High-Pressure Probing of a Changeover in the Charge-Transfer Mechanism for Intact Cytochromec at Gold/Self-Assembled Monolayer Junctions (original) (raw)
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Gated Electron Transfer of Yeast Iso-1 Cytochrome c on Self-Assembled Monolayer-Coated Electrodes
The Journal of Physical Chemistry B, 2008
Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) consisting either of 11-mercaptoundecanoic acid (MUA) or of 1:1 mixtures of MUA and either 11-mercaptoundecanol (MU) or 7-mercaptoheptanol (MH). The redox potentials and the apparent rate constants for the interfacial redox process as well as for the protein reorientation were determined by stationary surfaceenhanced resonance Raman (SERR) and time-resolved SERR spectroscopy, respectively. For YCC immobilized on MUA and MUA/MU at pH 7.0 and 6.0, the negative shifts of the redox potentials with respect to that for the protein in solution can be rationalized in terms of the potential of the zero-charge determined by impedance measurements. The apparent electron transfer rate constants of YCC on MUA/MU and MU/MH at pH 6.0 were determined to be 8 and 18 s -1 , respectively. A decrease of the relaxations constants by a factor of ca. 2 was found for pH 7.0, and a comparable low value was determined for a pure MUA even at pH 6.0. In each system, the rate constant for protein reorientation was found to be the same as that for the electron transfer, implying that protein reorientation is the rate limiting step for the interfacial redox process. This gating step is distinctly slower than that for horse heart cytochrome c (HHCC) observed previously under similar conditions (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). The different rate constants of protein reorientation for both proteins and the variations of the rate constants for the different SAMs and pH are attributed to the electric field dependence of the free energy of activation which is assumed to be proportional to the product of the electric field strength and the molecular dipole moment of the protein. The latter quantity is determined by molecular dynamics simulations and electrostatic calculations to be more than 2 times larger for YCC than for HHCC. Moreover, the dipole moment vector and the heme plane constitute an angle of ca. 10 and 45°in YCC and HHCC, respectively. The different magnitudes and directions of the dipole moments as well as the different electric field strengths at the various SAM/protein interfaces allow for a qualitative description of the protein-, SAM-, and electrode-specific kinetics of the interfacial redox processes studied in this and previous works.
Electron transfer dynamics of cytochrome c bound to self-assembled monolayers on silver electrodes
Bioelectrochemistry, 2002
Cytochrome c (Cyt-c) was electrostatically immobilised on Ag electrodes coated with self-assembled monolayers (SAM) that are formed by w-carboxyl alkanethiols with different alkyl chain lengths (C x). Surface enhanced resonance Raman (SERR) spectroscopy demonstrated that electrostatic binding does not lead to conformational changes of the heme protein under the conditions of the present experiments. Employing time-resolved SERR spectroscopy, the rate constants of the heterogeneous electron transfer (ET) between the adsorbed Cyt-c and the Ag electrode were determined for a driving force of zero electronvolts. For SAMs with long alkyl chains (C 16 , C 11), the rate constants display a normal exponential distance dependence, whereas for shorter chain lengths (C 6 , C 3 , C 3), the ET rate constant approaches a constant value (ca. 130 s À1). The onset of the non-exponential distance-dependence is paralleled by an increasing kinetic H/D effect, indicating a coupling of the redox reaction with proton transfer (PT) steps. This unusual kinetic behaviour is attributed to the effect of the electric field at the Ag/SAM interface that increasingly raises the energy barrier for the PT processes with decreasing distance of the adsorbed Cyt-c from the electrode. The distance-dependence of the electric field strength is estimated on the basis of a simple electrostatic model that can consistently describe the redox potential shifts of Cyt-c as determined by stationary SERR spectroscopy for the various SAMs. At low electric fields, PT is sufficiently fast so that rate constants, determined as a function of the driving force, yield the reorganisation energy (0.217 electronvolts) of the heterogeneous ET.
Langmuir, 2006
Cytochrome c was electrostatically immobilized onto a COOH-terminated alkanethiol self-assembled monolayer (SAM) on a gold electrode at ionic strengths of less than 40 mM. Scanning electrochemical microscopy (SECM) was used to simultaneously measure the electron transfer (ET) kinetics of the bimolecular ET between a solution-based redox mediator and the immobilized protein and the tunneling ET between the protein and the underlying gold electrode. Approach curves were recorded with ferrocyanide as a mediator at different coverages of cytochrome c and at different substrate potentials, allowing the measurement of k BI) 2 × 10 8 mol-1 cm 3 s-1 for the bimolecular ET and k°) 15 s-1 for the tunneling ET. The kinetics of ET was also found to depend on the immobilization conditions of cytochrome c: covalent attachment gave slightly slower tunneling ET values, and a mixed CH 3 /COOH-terminated ML gave faster tunneling ET rates. This is consistent with previous studies and is believed to be related to the degree of mobility of cyt c in its binding configuration and its orientation with respect to the underlying electrode surface.
Journal of Physical Chemistry B, 2003
The electron exchange kinetics of horse heart cytochrome c (Cyt c) at 4,4′-bipyridyl-and 4,4′-bipyridyldisulfide-modified Au electrodes has been studied for the first time under variable pressure conditions (up to 150 MPa), by using fast scanning cyclic voltammetry. A positive activation volume of +6.1 ( 0.5 cm 3 mol -1 was determined from the pressure dependence of the heterogeneous standard rate constant in both cases. This value is similar to that for the homogeneous Cyt c self-exchange process, predicted from the cross-reaction treatment. A careful analysis based on an extended version of the contemporary charge-transfer theory indicates that the process most probably takes place through an adiabatic ("protein friction") charge-transfer mechanism in which the positive volume of activation results from the pressure-induced increase of the protein's intrinsic viscosity (decrease of the characteristic relaxation mobility), which is also in remarkable agreement with earlier results from studies in which the viscosity was varied directly. This approach allows for variation of the internal protein viscosity without significant alteration of the properties (viscosity, diffusion coefficients) of the aqueous medium.
Electric-field effects on the interfacial electron transfer and protein dynamics of cytochrome c
Journal of Electroanalytical Chemistry, 2011
Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectroscopy have been employed to study the interfacial redox process of cytochrome c (Cyt-c) immobilised on various metal electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated mercaptanes. The experiments, carried out with Ag, Au and layered Au-SAM-Ag electrodes, afford apparent heterogeneous electron transfer constants (k relax ) that reflect the interplay between electron tunnelling, redox-linked protein structural changes, protein re-orientation, and hydrogen bond re-arrangements in the protein and in the protein/SAM interface. It is shown that the individual processes are affected by the interfacial electric field strength that increases with decreasing thickness of the SAM and increasing difference between the actual potential and the potential of zero-charge. At thick SAMs of mercaptanes including 15 methylene groups, electron tunnelling (k ET ) is the rate-limiting step. Pronounced differences for k ET and its overpotential-dependence are observed for the three metal electrodes and can be attributed to the different electric-field effects on the free-energy term controlling the tunnelling rate. With decreasing SAM thickness, electron tunnelling increases whereas protein dynamics is slowed down such that for SAMs including less than 10 methylene groups, protein re-orientation becomes rate-limiting, as reflected by the viscosity dependence of k relax . Upon decreasing the SAM thickness from 5 to 1 methylene group, an additional H/D kinetic isotope effect is detected indicating that at very high electric fields rearrangements of the interfacial or intra-protein hydrogen bond networks limit the rate of the overall redox process.
Electrochimica Acta, 2005
Surface structures controlled at the nanometer and single-molecule levels, with functions crucially determined by interfacial electron transfer (ET) are broadly reported in recent years, with different kinds of electrochemically controlled nanoscale/single molecule systems. One is the broad class of metallic and semiconductor-based nanoparticles, nano-arrays, nanotubes, and nanopits. Others are based on self-assembled molecular monolayers. The latter extend to bioelectrochemical systems with redox metalloproteins and DNA-based molecules as targets.
Procedia Engineering, 2012
A novel third generation biosensor was developed based on one-shot adsorption of chemically modified cytochrome c (cyt c) onto bare gold electrodes. The introduction of short-chain thiol derivatives (mercaptopropionic acid, MPA) on the lysine residues of cyt c enabled the very fast formation (<5 min) of an electroactive biological self-assembled monolayer (SAM) exhibiting a quasi-reversible electrochemical behavior and a fast direct electron transfer (ET). The high value estimated for the heterogeneous ET rate constant, k s = 1600 s-1 , indicates that short anchors might facilitate the ET via an efficient orientation of the heme pocket. In comparison, no direct ET was observed in the case of native and long-anchor modified (mercaptoundecanoic acid, MUA) cyt c adsorbed on gold. The so-made amperometric biosensor enabled real-time and non-invasive detection of extracellular H 2 O 2 released by unicellular aquatic microorganisms Chlamydomonas reinhardtii as a consequence of cadmium-induced oxidative stress. Motivation and results Over the last two decades amperometric biosensors based on the enzymatically-catalyzed reduction of H 2 O 2 have attracted a wide interest due to their accurate sensitivity and specificity. In the case of the so-called third generation biosensors which rely on direct ET the ability to achieve an efficient electrical communication underlies resolving orientation and distance issues between the electrode surface and the protein redox center. 1 It is known that cyt c exhibits a very low ET once in contact to solid bare electrodes which results in a poor electrochemical behavior. 2 Thus, a number of studies have focused on modifying the gold electrode surface in order to generate a suitable molecular environment that prevents from cyt c denaturing and enhances the ET rate constant. Here we report a chemically modified cyt c that forms adsorbed monolayer onto bare gold and exhibits very fast ET rate constant. The introduction of thiol derivatives-via chemical coupling on cyt c lysine residues-that act as anchor molecules on gold prevents protein denaturing and enables rational "tuning" of the ET rate with the chain length. As expected for native cyt c no faradaic peak developed on the capacitive current whereas well defined peaks are visible for cyt c-MPA which corresponds to the electrochemical oxidation and reduction of heme group (Fig. 1). However, cyt c-MUA that exhibits a longer anchor-spacer shows no direct ET albeit its electroactivity is restored as soon as small gold nanoparticles act as electron shuttle. In contrast, the cyt c-MPA layer shows a quasi-reversible electrochemical behavior indicating that fast interfacial ET mechanism is predominant due to a favorable protein orientation on gold. The voltammetric monitoring of MPA-cyt c chemisorption on bare gold emphasizes the rapidity of the electroactive protein layer formation with the maximum peak intensity reached within 5 minutes (20 cycles at = 0.05 V s-1), as shown in Figure 2A. Moreover, the analysis of the peaks intensity shows a reversible electrochemical behaviour with a value of Ia/Ic = 0.96. The calculation of the E FWHM = 86 mV which is below the theoretical value of 90.6 mV defines a nerstian monoelectronic process strongly adsorbed on the electrode surface. 3 The surface coverage () of electroactive MPA-cyt c on gold was estimated to be 4x10-12 mol cm-2 which corresponds roughly to 25 % of the theoretical value reported by Nakano et al. 4 for a fully packed cyt c surface coverage. It is also observable that the formal potential E°' =-48 mV (vs Ag/AgCl) is negative-shifted as compared to native cyt c in solution, as expected for a covalently immobilized protein. 5 Furthermore, cyclicvoltammetry was performed at different scan rates in order to study the ET mechanism taking place at a MPA-cyt c modified electrode. The voltammograms in Figure 2B show the influence of the scan rate both on the peak current intensity and the peak-to-peak separation. As depicted in Figure 2C the anodic and cathodic peaks currents are linearly proportional to the scan rate in the range between 0.01 and 0.6 V s-1 , which is expected for a surface-controlled electrochemical process. An estimated value of the heterogeneous ET rate constant, k s , has been calculated from the analysis of peak-to-peak separations fusing Laviron's method. Considering a charge transfer coefficient = 0.5 a high value of k s = 1600 s-1 was calculated, which is in good agreement with those reported for cyt c covalently attached onto a SAM of mercaptobutyric acid 6 or a mixed SAM of MPA/mercaptoethanol. 7