Electron Transfer Kinetics of Cytochrome C in the Submillisecond

Jan 15, 2009 - monolayer of 2-mercaptoethanol is measured using SEIRAS in the step-scan mode in an ATR configuration. Electron transfer is triggered b...
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J. Phys. Chem. C 2009, 113, 2256–2262

Electron Transfer Kinetics of Cytochrome C in the Submillisecond Time Regime Using Time-Resolved Surface-Enhanced Infrared Absorption Spectroscopy Ch. Nowak,† Ch. Luening,† D. Schach,† D. Baurecht,‡ W. Knoll,† and R. L. C. Naumann*,† Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and UniVersity of Vienna, Institute of Biophysical Chemistry, Althanstrasse 14, A-1090 Vienna, Austria ReceiVed: August 8, 2008; ReVised Manuscript ReceiVed: October 10, 2008

The two-layer gold surface developed before is used for time-resolved surface-enhanced infrared absorption spectroscopy (tr-SEIRAS). The electron transfer (eT) rate to cytochrome c adsorbed to a self-assembled monolayer of 2-mercaptoethanol is measured using SEIRAS in the step-scan mode in an ATR configuration. Electron transfer is triggered by periodic potential pulses applied to the gold surface which is used both as an electrode and a surface enhancing layer. Phase sensitive detection is used to separate and determine band parameters of the strongly overlapping absorption bands of the amide region. The surface enhancement effect of the two-layer gold surface was determined to be 128. Rate constants for eT under conditions of the experiment were determined by tr-SEIRAS to be kox ) 1810 ( 239 s-1 and kred ) 1880 ( 232 s-1 for oxidation and reduction, respectively. This was deduced from the time-dependent change of one particular amino acid (His18) directly ligated to the porphyrine ring of the heme center. Different groups of the peptide backbone were found to be excited at different excitation frequencies. Introduction Electron transfer (eT) kinetics at metal-solution interfaces can be investigated by electrochemical methods with time resolutions down to nanoseconds depending on the size of the electrodes.1 However, data thus obtained do not provide information on the molecular species involved in the eT. Spectro-electrochemistry is a way out of this dilemma, although there are only a few spectroscopic techniques available to meet the requirements regarding time resolution and sensitivity. Surface-enhanced infrared absorption spectroscopy, SEIRAS,2-5 has the potential to meet these requirements, particularly as it is applied in the ATR-FTIR mode where the bulk of the electrolyte adjacent to the metal surface is not a problem and, moreover, data acquisition is facilitated by Fourier transform infrared (FTIR) interferometry. With the help of the step-scan method, time resolutions down to the nanosecond time scale can be obtained.6,7 In situ monitoring of electrochemical kinetics down to the submillisecond scale was achieved as early as 1994 using time-resolved (tr)-SEIRAS.8 Interferograms were taken as a function of time following a series of potential pulses. This method was later refined by introducing the lock-in amplifier technique in conjunction with small amplitude excitation to overcome the limitation due to charging currents.9 Nevertheless, only a few applications of this technique can be found in the literature.8-11 Possible reasons are sensitivity limitations or insufficient stability of the electroactive species under investigation. The systems under study have to endure a large number of excitation pulses to produce a sufficiently large quantity of data. This makes high demands on the fabrication of the metal film which serves both as the surface-enhancement layer and the electrode. In our attempts to investigate the redox kinetics of heme proteins, we developed a two-layer gold film with an * Corresponding author. Tel.: +49 6131 379 157. Fax +49 6131 379 100. E-mail: [email protected]. † Max Planck Institute for Polymer Research. ‡ University of Vienna.

improved enhancement factor and stability for use with SEIRAS.12 In the present investigation, this system was applied for time-resolved studies of cytochrome c (cc) as an otherwise electrochemically and spectroscopically well-characterized system. Cc in direct contact with an electrode was found to rapidly denature, making the adsorption to self-assembled monolayers (SAMs) a necessary prerequisite for electron transfer (eT). Electrochemical studies were thus performed on cc adsorbed on SAMs terminated by pyridine groups,13 hydroxyl14,15 groups, and carboxyl groups.16,17 Cc is adsorbed by these SAMs in different orientations depending on the driving forces of absorption. Carboxyl groups are considered to drive electrostatic attraction of the positively charged binding domain of cc, which leaves the heme cleft directed toward the electrode. Pyridine moieties promote coordinative binding with the porphyrine ring of the heme cofactor positioning the heme cleft at an angle of 90° with respect to the surface. The inverted orientation of cc with the heme cleft pointing away from the electrode is provided by hydroxyl group terminated SAMs onto which cc binds by hydrogen bonding. Redox transitions of cc on all of these modified surfaces was investigated by SEIRAS thus providing spectra of final and intermediate states of reduction and oxidation.18-20 Time-resolved studies based on spectro-electrochemistry of cc were focused mainly on surface-enhanced Raman spectroscopy (SERRS). For example, cc on carboxyl terminated and pyridine group terminated SAMs was investigated by tr-SERRS.21,22 Tr-IR spectroscopy was applied to temperature-jump studies of cc in solution.23 In the present investigation we were particularly interested in tr-SEIRAS on the mercaptoethanol-modified surface to which cc is adsorbed in the reverse orientation with the heme cleft pointing to the outside of the monolayer. Such monolayers of cc were shown previously to be able to bind cytochrome c oxidase in a functionally active form.14 In order to perform tr-SEIRAS on such monolayers, however, a good S/N ratio is a necessary requirement. One way to achieve this goal is the application of modulated excitation and phase-sensitive detection using a lock-

10.1021/jp8071052 CCC: $40.75  2009 American Chemical Society Published on Web 01/15/2009

Electron Transfer Kinetics of Cytochrome C

Figure 1. Electrochemistry-controlled ATR-FTIR setup. Potentials are applied in a square wave function to the immobilized cc by the potentiostat controlled by the function generator, which also triggers the spectrometer. The phase-sensitive detection (PSD) of the timeresolved spectra is performed by software after the measurement.

in amplifier.9 As an equivalent to this method, the mathematical method of phase-sensitive detection was applied to analyze the time-resolved spectra.24

J. Phys. Chem. C, Vol. 113, No. 6, 2009 2257

Figure 2. Principle of tr-ATR-FTIR spectroscopy triggered by square wave periodic excitation of reduction and oxidation of the protein. (a) Potentials applied to the cc on the gold film. (b) IR measurement, triggered by the positive edge of the exciting potential. (c) Corresponding state of the protein during the excitation cycle.

Experimental Methods Sodium dithionite was purchased from Fluka Biochemika, Buchs, Switzerland. For all other chemicals: see ref 12. The sample preparation was done according to the optimum conditions obtained in ref 9: briefly, a 25 nm gold film was deposited by electrothermal evaporation onto the flat surface of the ATR-crystal which was then immersed into 50 mL of an aqueous solution of hydroxylamine hydrochloride (NH2OH · HCl) (0.4 mM) to which 500 µL of an aqueous solution of gold(III) chloride hydrate (AuCl3) (0.3 mM) was added. After 2 min, the same amount of AuCl3 solution was added. This process was repeated 5 times so that the sample was kept in the growth solution for a total of 10 min. The samples were removed from the growth solution, rinsed with water, and dried in a stream of nitrogen. Thereafter, the samples were immersed for 15 min into an aqueous solution of 2-mercaptoethanol (ME) (1 mM), rinsed with water, and subsequently cyt c (cc) was adsorbed for 60 min from a solution of cc (0.35 mM) in the PBS buffer (20 mM K2HPO4/10 mM NaClO4/pH ) 7). Time-Resolved (tr) Surface-Enhanced IR Absorption (SEIRA) Spectroscopy. The spectro-electrochemical cell and the FTIR spectrometer were the same as before.12 For tr-SEIRA measurements, the ATR-FTIR setup was equipped with a function generator which triggered the potentiostat of the Autolab as well as the spectrometer (Figure 1). The spectrometer was operated in the step-scan mode controlled by electrochemical excitation. Potentials were periodically applied to the gold film in a square wave function. The time-resolved FTIR measurement was triggered by the sudden potential change at the start of each period to record a succession of spectra that indicate the kinetics of the redox transition as a function of time (Figures 2). Spectra were analyzed by the software package OPUS 6 (Bruker, Karlsruhe). For static measurements, the mirror velocity was 120 kHz; the phase resolution was 128 cm-1; and 5555 scans were taken for one spectrum during a measurement time of 10 min. In the step-scan (SS) mode, an excitation frequency of 500 Hz was applied, using 40 time slides at a time resolution of 50 µs. All spectra were measured with parallel polarized light.

Figure 3. (a) Potentiostatic titration of cc by ATR-IR-spectroscopy. Potential differences applied across the immobilized protein were varied stepwise from the fully reduced to the fully oxidized state. Difference spectra are calculated vs the reduced state (spectrum at -100 mV). black s, 0 mV; red s, +50 mV; blue s, +100 mV; teal s, +150 mV; pink s, +200 mV; light green s, +250 mV; navy s, +300 mV; dark red s, +350 mV; dark pink, s +400 mV; green s, +450 mV. Assignment: amide I β-turn type III (reduced state): 1692 cm-1; amide I β-turn type III (oxidized state): 1672 cm-1; amide I β-turn type II (reduced state): 1662 cm-1; amide I β-turn type II (oxidized state): 1658 cm-1; amide I extended β-strand (reduced state): 1624 cm-1; amide II β-turn type III (oxidized state): 1552 cm-1.

Electrochemical Measurements. Electrochemical measurements were performed as described in ref 12 using an Autolab instrument (PGSTAT302). Principle of tr-SEIRAS Triggered by Electrochemical Potentials. Modulated electrochemical excitation was applied to the monolayer of cc on the gold film in the form of a square wave function varying the potential between + 500 and -100 mV, thereby changing the state of cc from the fully oxidized to the fully reduced form (Figure 2). The time-resolved FTIR measurement was triggered simultaneously with the fast potential change at the start of each period to record a succession of spectra that indicate the kinetics of the redox transition as a function of time. These functions were analyzed in terms of single exponentials considering a monomolecular reaction. The frequency of the electrochemical excitation depends on the kinetic parameters of the redox transition to be observed. Phase-Sensitive Detection. The analytical procedure of phase-sensitive detection (PSD)24 was applied to the time-

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Nowak et al. ◦

A90 ˜ ) ) Ak(ν˜ ) sin[φk(ν˜ )] ) k (ν

2 T

∫0T A(ν˜ , t) cos(kωt)dt

(k ) 1, 2, ...) ◦ A0k (ν˜ ) ) Ak(ν˜ )

cos[φk(ν˜ )] )

2 T

∫0T A(ν˜ , t) sin(kωt)dt

(k ) 1, 2, ...)

(2)

In eq 2, Ak(ν˜ ) is the modulation amplitude of the absorbance and φk(ν˜ )is the corresponding phase lag. These are the main parameters important for the interpretation of the modulation experiment and can be determined from the measured phase˜ ) and A0° ˜ )Ak(ν˜ ) using eq 3 resolved spectra A90° k (ν k (ν

√

◦

◦

Ak(ν˜ ) ) A0k (ν˜ )2 + A90 ˜ )2 k (ν ◦

A90 k sin φk ) , Ak

◦

A0k cos φk ) (3) Ak

PSD

Modulation spectra Aφk k (ν˜ ) can be calculated for any phase angle φPSD by using eq 4 k n

PSD

Figure 4. Heme structure of the cc molecule demonstrating the significance of axial ligand His-18. The prominent band at 1692 cm-1 is assigned to amide I β-turn type III vibration of this amino acid.

resolved spectra coadded during the excitation cycles, in order to eliminate all absorbance changes that do not periodically change with the excitation frequency. Moreover, PSD was used to separate the absorbance changes that are in phase with the exciting frequency from those that are out of phase. The result was a discrimination of all background signals which do not change with the excitation frequency, i.e., peaks that do not depend on the potential modulation, noise of all other frequencies, etc. This effectively helped to improve the S/N ratio of the measured spectra in such a way that a weak system response can thus be isolated from a large background absorption. In our case, PSD was used to identify and separate peak parameters of absorption bands that are relevant for the process under investigation. These parameters were subsequently used to analyze the actual time-resolved spectra. Principle of PSD.24 In reversible reactions, a periodic excitation leads to a periodic response of the time-dependent absorbance A(ν˜ ,t). To extract the periodic response at a given wavenumber, this time-dependent absorbance is multiplied by a sinusoidal function sin(kωt + φPSD k ) followed by a normalized integration over the modulation period PSD

Aφk k (ν˜ ) )

2 T

∫0T A(ν˜ , t) sin(kωt + φPSD k )dt

(1)

φPSD is the operator-controlled phase angle. Applying eq 1 to k all wavenumbers ν˜ of the spectrum leads to a vector PSD where PSD the absorbance spectra A(ν˜ ,t) and Aφk k (ν˜ ) are treated like vectors (vector PSD). A(ν˜ ,t) are the time-resolved absorbance spectra PSD measured during an excitation period, and Aφk k (ν˜ ) is referred to as a phase-resolved modulation spectrum or a phase-resolved absorbance spectrum associated with the frequency kω (k ) 1 corresponds to the fundamental, i.e., to the frequency of PSD ) 0° and excitation ω) and PSD phase setting φPSD k . For φk PSD φkPSD φk ) 90°, Ak (ν˜ ) is equivalent to the Fourier component of the cosine and sine function, respectively (eq 2).

Aφk k (ν˜ ) )

∑

2 ki s A(ν˜ , ti) sin 2π + φPSD k 3N i)0 i n

(

)

(4)

The mathematical procedure of the PSD was implemented into the OPUS software by macros using the algorithm described in ref 24. Results and Discussion The absorption of cc to the mercaptoethanol-modified twolayer gold surface was shown before to take 60 min as indicated by SEIRAS.12 This indicates a slow adsorption process in accordance with ref 18. Saturation was measured by the amplitude of the most prominent bands at 1661 and 1551 cm-1, which represent the amide I and II vibrations of the protein (Figure 5 of ref 12). Spectral changes were then recorded as a function of applied potential. To this end, difference spectra were taken at different potentials with the reduced state (-103 mV vs SHE) taken as the reference (Figure 3). The potential was varied in steps of 50 mV between +497 and -103 mV vs SHE. Changes in the spectra were observed at wave numbers in the range of 1500-1800 cm-1. The decrease of the strong band at 1692 cm-1 had been assigned to the amide I band of the β-turn type III of amino acids 14-19 (including His-18) and 67-70 of reduced cc.18 His-18 is the axial ligand of the porphyrine ring of the heme structure inside the cc (Figure 4). Hence the decrease of this band was considered to directly reflect the transition of cc into the reduced state. It was correlated with an increase of the band at 1672 cm-1 which had been attributed to the same type of amide I band of oxidized cc (for the band assignments of cyt c see ref 18).1 The strong positive band at 1658 cm-1 and the negative band at 1667 cm-1 were assigned to an amide I band of the β-turn type II of amino acids 32-35 and 35-38 of oxidized and reduced cc, respectively (with slight shifts of the frequencies vs ref 18). The positive band at 1552 cm-1 indicated an amide II band of the β-turn type III type of oxidized cc, which is more remote from the heme structure. The small negative band at 1624 cm-1 indicated the extended β-strand of cc in the reduced form. These changes were very similar to the spectra observed on the classical SEIRA surface, at least as far as the band positions are concerned. The relative intensities of the bands measured on the two-layer gold surface, however, are more similar to the ones on mercaptopropionic acid rather

Electron Transfer Kinetics of Cytochrome C

J. Phys. Chem. C, Vol. 113, No. 6, 2009 2259

Figure 5. (a) Absorbance of the amide I β-turn type III band at 1692 cm-1 vs the potential in mV vs SHE fitted to the sigmoidal curve of a “Nernst”-plot. The inflection point is calculated to be 217 mV. (b) The absorbance of the amide II β-turn type III band at 1552 cm-1 vs the potential in mV vs SHE fitted to the sigmoidal curve of a “Nernst”-plot.

Figure 6. Time-resolved spectra of cc measured in the step-scan mode between -100 mV and +500 mV with a excitation frequency of 500 Hz, using 40 time slides at a time resolution of 50 µs. black s, 125 µs; red s, 325 µs; blue s, 525 µs; teal s, 725 µs; purple s, 925 µs; light green s, 1125 µs; navy s, 1325 µs; dark pink s, 1525 µs; pink s, 1725 µs; green s, 1925 µs. Assignment: amide I β-turn type III (reduced state): 1692 cm-1; amide I β-turn type II (reduced state): 1663 cm-1; amide I unordered (reduced state): 1644 cm-1; amide I extended β-strand (reduced state): 1624 cm-1.

than the ME-modified classical SEIRA surface reported in ref 18. This might be due to different morphologies of the two gold layers. Which of the bands is more or less correlated with the redox transition can be deduced from a plot of the amplitudes vs potential. For example, the band at 1692 cm-1 yielded a sigmoidic curve, which can be fitted to the Nernst equation with a slope of 36 mV and an inflection point at 217 mV in reasonable agreement with the theoretical slope of 25 mV and the standard redox potential of cc, E0 ) 230 mV (Figure 5A).14 The band at 1552 cm-1 yielded also a sigmoidic function with an inflection point at 220 mV, but a shallow slope of 130 mV (Figure 5B). This is in agreement with a band, the peptide group of which is located at a greater distance from the redox center and is therefore less correlated with the redox transition. An entirely unspecific orientational or other change of a peptide in an electric field wo uld be indicated by a straight line as was observed for molecules, which do not undergo any faradaic process.6 Such difference spectra were then used to calculate the enhancement factor of the two-layer gold surface according to a procedure introduced by Osawa.3,9 To this end, the absorbance of the band at 1692 cm-1 of reduced-minus oxidized difference

Figure 7. Time-resolved spectra of cc measured in the rapid scan mode between -100 mV and +500 mV with a excitation frequency of 0.7 Hz. brown s, 44.5 ms; black s, 89 ms; blue s, 133.5 ms; teal s, 178 ms; pink s, 222.5 ms; red s, 267 ms; navy s, 311.5 ms; blue s, 356 ms; light gree s, 400.5 ms; green s, 445 ms; purple s, 489.5 ms. Assignment: amide I β-turn type III (reduced state): 1692 cm-1; amide I β-turn type III (oxidized state): 1672 cm-1; amide I β-turn type II (reduced state): 1663 cm-1; amide I β-turn type II (oxidized state): 1658 cm-1; amide I extended β-strand (reduced state): 1624 cm-1; amide II β-strand (reduced state): 1552 cm-1; tyrosine in-plane ring vibration (reduced state): 1519 cm-1.

spectra of cc in solution (4 mM) was measured on the bare silicon crystal before and after cc was reduced by dithionite. The absorbance per molecule in the bulk was calculated to be 3.4 × 10-19 a.u. from the absorbance of the difference spectrum (6 × 10-5 a.u.) and the number of molecules (1.759 × 1014) within the ATR-active volume (73 nL) considering an operative distance of the evanescent wave of 940 nm and the electrode area of 0.785 cm2. The absorbance per molecule on the twolayer gold surface was calculated to be 4.36 × 10-17 a.u. from the absorbance of the difference spectrum (1.95 × 10-3 a.u.) normalized by the surface coverage of cc obtained from cyclic voltammograms (Γ ) 7.46 × 10-11 mol cm-2 ) 4.47 × 1013 molecules cm-2). The ratio of the two absorbance values yielded an enhancement factor of 127.8, 5.8 times the value of 22 found for the classical gold surface obtained by electroless deposition.19 This is certainly in accordance with the overall 5-fold increase of the total absorbance in the SEIRA spectra of the amide I band of cc on the two-layer reported in ref 9 vs the classical SEIRA-active gold surface. Finally, potential-controlled time-resolved (tr)-SEIRA measurements were conducted in the step-scan mode with a square

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Figure 8. Time-resolved spectra of cc measured in the fast rapid scan mode between -100 mV and +500 mV with an excitation frequency of 1.85 Hz. brown s, 33.7 ms; black s, 67.4 ms; blue s, 101.1 ms; teal s, 134.8 ms; pink s, 168.5 ms; red s, 202.2 ms; navy s, 235.9 ms; blue s, 269.6 ms; light green s, 303.3 ms; green s, 337 ms; purple s, 370.7 ms. Assignment: amide I β-turn type III (reduced state): 1692 cm-1; amide I β-turn type II (reduced state): 1663 cm-1; amide I β-turn type II (oxidized state): 1658 cm-1; amide I extended β-strand (reduced state): 1624 cm-1; amide II β-strand (reduced state): 1552 cm-1.

Figure 9. Phase-resolved ATR-FTIR spectra calculated with an angular resolution of 30°. The marked bands are used for the fitting. The excitation frequency was 500 Hz. black s, 0°; red s, 30°; blue s, 60°; teal s, 90°; purple s, 120°; green s, 150°; blue s, 180°; dark red s, 210°; pink s, 240°; green s, 270°; navy s, 300°; orange s, 330°.

wave excitation frequency of 500 Hz (Figure 6). Regarding these excitations, one should bear in mind that changes (conformational or orientational) of different peptide groups are indicated by tr-SEIRAS at different exciting frequencies. For example, the band at 1692 cm-1 had the highest amplitude at an exciting frequency of 500 Hz. This band had been shown above to directly reflect the redox transition of cc, since it indicates a conformational or orientational change of the amino acid directly attached to the central iron atom of the porphyrin ring (Figure 4). The frequency of the tr-SEIRA spectrum with the highest amplitude therefore was considered to match the frequency of the redox transition. Changes of other peptide groups were excited at much lower frequencies. For example, the band at 1552 cm-1 was excited at 1.85 Hz. Figure 7 shows a fast rapid scan measurement at this frequency. This band had been shown above to be related to a remote peptide group. It was more prominent at even lower frequencies, for example by a rapid scan measurement taken at 0.7 Hz (Figure 8). Changes of peptide groups represented by these bands were regarded as not directly correlated with the redox process.

Nowak et al.

Figure 10. Sample of the curve fitting of a phase resolved spectrum that was obtained from the PSD of time-resolved absorbance spectra (500 Hz, φ ) 0°). The fitting parameters of separated bands are subsequently used to analyze time-resolved data. black s, PSD 0° spectra; red s, spectrum fit; blue s, 1692 band fit; teal s 1663, band fit; purple s, 1644 band fit; light gree s, 1625 band fit.

Figure 11. Fitting of time-resolved spectra using band parameters obtained from PSD spectra. black, s time-resolved spectrum; red s, fit with PSD parameters; green s, band 1692 cm-1; purple s, band 1663 cm-1; teal s, band 1644 cm-1; blue s, band 1625 cm-1.

Further to the tr-SEIRA spectra obtained at 500 Hz (Figure 6), time-dependent changes of the bands not only at 1692 cm-1 but also at 1663, 1644, and 1625 cm-1 are seen. They were depicted in the context of the entire spectral range to demonstrate their amplitudes to clearly exceed the noise level of adjacent frequencies. However, the isolation of spectral changes out of a noisy background due to external perturbation can be improved even more by the application of phase-sensitive detection (PSD). Figure 9 shows the same spectral range treated by PSD. The bands changing with time can now be discriminated more effectively. Although a purely mathematical procedure, PSD has an effect similar to the analysis of the detector signal with the help of a lock-in amplifier, suggested to be applied for trSEIRAS earlier.9 Signals were obtained that are in phase or out of phase with the exciting perturbation. Signals can thus be recorded at different phase angles. Only the signals that change with the phase angle need to be considered to depend on the excitation, whereas noise is not phase-shifted and therefore suppressed compared to the original spectrum. This is clearly demonstrated in Figures 6 and 9. By curve fitting, using Lorentzian curves, we used PSD spectra to obtain the band parameters of absorbing components such as frequency, bandwidth, and amplitude. An example of a deconvoluted PSD spectrum is shown in Figure 10. These band parameters were

Electron Transfer Kinetics of Cytochrome C

J. Phys. Chem. C, Vol. 113, No. 6, 2009 2261 and reductive branch, respectively. The overall error includes the error due to sample preparation, the repeatability of the measurement, as well as the fitting. The charging current is fast compared to kox and kred and hence can be neglected. The rate constant of charging is approximately 3300 s-1 (calculated from the capacitance and resistance of the double layer of 10 µF cm-2 and 30 Ω cm, respectively). Conclusion

Figure 12. Peak area of the band at 1692 cm-1 plotted as a function of time. Monoexponentials were fitted to the data, and the corresponding time constants are k0,red ) 1880 ( 232 s-1 for the reductive branch and k0,ox ) 1810 ( 239 s-1 for the oxidative branch.

then applied to analyze the area of the bands of the time-resolved spectra (Figure 11). This analysis would otherwise be hard to achieve. It is greatly facilitated as illustrated by the example of a deconvoluted tr-spectrum shown in Figure 12. Finally, the area of the band at 1692 cm-1 obtained from tr spectra was plotted as a function of time for the reductive and oxidative branch of the square-pulse excitation (Figure 13). Measurements were carried out on four samples measured three times each. The data sets were fitted to the monoexponential functions

∆Aox(t) ) ∆Amax ,ox{1 - exp-kox(t - t0,ox)} + ∆A0,ox

(5) ∆Ared(t) ) ∆Amax,red exp-kred(t - t0,red) + ∆A0,red (6) for the oxidative and reductive branch, respectively, yielding a rate coefficient of kox ) 1810 ( 239 s-1 for the oxidative branch and kred ) 1880 ( 232 s-1 for the reductive branch (Figure 10). ∆Aox(t) and ∆Ared(t) are the absorbances as a function of time; Amax,ox and Amax,red are the maximal amplitudes of the absorbance; ∆A0 are the amplitudes at the starting point of oxidation/reduction; and t0 are the start times for the oxidative

Cyclic voltammetry has shown that the reduction and oxidation of cc was controlled by diffusion. However, to a certain extent the protein was also adsorbed to the surface as revealed by the dependence of the amplitude of the SEIRA spectra on the time of immersion. The electrostatic nature of the adsorption was also indicated by the dependence of the ionic strength of the buffer solution.14 The SEIRA spectra were therefore assumed to reflect predominantly the adsorbed species considering the effective volume due to the evanescent wave of the surface plasmon and the concentration of cc in solution (0.35 mM). This concentration would be much too small to be indicated by IR spectroscopy in the ATR configuration (see the evaluation of the enhancement factor). Reduced-minus-oxidized difference spectra enabled us to apply the method developed by Osawa to quantify the surface enhancement effect.3,4,19 The absorbance of cc molecules in the bulk was compared to the absorbance of cc molecules on the two-layer gold surface. The improved surface enhancement effect reported in ref 12 could be confirmed. This allowed us to perform tr-SEIRAS measurements with a good S/N ratio. The S/N ratio was further improved by the application of PSD.24 Given the high stability of the two-layer gold surface, longterm measurements required for tr-SEIRAS were not a problem. Apart from the extent of the enhancement effect, reducedminus oxidized difference spectra are fully compatible with similar measurements on the classical SEIRAS surface except for slight variations of relative band amplitudes, which are considered to be due to different surface morphologies. Most importantly, the ratio of the absorbance amplitude of these spectra to the difference spectrum of the fully oxidized protein vs the ME-modified surface, e.g., of the amide I band, is equal to 1:10 in both cases irrespective of the kind of gold surface.

Figure 13. Different orientations of cc absorbed to differently modified gold surfaces. A: cc absorbed on a SAM of MUA (11mercaptoundecanoicacid). B: cc absorbed on a SAM of 2-mercaptoethanol.

2262 J. Phys. Chem. C, Vol. 113, No. 6, 2009 The 1:10 ratio is therefore concluded to be inherent in the enhancement of IR absorption on colloidal surfaces under the influence of an electric field. The ME-modified gold surface was claimed previously to adsorb cc in the inverse orientation with the heme cleft directed away from the surface (Figure 12). In this orientation, electrochemical eT should be relatively slow since the pathway through the heme cleft toward the electrode is blocked. Cyclic voltammetry yielded a rate coefficient of 20 s-1 on conventional gold electrodes.14 Higher rate constants were obtained previously only for cc on carboxyl or pyridyl group terminated SAMs which assemble on the surface in the right orientation thus facilitating the eT (Figure 12).21,22 The relatively high rate constant of the inverse orientation that we found here can only be explained in terms of the morphology of the newly developed two-layer gold surface. Given that the cc molecules adsorb to the side walls of the NPs, they can also touch the horizontal parts of the surface providing more than one pathway for eT. Other possibilities are tilt angles different from the ones on flat surfaces. The rate constants were derived from time-dependent changes of an amino acid (His-18) directly ligated to the porphyrine ring of the heme center. Other groups of the peptide backbone were found to be excited at much lower frequencies. This was considered in terms of conformational transitions of peptide groups located at a greater distance from the redox center, which are not directly correlated with the redox process. These findings are expected to have important implications also for more complex proteins. Acknowledgment. Extensive discussions with Dieter Walz from the Biozentrum, Basel, Switzerland are gratefully acknowledged. References and Notes (1) Bard, A. J.; Faulkner, L. R. Electrochemical methods and Applications; John Wiley & Sons: New York, 2001; Vol. Second edition.

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