Influence of the Molecular Structure of Carboxyl-Terminated Self

Dec 23, 2007 - Multifunctional Cytochrome c: Learning New Tricks from an Old Dog. Damián Alvarez-Paggi , Luciana Hannibal , María A. Castro , Santia...
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J. Phys. Chem. C 2008, 112, 813-819

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Influence of the Molecular Structure of Carboxyl-Terminated Self-Assembled Monolayer on the Electron Transfer of Cytochrome c Adsorbed on an Au Electrode: In Situ Observation by Surface-Enhanced Infrared Absorption Spectroscopy Xiue Jiang, Kenichi Ataka,* and Joachim Heberle Bielefeld UniVersity, Department of Chemistry, Biophysical Chemistry (PC III), Bielefeld, Germany ReceiVed: August 14, 2007; In Final Form: October 15, 2007

Surface-enhanced infrared adsorption spectroscopy (SEIRAS) was employed for the in situ observation of structural changes that occur in a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) bound on a gold surface. The observed SEIRA spectra reveal a deprotonation of the carboxyl head group of the MUA-SAM layer after adsorption. An analysis of the vibrational spectra suggests that the deprotonation process occurs when the adsorbed MUA molecules reach a critical mutual distance. MUA-SAMs promote direct electron transfer between the metal electrode and cytochrome c, the electron mediator between the integral membrane protein complexes of the respiratory chain. The results show that the coverage of cytochrome c increases with the coverage of deprotonated MUA on the surface. On the other hand, the electron transfer of cytochrome c is optimized only when a moderate amount of the carboxyl head group is deprotonated. The electron transfer of cytochrome c is suppressed with a further increase of the deprotonated MUA. The relationship between the surface structure of the MUA layer and the electron transfer of cytochrome c is discussed on the basis of the spectroscopic data.

Introduction Electrochemically induced oxidation and reduction process of a monolayer of cytochrome c (cyt c), a protein that mediates single electron transfer between the integral membrane protein complexes of the respiratory chain, is considered to be a good model system for studying electron transfer within and between proteins.1 Electrons are directly injected to and/or retracted from cyt c after contact has been established with the electrode. Such contact can be made by functionalizing the metal electrode surface by chemical modification. A typical example of the chemical modification is self-assembled monolayer (SAM) approach. The SAM generally consists of a methylene chain with a thiol group on one end that quasi-covalently binds to the metal surface. On the other end, a functional group makes the contact to the protein. This method provides an attractive method to immobilize proteins into arrays, because of the possibility to form organized films with diverse chemical functionality on their surface. Carboxyl-terminated SAM is often used for the surface modifier to promote electron transfer between metal electrodes and cyt c.2-4 The negatively charged carboxyl head group of the SAM forms electrostatic interactions with the positively charged lysine rich domain around a heme-exposed cleft.4-6 This interaction sets up a proper orientation to facilitate electron transfer from heme iron to the metal electrode. Thus, the strength of the negative charge of the SAM surface plays an important role in manipulating electron transfer by controlling the nature of the immobilization. A molecular simulation study predicts that the electron transfer between cyt c and carboxyl-terminated SAM is influenced by dissociation of a proton from the carboxyl groups due to its electronegativity.7 A similar conclusion has * Author to whom correspondence should be addressed. E-mail: [email protected].

been drawn from the comparison among different SAM head groups with various extent of negative charges.8,9 The structure of the carboxyl-terminated SAM has been investigated by means of electrochemistry,10 scanning tunneling microscopy,11,12 and infrared spectroscopy.13-19 STM studies suggest that carboxylterminated SAM exist in two characteristic structures when adsorbed as a monolayer on Au(111) surface, which have been assigned to (3 × 3) and (p × x3, p ) 5, 6, 8, and 10) structures.12 The nature and the origin of these different structures are, however, not clear. Surface IR studies suggest that the carboxyl head group of the SAM layer forms a strong hydrogen-bonded network among the adjacent head groups.14 Despite these structural studies, the relation between surface structure of the carboxyl-terminated SAM and the reactivity of cyt c has not been studied in detail. The preparation procedure of the SAM layer for effective electron transfer to cyt c is discussed controversially. Several reports claim that the Au electrode has to be immersed overnight or longer in the solution containing the surface modifier although the adsorption process is finished within 1 h.5,9,18,20 Others emphasize the necessity of electrochemical or thermal pretreatment of the SAM-modified electrode prior to the adsorption of cyt c.21 An effect of the roughness or low-index face of the metal surface on electron-transfer reactivity has also been suggested.22 These empirical claims are based on the observation of the electron-transfer redox current, which is a consequence of the interfacial cyt c/SAM construction rather than based on the structural analysis of the SAM layer itself. As a result of this mixed information, the electron-transfer promotion effect of cyt c is often not reproducible. The problem is that the change of the structure and chemical nature of the SAM during preparation is not considered. On the other hand, the in-situ detection of subtle structural changes occurring on the monolayer is still technically challenging.

10.1021/jp076525w CCC: $40.75 © 2008 American Chemical Society Published on Web 12/23/2007

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Jiang et al.

We employed surface-enhanced infrared absorption spectroscopy (SEIRAS) for the structural analysis of the SAM and adsorbed cyt c layer. SEIRAS possesses sufficient sensitivity to detect weak signals on the level of monolayer and yet provides a wealth of molecular information.23-25 In this work, we investigated the formation process of the SAM of 11mercaptoundecanoic acid (MUA). The high sensitivity of SEIRAS allows the observation of changes in the chemical nature of the carboxyl head group of the MUA during formation of SAM layer. This characteristic of MUA SAM significantly affects the subsequent interfacial structure and electron-transfer properties of cyt c. The result suggests that the carboxyl head group of MUA varies its molecular structure depending on the incubation time, which significantly affects the adsorptivity and the electron-transfer properties of cyt c. Experimental Methods FT-IR Spectroscopy To Monitor the Assembly of 11Mercaptoundecanoic Acid on the Gold Surface. 11-Mercaptoundecanoic acid (MUA, Aldrich) and horse heart cytochrome c (Sigma) were used without further purification. The experimental setup and procedures for SEIRAS and potential-induced FT-IR difference spectroscopy have been described elsewhere.24,25 Briefly, a thin gold film with thickness of ca. 200 nm was formed on the flat surface of a half-cylindric silicon prism by chemical deposition. After electrochemical cleaning, the gold-coated prism was mounted in a glass cell. The IR beam from the interferometer of the FT-IR spectrometer (Bruker IFS 66V/S, Rheinstelten Germany) was coupled into the silicon prism at an incident angle of 60°, and the reflected beam was recorded with a liquid nitrogen-cooled MCT. In order to observe the self-assembly process, a reference spectrum was recorded with the bare Au surface immersed in 500 µL ethanol. Then, 0.1 mM MUA (in 500 µL ethanol) was added and IR spectra were recorded of the process. A series of 15 spectra with intervals of 10, 60, and 300 s, respectively, have been recorded. Adsorption and Electrochemical Properties of Cytochrome c on the MUA SAM. After the MUA SAM was formed on the gold surface, the sample prism was rinsed and covered by 10 mM phosphate buffer solution (pH ) 7.0). After a reference IR spectrum was recorded under these conditions, cyt c dissolved in the same buffer solution was added to achieve a final concentration of 2 µM. A series of IR spectra were recorded during the adsorption process of cyt c. The gold film was used as a working electrode, connected via a copper plate to the potentiostat (Autolab PGSTAT 12, Metrohm, Filderstadt, Germany). Ag/AgCl/3 M KCl and Pt mesh were used as a reference and counter electrodes, respectively. All potentials refer to the Ag/AgCl reference electrode. Cyclic voltammograms were recorded parallel to the IR spectra. A single-beam background IR spectrum was taken at -0.2 V, i.e., the potential where the adsorbed cyt c is fully reduced. Then, the potential was increased to 0.2 V, where the adsorbed cyt c is completely oxidized, and a sample spectrum was acquired. Typically, 1280 scans were averaged for each pair of background and sample spectra. The whole procedure was repeated six times, and the difference spectra were averaged to improve the signal-to-noise ratio. Results Formation of the MUA SAM on the Gold Electrode. Figure 1 shows the SEIRA spectra of MUA during its adsorption to the gold electrode. The abscissa is divided into two ranges at particular interest, namely the C-H stretching region at around 3000 cm-1 and the fingerprint region at 1800-1200

Figure 1. SEIRA spectra of MUA adsorbed on the Au surface from an ethanol solution at different times after addition of MUA.

cm-1. A strong positive band appears at 1720 cm-1 immediately after the addition of MUA (Figure 1, 0.5 min). The peak position of this band shifts down to 1708 cm-1 after an adsorption time of 4 h. The shape of the band at 1708-1720 cm-1 is apparently not symmetrical and contains a shoulder peak at around 1738 cm-1. Both bands at 1708-1720 cm-1 and the shoulder at 1738 cm-1 are assigned to the CdO stretching mode, ν(CdO), of the COOH carboxyl head group of MUA with two different laterally hydrogen-bonding order.16,19,26 Concomitant with the appearance of the ν(CdO) band, a positive band appears at 1410-1415 cm-1 (Figure 1, 17.5 min). This band is assigned to the C-O stretching mode of the carboxyl head group which is coupled to the O-H bending mode.13,19,26 In the later stage of adsorption (after ca. 40 min), a band at 1420-1425 cm-1 appears, which overlaps with the band at 1410-1415 cm-1, together with a shoulder shifting from 1465 to 1454 cm-1. These bands are both assigned to symmetric stretching modes of the COO- carboxylate groups, νs(COO-). Distinction between the 1410-1415 cm-1 and 1420-1425 cm-1 bands is based on the different kinetics (Vide infra). As the νs(COO-) emerges, two additional bands appear at 1550-1555 and 1590-1595 cm-1 that result from asymmetric stretching mode of the carboxylate group, νas(COO-). The appearance of these bands indicates that the carboxylic acid groups of MUA dissociate to form mercaptoundecanate (MU). The two different peak positions for the νs(COO-) as well as for the νas(COO-) suggest the existence of, at least, two different structures of the adsorbed MUs. The left panel in Figure 1 shows the bands at 2920 and 2850 cm-1, which are attributed to the asymmetric νas(CH2) and symmetric νs(CH2) stretching vibrations of the CH2 groups of alkyl chain, respectively.26 The negative peak at 2973 cm-1 is assigned to the CH3 antisymmetric stretching mode of ethanol, which is removed from the Au surface by the adsorption of MUA. Figure 2 shows the peak heights of the aforementioned bands plotted versus the adsorption time. The intensity of ν(CdO) at 1708-1720 cm-1 (open circle) shows three stages in which it (i) increases monotonically in the first 10 min, (ii) remains constant between 10 min to 40 min, and then (iii) gradually decreases after 40 min. The band at 2850 cm-1 (νs(CH2)) and the 2920 cm-1 (νas(CH2)) (filled triangle and circle, respectively) concomitantly increase in intensity along with the appearance

Electron Transfer of Cytochrome c on an Au Electrode

Figure 2. Plot of the intensities of the MUA bands vs adsorption time: (O) ν(CdO) at 1706-1720 cm-1, (left triangle) νs(COO-) at 1415-1425 cm-1, (]) νs(COO-) at 1454-1465 cm-1, (4) νas(COO-) at 1550-1555 cm-1, (3) νas(COO-) at 1590-1595 cm-1, (2) νs(CH2) at 2850 cm-1, (b) νas(CH2) at 2920 cm-1, (+) ν(CdO) at 1712 cm-1 from MUA/HT mixed monolayer, respectively.

of the ν(CdO) in the first 10 min. After ca. 10 min, the intensities remain unchanged until 200 min followed by a second intensity increase. Intensity ratio of νs(CH2) and νas(CH2) remains constant after 10 min, which suggests that orientation of the alkyl chain is not altered (see Supporting Information).17 Longer adsorption kinetics could not be monitored because of the inevitable drift of the spectrometer at times by 4 h. The band at 1410-1415 cm-1 appears concomitantly with the appearance of the ν(CdO) and the ν(CH2) bands, which increases up to 10 min. Its intensity stays nearly unchanged until ca. 40 min, and then it starts to increase again. This reincrease is due to an appearance of a new band at 1420 cm-1 assigned to the νs(COO-) overlapped with the existing band at 1410 cm-1. The appearance of νs(COO-) at 1420 cm-1 corresponds to the decrease of the CdO band at 1708-1720 cm-1. The time in which the CdO band decreases and the νs(COO-) band reincreases also corresponds to the appearance of an other νs(COO-) component at 1465 cm-1 and νas(COO-) bands at 1550-1555 and 1590-1595 cm-1. For the analysis of the adsorption kinetics of MUA, ν(CdO) or ν(COO-) bands are not appropriate makers for the determination of a simple adsorption since the COOH/COO- head group shows a change in their chemical nature during the adsorption process. On the other hand, the intensities of ν(CH2) bands reflect the total amount of the adsorbed MUA/MU. A constant intensity of the ν(CH2) band suggests that the adsorption of MUA is saturated after 10 min, and the coverage remains constant up to 200 min. This result implies that the observed deprotonation of the COOH head group after 40 min mostly starts among the adsorbed species at times after MUA adsorption reaches saturation coverage. Effect of the Structure of the MUA Layer on the Adsorption and Electrochemical Properties of cyt c. The electrochemical behavior of cyt c when adsorbed to MUA is compared at different preparation times in order to gauge the effect of the structural change of the carboxyl head group on the electron transfer from the modified Au electrode to cyt c. Three typical MUA SAM preparation times are chosen: (a) at 17 min after adsorption where the surface is fully covered by MUA but the majority of the adsorbate exists in its protonated form (dissociation degree, R ) 0), denoted as Cc17-MUA, (b) at 90 min after the adsorption where 32% of the adsorbate are deprotonated (R ) 0.32), denoted as Cc90-MUA, (c) 4 h after the adsorption where 73% of the adsorbate are deprotonated (R

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Figure 3. Cyclic voltammograms of cytochrome c adsorbed on the MUA/Au surface after 17 min (broken curve), 90 min (solid curve), and 4 h (dotted-broken curve) of preparation of the MUA layer. The scan rate is 50 mV/s. The electrolyte solution contains 2 mM cyt c in 10 mM phosphate buffer solution (pH 6.8).

) 0.73), denoted as Cc4h-MUA. The surface ratio of MU in the SAM layer is calculated from the peak area of ν(CdO) and νas(COO-) bands by using an extinction coefficient of ν(Cd O)/νas(COO-) ) 280/38027 (based on the hypothesis that the peak area of the respective bands is a mixture of several different orientations, which may compensate for the contribution of the surface selection rules). Figure 3 shows the cyclic voltammograms of cyt c adsorbed on the MUA SAM formed at various adsorption times. It is evident that both Cc90-MUA (solid curve in Figure 3) and Cc17MUA/Au (broken curve in Figure 3) electrodes lead to a pair of redox peaks with the formal potential (E0 ) (Ep,a + Ep,c)/2) at 0.01 V, with a peak-to-peak separation of 0.023 V. Integration of the peak currents gave a surface coverage of electroactive cyt c of 7.7 pmol cm-2 (C90-MUA) and 4.0 pmol cm-2 (Cc17MUA), respectively, calculated with a geometric surface area of 1.77 cm2 with a roughness factor of 2.5. Although the determined coverage is low as compared to the fully packed cyt c layer (15 pmol cm-2), the observed formal potentials correspond to those reported in the literature.2 On the other hand, the cyclic voltammogram of the Cc4h-MUA/Au (dotted-broken curve in Figure 3) shows no obvious redox peaks. Very small redox peaks are observed on Cc4h-MUA/Au when differential pulse voltammetry is applied (data not shown). This suggests that either a very low amount of cyt c is adsorbed or it adsorbed in an electrochemically inactive form, e.g., denatured or in wrong orientation relative to the MUA/Au surface. This issue has been checked by SEIRAS which selectively monitors the adsorption process of cyt c on MUA/Au surfaces. Figure 4 shows the SEIRA spectra of cyt c adsorbed on the MUA SAM at different preparation times. As cyt c was added to the solution, an immediate increase of two bands at 1659 and 1551 cm-1 is observed. These bands are assigned to the amide I and II modes of cyt c.28 Representative spectra were chosen from the one after the band intensities of adsorbed cyt c reaches maximum, i.e., coverage of cyt c is saturated on the respective surfaces. This result suggests that cyt c can adsorb on all different types of MUA layers, although the intensities depend on the preparation time of MUA. Peak positions of the amide bands are also identical for all MUA layers and corresponds well to the spectrum of cyt c in aqueous solution,29 which suggests that significant structural changes do not take

816 J. Phys. Chem. C, Vol. 112, No. 3, 2008

Figure 4. SEIRA spectra of cyt c adsorbed on MUA/Au surface at preparation times of (a) 4 h, (b) 90 min, and (c) 17 min. All spectra are taken at 37 min after the addition of cyt c to the solution at each surface. Spectra are normalized to the intensity of the ν(CdO) band of MUA (see Supporting Information for details).

place during the adsorption. The coverage of adsorbed cyt c on different MUA/Au surfaces can be calculated according to the ratio of the magnitude of amide I combination with the electrochemical data (Vide infra). Differences in the enhancement factor that arise from the morphology of the Au surface are normalized by the intensity of the ν(CdO) band at the adsorption time of 17 min for each spectrum (see Supporting Information for details). The relative intensity ratio of the amide I bands of Cc4h-MUA:Cc90-MUA:Cc17-MUA is 4.8:2:1. The amide I intensity ratio of 2 between Cc90-MUA and Cc17MUA is consistent with the ratio of the surface coverage determined from cyclic voltammetry (7.7 pmol cm-2/4.0 pmol cm-2 ) 1.93). Considering the amide I intensity ratio between Cc4h-MUA and Cc90-MUA of 4.8:2, the surface coverage of Cc4h-MUA is determined to be ca. 18 pmol cm-2. This nearly corresponds to a value of a densely packed monolayer of cyt c (15 pmol cm-2), which suggests that the Cc4h-MUA surface is covered by a fully packed monolayer of cyt c that, however, is not electrochemically active. Figure 5 shows the potential-induced redox difference spectra of adsorbed cyt c on the Cc4h-MUA (a), the Cc90-MUA/Au (b), and the Cc17-MUA/Au (c) surfaces, respectively. Although the spectra seem to be different, the difference spectra of Cc4hMUA and Cc90-MUA exhibit small bands which are characteristic to the redox difference spectrum of cyt c. These are negative peaks at 1693, 1663, 1642, 1627 cm-1, and positive peaks at 1673, 1660, cm-1. The negative and positive signs correspond to the reduced and oxidized state of cyt c, respectively. Details of these band assignments have been described elsewhere.24 On the other hand, all spectra show additional positive peaks at 1737, 1557, and 1410 cm-1 and a negative broad peak overlapping with the cyt c band at 1690 cm-1. These bands are assigned to vibrational modes of the MUA layer. This can be verified by the fact that those bands appear in the potential difference spectrum of the MUA layer without cyt c (Figure 5d). The bands at 1737/1690 cm-1 are assigned to the CdO stretching mode of the carboxyl head group of MUA, whereas the bands at 1554 and 1410 cm-1 are assigned to symmetric

Jiang et al.

Figure 5. Potential-induced SEIDA spectra of cyt c adsorbed on MUA SAM at preparation times of (a) 4 h, (b) 90 min (c) 17 min. (d) SEIDA spectrum of the pure MUA SAM prepared at 90 min of adsorption time. Reference and sample spectra are recorded at -0.2 and +0.2 V, respectively.

and asymmetric stretching modes of the COO- group of the MU. These bands are slightly shifted to higher wavenumber in Cc4h-MUA at 1578 and 1416 cm-1, respectively. This result suggests that the adsorbed MUA layer exhibits a reversible protonation/deprotonation of the carboxyl head group induced by the potential excursion. The spectrum of Cc17-MUA exhibits bands other than the reduced/oxidized cyt c and the MUA underlayer. These bands appear as broad positive peaks at 1658 and 1557 cm-1. Although the latter band overlapped with the MUA band, subtraction of the MUA spectra (Figure 5d) reveals these two characteristic peaks (data not shown). They are attributed to the amide I and II bands, respectively, of cyt c which further adsorbed on the MUA/Au surface during potential excursion. Discussion Time-Dependent Structural Changes of MUA Adsorbed on the Gold Electrode. Our results demonstrate that the deprotonation process takes place from a saturated MUA layer. Similar deprotonation phenomena have been observed when the dried MUA SAM film is exposed to higher pH solution (pH > ∼8),30 ethanol,16 or vapor-phase amine-functionalized molecules.18,19 The deprotonation may rise from a change in the dielectric environment surrounding the carboxyl head group, which leads to a shift of pKa for surface MUA.31 Continual change of the dielectric environment upon the adsorption reflects gradual shift of ν(CdO) band from 1724 to 1712 cm-1, which indicates strengthening of a hydrogen bond between adjacent carboxyl head groups.32 The deprotonation takes place after the fully packed monolayer is formed, where a mutual distance is close enough to eliminate excess protons that could not fit into the hydrogen-bonding network of the carboxyl head groups. A control experiment was handled with a mixed SAM layer of MUA and hexanethiol (HT). The intensities of the ν(CdO) band from the mixed layer is plotted in Figure 2 (+). The intensity as well as the peak position (+ in Figure 6) remains unchanged after MUA reaches saturation coverage. Moreover, there is no

Electron Transfer of Cytochrome c on an Au Electrode

Figure 6. Plot of the peak position of selected MUA bands vs adsorption time: (O) ν(CdO) at 1706-1720, cm-1, (+) ν(CdO) at 1712 cm-1 from MUA/HT mixed monolayer, (3) νas(COO-) at 15901597 cm-1, (4) νas(COO-) at 1545-1557 cm-1, (left triangle) νs(COO-) at 1456-62 cm-1, (]) νs(COO-) at 1410-1425 cm-1, respectively.

trace for the appearance of νas(COO-) bands in the spectra of the mixed layer (data not shown). These results clearly show that the deprotonation does not take place from the MUA molecules in the MUA/HT mixed SAM layer. Existence of the HT layer prevents the approach of MUA molecules to a critical mutual distance to cause the dielectric change for a sufficient pKa shift.31 As a consequence, the deprotonation is suppressed on the mixed monolayer. The deprotonated, thus negatively charged MU group strongly attracts hydrogen atoms from the residual MUA, which consequently induces a further downshift of the ν(CdO) band. An interaction between MUA and MU is suggested from the fact that the peak positions for the ν(COO-) bands shift concomitantly with the downshift of ν(CdO). Figure 6 clearly shows a downshift of νas(COO-) and νs(COO-) to 1590 and 1456 cm-1, respectively, and an upshift of νas(COO-) and νs(COO-) to ca. 1554 and 1425 cm-1, respectively, with the increase of the MU concentration on the surface until ca. 100 min. Synchronized shifts for two different νas(COO-) and νs(COO-) couples suggest the existence of, at least, two different structures within the MU layer. The shifts of MU bands indicate that lateral interactions between MUA and MU head groups, or among MUs, are constantly changing during the deprotonation process, which implies that the MUA/MU layer undergoes a reconstruction of the two-dimensional surface structure. It is interesting to note that the total surface coverage of the MUA/MU layer is slightly increased after the deprotonation process occurred (ca. 4 h.). This can be explained by the differences in surface packing densities on the MUA and the MU layer. It has been reported that a carboxyl-terminated thiol adsorbed on the surface of Au(111) provides the two structures with different local coverage. Sawaguchi et al.12 investigated two-dimensional structure of MUA with in situ STM and obtained hexagonally arranged (3 × 3) domains together with a stripe structure. Similar structures have also been observed for mercaptopropionic acid (MPA, HS(CH2)2COOH) adsorbed on Au(111), where the latter structure has been assigned to (p × x3, p ) 5, 6, 8, and 10).11 The idea of a coexistence of

J. Phys. Chem. C, Vol. 112, No. 3, 2008 817 MUA and MU was not mentioned in the 2D structural assignment since STM cannot distinguish between the two molecular structures. However, if we speculate that the incommensurate hexagonal and the commensurate stripe structures arise from MUA and MU, respectively, a change of the chemical nature from MUA to MU at the surface must be accompanied with a change in the 2D structure. This incommensurate-tocommensurate phase change creates an additional access surface for the adsorption of MUA from the solution. This leads to the slight increase of the total coverage of MUA after MUA/MU deprotonation. Interestingly, the peak position of the ν(COO-) bands becomes constant when the reincrease of the surface coverage starts (>100 min). A constant peak position for the ν(COO-) bands suggests formation of a stable hydrogenbonding network for the tightly packed structure of the MU layer. This observation may also fit to the model of a incommensurate/commensurate 2D phase change of MUA/MU layers. Effect of the Chemical Structure of the MUA SAM Layer on the Redox Properties of cyt c. SEIRA spectroscopy demonstrated that the longer the incubation of MUA on the Au electrode, the more dominant of MU, thus the more negatively charged the surface is. The order of the negativity of the surface is Cc4h-MUA > Cc90-MUA > Cc17-MUA. This order tallies with the number of cyt c adsorbed on the MUA/Au surface. This suggests that the negativity of the MUA/Au surface relates to the adsorptivity of cyt c. It has been suggested that an electrostatic interaction between the positively charged lysine domain surrounding the heme cleft and the negatively charged surface of the binding partner is the major driving force for the binding of the protein.4,6 Since cyt c has excess positive surface charge (a net charge of +6),1 the increase of negative charge at the MU-covered surface enhances the attraction of cyt c. The observed redox activity corresponds neither to the coverage of cyt c nor to the surface ratio of MUA/MU. The electrochemical activity is rather enhanced when the surface comprises a moderate mixture of MUA and MU. Thus, the order in redox activity of cyt c is Cc90-MUA>Cc17-MUA>Cc4hMUA. It is clear that the low redox activity on Cc17-MUA is due to the low coverage of cyt c. The low intensities of amide bands from the spectrum of Cc17-MUA (Figure 4) imply that cyt c does not preferentially adsorb on the MUA-dominated surface because of a lack of sufficient negative charge. Cc4hMUA does not show significant electrochemical activity, although the electrode is fully covered by cyt c. Since Cc4hMUA is predominantly protonated, the adsorption of cyt c on the MU surface seems to stall the redox activities of the latter. Zhou et al.7 simulated the orientation of the heme plane and distribution at different degrees of deprotonation of the carboxyl head group. They suggested that the orientation angle comes closer to 90° with a narrower orientational distribution as the proton dissociation increases. In our sample, the surface ratio of MU in the SAM layer is 32% and 73% for Cc90-MUA and Cc4h-MUA, respectively. By comparison with the simulation, we presume that cyt c is tightly bound to the MU/MUA surface with a monodispersed orientational distribution at the Cc4hMUA. Such tight binding of cyt c is unfavorable for electron transfer to the electrode surface. A similar example is reported in the case of cyt c adsorbed on sulfonate-terminated SAMs which provides higher negative charge than carboxyl-terminated SAMs. Chen et al. found that cyt c shows no redox reaction on SO3--terminated SAMs although surface plasmon resonance experiments indicate full surface coverage by cyt c.9 Du et al.33,34 obtained a significantly narrower orientation distribution of cyt

818 J. Phys. Chem. C, Vol. 112, No. 3, 2008 c on sulfonate-terminated SAMs than the distributions measured previously on other types of SAMs. These results are explained by a confinement of cyt c to a “preferred” orientation that places the heme group deviated from its position to engage in the electron transfer. In order to make electron transfer, cyt c undergoes a repositioning of the heme relative to the electrode surface by changing its orientation or conformation.4,35-38 However, the highly negatively charged surface restrains such repositioning because of strong electrostatic interaction. Cc90-MUA provides an intermediate negativity between Cc17-MUA and Cc4h-MUA. The results of the molecular dynamics simulation by Zhou et al.7 show a slightly broader orientational distribution of cyt c for the Cc90-MUA surface than for the Cc4h-MUA surface. This suggests that cyt c adsorbed on Cc90-MUA provides more freedom for a repositioning movement of the heme than the highly negatively charged surface. Yet, Cc90-MUA provides sufficient negativity to attract cyt c to the electrode surface. In summary, the electrode has to comprise sufficient negative charge to attract cyt c to the vicinity of the surface. At the same time, the negative charge must not be excessive to hamper the repositioning movement of heme in order to engage in electron transfer. Zhou et al.7 also suggested that strong electrostatic interaction induced on highly proton-dissociated surfaces may cause a distortion of the structure of cyt c based on their calculation of the root-mean-square deviation. They calculated that a large conformational change of 65% enlargement occurs when cyt c is adsorbed on the surface where 50% of the protons are dissociated from the carboxyl head group. They attributed this structural distortion as the cause of the denaturation process that prevents the redox reaction of cyt c. However, according to our secondary structural analysis of the IR spectra of the adsorbed cyt c, we do not find significant structural changes of cyt c when adsorbed to MUA with various degrees of dissociation of the carboxyl head group. Therefore, we conclude that the deactivation of redox properties of cyt c on a highly charged surface is due to the hindrance of the repositioning movement of the heme. Conclusions In this work, the structural changes during the MUA SAM formation on a gold substrate were investigated by means of SEIRAS. Vibrational spectra reveal that the adsorbed MUA SAM exhibits chemical structures that vary in the relative content of protonated/deprotonated carboxyl head groups. From the low frequency of the ν(CdO) vibration, it is suggested that the carboxyl head group forms a strong hydrogen-bonding network with the adjacent MUAs. Dissociation of the proton from the carboxyl head group occurs after the gold substrate is fully covered by MUA when the distance between adjacent MUAs are sufficiently close. The deprotonation process does not take place when the MUA SAM is diluted by the addition of HT, which prevents the approach of adjacent MUAs to a critically close distance. The effect of the chemical nature of the carboxyl head group on the electrochemical activity of cyt c adsorbed on the MUA SAM layer has also been detected. Cyt c becomes most redox active when moderate dissociation of the carboxyl head group is achieved (R ) 0.3). When the ratio of deprotonated carboxyl head group is too low or too high, the electrochemical redox activity of adsorbed cyt c is suppressed. The former condition is understood by the lack of sufficient negative surface charge from the MUA group to attract the positively charged cyt c to

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