbed HHCC Monolayer. Cyclic voltammograms (CVs) were recorded with a CHI 830A electrochemical workstation (CH Instruments, Austin, TX). A Pt net auxiliary electrode and a Ag/AgCl reference electrode were used. All potentials were referred to the Ag/AgCl reference electrode. The Au film was used as a working electrode, connected via a copper plate to the electrochemical workstation. The PBS was purged with high-purity nitrogen for at least 10 min prior to each measurement to remove the dissolved oxygen. Cyclic votammograms were recorded parallel to the IR spectra in the potential range of 0.2 to +0.2 V with a scan rate of 50 mV/s. A singlebeam background IR spectrum was taken at 0.2 V, where the adsorbed HHCC is fully reduced. Then the potential was successively increased to +0.2 V, and a sample spectrum was acquired at each oxidation potential. A total of 512 scans were averaged for each pair of background and sample spectra. The whole procedure was repeated nine times, and the difference spectra were averaged to improve the signal-to-noise ratio.

Figure 1. Cyclic voltammograms of HHCC adsorbed to an HHCC/ MUA/Au electrode (red) and an HHCC/Au electrode (black) in 10 mM phosphate buffer solution (pH 7.2) at a scan rate of 50 mV/s.

Figure 2. Set of kinetic SEIRA spectra of 2 μM HHCC adsorbed to the MUA-modified Au surface (a) and the bare Au surface (b) in 10 mM phosphate buffer solution (pH 7.2) at 30, 60, 100, 213, 573, 1023, 2823, and 5223 s (from bottom to top).

a pair of redox peaks with a formal potential of 0.01 V (E° = (Ep,a + Ep,c)/2) and a peak-to-peak separation of 0.02 V (red). Integration of the peak currents gave a surface coverage of electroactive HHCC of 9.8 pmol cm 2, calculated with a geometric surface area of 1.77 cm2 with a roughness factor of 2.5. Although the determined coverage is lower as compared to that of the fully packed HHCC layer (15 pmol cm 2), the observed formal potential corresponds to those reported in the literature.6 However, as

’ RESULTS AND DISCUSSION Electrochemical Properties of Adsorbed HHCC. Figure 1 shows the electrochemical behaviors of HHCC adsorbed on the bare (black) and the MUA-modified (red) Au films. It is evident that HHCC adsorbed on the MUA-modified Au electrode shows 638

dx.doi.org/10.1021/jp2063782 |J. Phys. Chem. C 2012, 116, 637–642

The Journal of Physical Chemistry C

ARTICLE

Figure 3. SEIRA spectroscopy study of the orientation of HHCC adsorbed on different surfaces. Surface coverage and ratio of the amide I band to amide II band of HHCC adsorbed on the MUA-modified Au surface (red circle) and the bare Au surface (blue triangle) versus adsorption time.

in many reports, the cyclic voltammogram of HHCC adsorbed on the bare Au electrode shows no obvious redox peaks (black). Very small redox peaks were observed when differential pulse voltammetry was applied (data not shown). This has been explained by adsorption-induced conformational changes of the protein on the bare gold surface, protein unfolding, or even protein denaturation,22 surface blocking by the adsorption of inactive molecules,14 and the conformation and orientation changes of the heme.16 18 To understand this phenomenon intrinsically, this issue has been checked by SEIRA spectroscopy, which selectively monitored the adsorption process of HHCC on the bare and the MUA-modified Au surfaces on the molecular level. Adsorption Properties of HHCC. Figure 2 shows the SEIRA spectra of HHCC adsorbed on the MUA-modified (a) and the bare (b) Au surfaces at different times. After addition of HHCC into the solution, an immediate increase of two bands at 1659 and 1551 cm 1 was observed on both surfaces. These bands have been assigned to the amide I and II modes of adsorbed HHCC from the CdO stretching vibration and the in-plane N H bend and C N stretch, respectively. The overall shape and the maxima of the amide I band are determined by the secondary structure of the protein.23 25 The identical peak position and nearly the same shape of the amide I band suggest that the adsorption of HHCC on the bare Au electrode should not result in serious conformational changes, protein unfolding, and protein denaturation. Therefore, the electrochemical deactivation of HHCC adsorbed on the bare Au electrode should come from either subtle structural changes or a wrong orientation. Figure 3 shows the time-dependent amide I/II ratio and the surface coverage of HHCC adsorbed on the MUA-modified (red circles) and the bare (blue triangles) Au surfaces. The timedependent surface coverage derived from the integrated area of the amide II band also reflects the kinetics of adsorbed HHCC. It is evident that the binding isotherm is fitted assuming Langmuirtype adsorption with adsorption time constants τ of 304 and 696 s for HHCC adsorbed on the bare (blue triangles) and the MUA-modified (red circle) Au surfaces, respectively. Adsorption of HHCC on the bare Au surface is faster than on the MUAmodified Au surface, which might come from the different interaction modes between HHCC and the two surfaces. This is

Figure 4. Curve-fitting analysis of the secondary structure of HHCC adsorbed on different surfaces. The results of the quantitative curvefitting analysis for the amide I region of HHCC adsorbed on the MUA/ Au (a) and the bare Au (b) surfaces are shown as thin lines, and their sum total (gray dotted line) reproduces the original data (thick line) well. (c) Fraction of the secondary structure of HHCC adsorbed on the MUA/ Au (black) and the bare Au (gray) surfaces, as determined by quantitative curve-fitting analysis. Spectra were taken at 87 min after addition of protein to the solution.

further supported by different changes of the amide I/II ratio. With adsorption of HHCC on the bare Au surface, the amide I/II ratio remains nearly constant, while with adsorption of HHCC on the MUA-modified electrode, this ratio remains constant only before 1600 s, the time corresponding to saturation adsorption, and then slightly increases. Overall, the amide I/II ratio of HHCC adsorbed on the MUA-modified electrode is more than 2.1, which is larger than 1.7, the amide I/II ratio of HHCC adsorbed on the bare Au surface. The ratio of the amide I band to amide II band has been used as a useful tool to qualitatively assess orientation changes.25,26 Due to the surface selection rule of SEIRA spectroscopy, only molecular vibrational peaks giving rise to a dynamic dipole moment perpendicular to the surface will be enhanced. A bigger ratio of the amide I band to amide II band indicates that predominant α-helices of HHCC are perpendicular to the gold surface when adsorbed to the MUA-modified electrode. The amide 639

dx.doi.org/10.1021/jp2063782 |J. Phys. Chem. C 2012, 116, 637–642

The Journal of Physical Chemistry C

ARTICLE

Table 1. Positions (cm 1) and Fractional Areas (%) of the Amide I Bands of HHCC Adsorbed to the MUA-Modified Gold Surface or the Bare Gold Surface HHCC/MUA/Au band area HHCC/Au band area

assignment19

1680

28

1677

42

β-turn type III

1658

58

1658

42

α-helix

1644

2

1644

4

unordered

1637

7

1636

8

extended β-strand

1627

4

1625

4

extended β-strand

I mode of the helix is perpendicular and the amide II mode is parallel to the surface and, thus, result in a bigger amide I/II ratio. A smaller amide I/II ratio indicates a different orientation of adsorbed HHCC on the bare Au surface. The slight increase of the amide I/II ratio of adsorbed HHCC on the MUA/Au surface indicates a slight change in the orientation of HHCC on the surface. The adsorbed protein can alter its orientation according to the change of the local surrounding conditions.27 With rising surface densities, the repulsive forces among adsorbed proteins facilitate the orientation changes by rotation.27 However, this seems to be inhibited when HHCC is adsorbed on the bare Au surface. The rotation ability of adsorbed HHCC on the surface is important for electron transfer. Zhou et al.28 simulated the orientation distribution of the heme plane at different degrees of deprotonation of the carboxyl headgroup. They pointed out that the orientation angle comes closer to 90° with a narrower orientation distribution as the proton dissociation increases. To cause electron transfer, HHCC undergoes a repositioning of the heme relative to the electrode surface by changing its orientation or conformation; however, a surface with a highly negative charge restrains such repositioning because of strong electrostatic interactions, which deactivates the redox properties of adsorbed HHCC on a highly charged surface.11 The subtle structural changes upon adsorption of HHCC on the bare Au surface were identified by the amide I band analysis. Figure 4 shows the amide I regions of HHCC adsorbed on the MUA-modified (a) and the bare (b) Au surfaces at 87 min and the corresponding Gaussian curve-fitting analysis. Most of the peak positions and bandwidths were found in the second-derivative spectra. A peak for an unordered structure at ∼1644 cm 1 was added, although it was difficult to distinguish in the secondderivative spectra since it should be present.29 It can be seen that the sum of the fitted underlying component bands yields a simulated amide I band (red line) that is in very good agreement with the experimental curve (black line). The overall shapes of the amide I band adsorbed on both surfaces are similar, and the curve fitting also produces similar component bands. The assignment of those bands is listed in Table 1. Peaks with maxima at 1658, 1644, higher than 1670, and lower than 1637 cm 1 have been assigned to the α-helix, unordered structure, β-turn type III, and extended β-strand, respectively.19 After classification of the same structure, the fraction of each structure is shown in Figure 4c. Adsorption of HHCC on the bare Au surface shows a higher ordered structure with an α-helix of 42%, although this is smaller than the value of HHCC adsorbed on the MUA-modified Au surface, 58%. This difference was induced by the surface selection rule of SEIRA spectroscopy, supported by nearly the same fraction of the unordered structure on the two surfaces. Adsorption of HHCC on the MUA-modified Au surface results in perpendicular orientation of most of the α-helices relative to the

Figure 5. SEIRA spectroscopy study of the electron transfer of adsorbed HHCC on different surfaces. (a) Set of potential-induced SEIRA difference spectra of adsorbed HHCC on the MUA/Au (red) and the bare Au (black) surfaces between the fully reduced state ( 0.2 V) and various oxidation potentials. The arrow corresponds to an absorbance ΔA of 10 3. (b) Intensities of the band at 1693 cm 1 of the difference spectra displayed in part a plotted as a function of the applied potential.

gold surface. Obviously, the hindrance of electron transfer of adsorbed HHCC on the bare Au surface is not induced by serious structural changes. Potential-Induced SEIRA Difference Spectra of Adsorbed HHCC. Figure 5 shows a series of SEIRA difference spectra of HHCC adsorbed on the MUA/Au (red) and the bare Au (black) surfaces between the fully reduced state and various degrees of oxidation. The reference spectrum was taken at 0.2 V, and sample spectra were acquired at successively increased potentials. The appearance of a negative peak at 1693 cm 1 indicates a change in hydrogen bonding of the β-turn from that in the reduced form.30,31 The intensity of the band is plotted versus the applied potential (Figure 5b) upon the oxidation of adsorbed HHCC at the MUA/Au (black) and the bare Au (black) electrodes. A sigmoid dependence of the absorbance change of the band (red) suggests a direct electron transfer to the MUA/ Au electrode,31 whereas a nearly linear dependence (black) suggests neglectable electron transfer to the bare Au electrode. Figure 6 shows the potential-induced difference spectra of adsorbed HHCC on the MUA-modified (red) and the bare (black) Au surfaces between the fully oxidized (+0.2 V, positive peaks) and the reduced ( 0.2 V, negative peaks) states. Neither baseline correction nor any smoothing procedure has been applied. The spectral features of HHCC adsorbed on the MUA-modified Au surface (red line) are essentially the same as the reported results.19 The amide I bands are observed at 1693, 1666, 1642, and 1627 cm 1 for the reduced state of HHCC (negative peaks) 640

dx.doi.org/10.1021/jp2063782 |J. Phys. Chem. C 2012, 116, 637–642

The Journal of Physical Chemistry C

ARTICLE

Figure 7. Surface adsorption models of HHCC derived from SEIRA difference spectra. (a) HHCC was adsorbed on an MUA monolayer via electrostatic interaction between terminal amine groups of Lys 13 and 72 and the carboxylate surface of MUA. (b) HHCC was adsorbed on a bare Au surface via sulfur-containing Cys 14 and 17. The HHCC structure was created by Swiss PDB Viewer 3.7 with crystallographic data taken from the Protein Data Bank (PDB entry 1HRC).

Figure 6. Redox-induced SEIRA difference spectra probing the orientation and conformation of HHCC adsorbed on the MUA/Au (red) and the bare Au (black) electrodes.

indicates that the heme orientations of adsorbed HHCC on both surfaces are different. The adsorption orientation and interaction sites of HHCC on a mercaptopropionic acid (MPA)-modified electrode have been reported.19 Since MUA has a structure similar to that of MPA, we proposed the adsorption mode of HHCC on the MUA-modified gold surface as shown in Figure 7a. In this orientation, the type III β-turns comprising residues 67 70 and 14 17 are close to the surface, and Tyr 48 and 67 are at the half-height of the protein; therefore, the peaks at 1519/1513 cm 1 are small. The terminal amine groups of Lys 13, 72, and 86 are the dominant electrostatic interaction sites between the protein and the carboxyl moiety. When HHCC is adsorbed on the bare Au surface, Tyr 74 or 97 should approach the surface as we discussed above. On the basis of the crystal structure of HHCC (PDB entry 1HRC), the adsorption orientation of HHCC on the bare Au surface can be deduced. If Tyr 74 approaches the surface, the extended β-strand should also be close to the surface, and then the peak intensity of the extended β-strand at 1627 cm 1 should be enhanced according to the optical near-field effect. However, this is not observed in the SEIRA difference spectrum. Therefore, we proposed that the Tyr 97 residue approached the bare Au surface as shown in Figure 7b. In this orientation, the Tyr 48 and 67 residues are on the top surface of the protein. Therefore, the vibration bands at 1518( )/1512(+) cm 1 nearly disappear. The adsorption of HHCC on the bare gold electrode through the cysteine residues that directly bind to the heme group has been reported.15 Therefore, cysteine 14 and 17 should be the interaction sites between HHCC and the bare Au surface. By such an interaction, the heme is dragged to the Au surface, which induces the enhanced vibration of the porphyrin ring at 1417 cm 1. In this orientation, the heme is flatter than its orientation on the MUAmodified electrode; therefore, some bands of the heme vibration nearly disappear. Such an orientation also results in residues 67 70 being far from the surface and most of the α-helices being parallel to the surface, which induces a decrease of the intensity at 1693 cm 1 and a smaller amide I/II ratio. The electron transfer of HHCC adsorbed on a metal electrode has been studied for a long time. The adsorption has been shown to result in serious conformational changes, protein unfolding, and even denaturation of the protein.22 Our results clearly indicate that the adsorption of HHCC on the bare Au surface nearly maintains the native structure. The hampered electron transfer mainly comes from the adsorption orientation. HHCC is adsorbed

and at 1673 and 1660 cm 1 for the oxidized state (positive peaks). The bands at 1693 and 1673 cm 1 are assigned to the type III β-turns comprising residues 14 17 and 67 70,19 and the bands at 1666 and 1660 cm 1 are assigned to the type II β-turns that are found at residues 32 35 and 35 38.19 In addition to the amide I region, the amide II region (1600 1500 cm 1) is also included. The difference bands at 1602(+)/1595( ) and 1580 (+) cm 1 are assigned to the heme vibration, ν(CβCβ) and ν(C αC m)asym.18,19 The bands at 1519( )/1513(+)/ 1504(+) cm 1 are assigned to the in-plane ring vibration of a tyrosine residue.19 The spectral region between 1430 and 1480 cm 1 may include signals arising from CH2 and CH3 bending modes of the amino acid side chains at 1434(+) cm 1, the ν(CαCm)sym stretching vibration of the heme moiety at 1465 cm 1,19 and the ν(pyr quarter ring) vibration at 1417 cm 1.18 The potentialinduced difference spectrum of HHCC adsorbed on the bare Au surface (black) shows some different features. The band at 1737 cm 1 assigned to the vibrational mode of the MUA layer disappears because of the absence of the assembled MUA. The peak intensity at 1693 cm 1 decreases, and the peaks at 1602(+)/ 1595( )/1580(+) and 1518( )/1512(+) cm 1 nearly disappear. However, the peak intensities at 1504, 1434, and 1417 cm 1 significantly increase. According to the surface selection rule and the optical near-field effect, the IR signal is the strongest in close proximity and perpendicular to the surface. Different orientations of adsorbed HHCC will lead to major alterations in the intensities of the vibrational modes of those residues that change the direction of the dipole moment and the position relative to the surface. The bands at 1518( )/1512(+) cm 1 assigned to Tyr 48 and 67 nearly disappear, while the peak intensity at 1504 cm 1 significantly increases. This peak has been assigned to the in-plane ring-breathing mode of the tyrosine residue.19 According to the primary structure of the HHCC, possible residues are only Tyr 74 and 97. Therefore, Tyr 74 or 97 must be closer to the surface when HHCC is adsorbed on the bare Au surface than on the MUA-modified Au surface. Meanwhile, the increase of some signals arising from the heme vibrations, ν(CαCm)sym and ν(pyr quarter ring), at 1465 and 1417 cm 1 together with the decrease of some peak intensities corresponding to the vibration of the heme, ν(CβCβ) and ν(CαCm)asym, at 1602/1595/1580 cm 1 641

dx.doi.org/10.1021/jp2063782 |J. Phys. Chem. C 2012, 116, 637–642

The Journal of Physical Chemistry C

ARTICLE

on the bare gold surface via cysteine 14 and 17, which is different from the active binding site to its cognate binding partner. In addition, the adsorbed HHCC should have the ability to rotate the heme to engage in electron transfer. However, the rotation ability has been confined for HHCC adsorbed on the bare Au surface. This might be the other reason for the deactivation of the redox properties of adsorbed HHCC.

(8) Yue, H. J.; Waldeck, D. H. Curr. Opin. Solid State Mater. Sci. 2005, 9, 28–36. (9) Xu, J.; Bowden, E. F. J. Am. Chem. Soc. 2006, 128, 6813–6822. (10) Niki, K.; Hardy, W. R.; Hill, M. G.; Li, H.; Sprinkle, J. R.; Margoliash, E.; Fujita, K.; Tanimura, R.; Nakamura, N.; Ohno, H.; Richards, J. H.; Gray, H. B. J. Phys. Chem. B 2003, 107, 9947–9949. (11) Jiang, X.; Ataka, K.; Heberle, J. J. Phys. Chem. C 2008, 112, 813–819. (12) Reed, D. E.; Hawkridge, F. M. Anal. Chem. 1987, 59, 2334–2339.  .; Novak, M. J. Electroanal. Chem. 1995, 383, 75–84. (13) Sz€ues, A (14) Allen, H.; Hill, O.; Hunt, N. I.; Bond, A. M. J. Electroanal. Chem. 1997, 436, 17–25. (15) Zhou, Y.; Nagaoka, T.; Zhu, G. Biophys. Chem. 1999, 79, 55–62. (16) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710–6721. (17) Niaura, G.; Gaigalas, A. K.; Vilker, V. L. J. Electroanal. Chem. 1996, 416, 167–178. (18) Yu, Q.; Golden, G. Langmuir 2007, 23, 8659–8662. (19) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2004, 126, 9445–9457. (20) Jiang, X.; Zaitseva, E.; Schmidt, M.; Engelhard, M.; Siebert, F.; Schlesinger, R.; Ataka, K.; Vogel, R.; Heberle, J. Proc. Nati Acad Sci U.S.A. 2008, 105, 12113–12117. (21) Jiang, X.; Engelhard, M.; Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2010, 132, 10808–10815. (22) Fedurco, M. Coord. Chem. Rev. 2000, 209, 263–331. (23) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168–8173. (24) Chittur, K. K. Biomaterials 1998, 19, 357–369. (25) Wu, Y. Q.; Murayama, K.; Czarnik-Matusewicz, B.; Ozaki, Y. Appl. Spectrosc. 2002, 56, 1186–1193. (26) Kim, H. S.; Hartgerink, J. D.; Ghadiri, M. R. J. Am. Chem. Soc. 1998, 120, 4417–4424. (27) Rabe, M.; Verdes, D.; Seeger, S. Adv. Colloid Interface Sci. 2011, 162, 87–106. (28) Zhou, J.; Zheng, J.; Jiang, S. Y. J. Phys. Chem. B 2004, 108, 17418–17424. (29) Buijs, J.; Norde, W. Langmuir 1996, 12, 1605–1613. (30) Osawa, M. Bull. Chem. Soc. Jpn. 1997, 70, 2861–2820. (31) Ataka, K.; Heberle, J. J. Am. Chem. Soc. 2003, 125, 4986–4987.

’ CONCLUSIONS In this work, our electrochemical results indicated that the redox activity of adsorbed HHCC on the bare Au surface was suppressed. The reason for that was studied by SEIRA spectroscopy on the molecular level. Our results indicated that adsorption of protein on both surfaces showed well-defined amide I and II bands with identical maximal absorption and similar shapes. Similar components of the secondary structure have been obtained by Gaussian curve-fitting analysis of the amide I band, which suggested the preservation of the ordered structure of HHCC even adsorbed on the bare Au surface. Different amide I/II ratios of adsorbed HHCC on both surfaces indicated different absorption orientations. The adsorbed HHCC on the MUAmodified Au surface could alter its orientation according to the surface densities, while this was confined on the bare Au surface. Adsorption of HHCC on the bare Au surface was caused by the interactions of cysteine 14 and 17 with the gold surface, and HHCC was adsorbed on the MUA-modified Au surface via the electrostatic interactions between Lys 13, 72, and 86 and the carboxylic groups of the MUA.19 Such different interaction sites resulted in most of the α-helices being parallel to the bare Au surface and perpendicular to the MUA-modified surface with different heme orientations and adsorption rates. Our results indicated that the hindrance of electron transfer of adsorbed HHCC on the bare gold electrode was caused by the absorption orientation and suppressed rotation of the adsorbed protein. ’ AUTHOR INFORMATION Corresponding Author

*Phone: +86 431 85262426. Fax: +86 431 85685653. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Youth Foundation of Jilin Province (Grant 201101081), President Funds of the Chinese Academy of Sciences, Startup Foundation for Scientific Research, and Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. We especially appreciate the support of Professor Erkang Wang, Shaojun Dong. ’ REFERENCES (1) Shleev, S.; Tkac, J.; Christenson, A.; Ruzgas, T.; Yaropolov, A. I.; Whittaker, J. W.; Gorton, L. Biosens. Bioelectron. 2005, 20, 2517–2554. (2) Rusling, J. F.; Forster, R. J. J. Colloid Interface Sci. 2003, 262, 1–15. (3) Davis, J. J.; Morgan, D. A.; Wrathmell, C. L.; Axford, D. N.; Zhao, J.; Wang, N. J. Mater. Chem. 2005, 15, 2160–2174. (4) Wong, T. S.; Schwaneberg, U. Curr. Opin. Biotechnol. 2003, 14, 590–596. (5) N€oll, T.; N€oll, G. Chem. Soc. Rev. 2011, 40, 3564–3576. (6) Clark, R. A.; Bowden, E. F. Langmuir 1997, 13, 559–565. (7) Tarlov, M. J.; Bowden, E. F. J. Am. Chem. Soc. 1991, 113, 1847–1849. 642

dx.doi.org/10.1021/jp2063782 |J. Phys. Chem. C 2012, 116, 637–642