Correlating molecular orientation distributions and electrochemical kinetics in subpopulations of an immobilized protein film.


Immobilized metalloproteins have potential applications in several emerging areas such as bioelectronics and biomimetic energy conversion.1-3 It is well recognized that immobilization of a metalloprotein may affect its electron transfer activity, because of an “incorrect” molecular orientation and/or altered conformation relative to the native state.4-8 Horse heart cytochrome c (cyt c) has been frequently used as a model to study these effects.5 Cyt c undergoes quasi-reversible electron transfer when immobilized on a variety of electrode surfaces, and current knowledge of the structural requirements for achieving protein-electrode communication are based primarily on studies of this protein.6,7 Most of these studies have been performed on Au electrodes modified with selfassembled monolayers (SAMs),8 although cyt c adsorbed directly to indium-tin oxide (ITO) is also electrochemically active.9 The influence of electrode-protein separation distance on the electron transfer rate constant (k0) of immobilized cyt c has been thoroughly investigated.6-8 Orientation is also predicted to play a significant role because the heme is not located at the center of the protein.5c,10-12 Thus a distribution of molecular orientations will generate a distribution of heme-electrode separation distances and electron-tunneling pathways, producing a distribution of k0 values. This concept has been invoked to explain the nonideal voltammetry of immobilized cyt c films,10 but has not been experimentally verified. The difficulty lies in measuring the molecular orientation distribution of a cyt c film, measuring a distribution of k0 values on that film, and establishing a correlation between the distributions. Dick et al.5c demonstrated that a more oriented cyt c film is more electrochemically reversible but their work did not address distributions of orientations and k0 values. Recently we used a combination of polarized internal reflection techniques to measure the tilt angle distribution of the heme molecular plane in a cyt c monolayer adsorbed to ITO (Figure 1).11 The very broad orientation distribution should give rise to a broad distribution of heme-electrode separation distances and k0 values. A complicating factor is that the total surface coverage, measured spectroscopically, is 22 pmol/cm2, but the electroactive surface coverage, measured electrochemically, is only 9.5 pmol/cm2.13 Thus the orientation distribution and k0 (measured electrochemically) cannot be correlated because the former is measured on the entire film whereas the latter is measured on only the electroactive portion (43%) of the film. Herein we describe an electroreflectance (ER) technique that allows measurements of k0 values for differently oriented subpopulations of the electroactive molecules in an immobilized cyt c film, without interference from the nonelectroactive subpopulation. To our knowledge, this is the first demonstration that distinct k0 values correlated to differently oriented molecules can be recovered from any redox-active molecular film. ER spectroscopy is an optical analog of electrochemical impedance spectroscopy in which changes in the reflectance of a redoxactive molecular film deposited on an electrode are measured as a function of the frequency at which the potential at the electrode is modulated.14,15 Most UV-vis ER studies reported to date have been performed using an external reflectance geometry at a Au electrode or in a transmission geometry using an optically transparent electrode. Attenuated total reflectance (ATR) is an alternative that provides a larger optical path length because the probe beam is internally reflected multiple times down the length of a waveguide electrode.16 In addition, measurements can be made in both transverse electric (TE) and transverse magnetic (TM) polarizations, which can provide information about molecular orientation. This approach, termed potential modulated ATR (PM-ATR), has been used to examine electro-optical and charge transfer processes in conducting polymer and Prussian blue films deposited on ITOcoated planar waveguides.17 Here PM-ATR was used to measure k0 values for cyt c adsorbed to ITO when probed with either TE or TM polarized light. ITOcoated planar waveguides were fabricated by sputtering a ca. 100 nm thick layer of ITO on 150 um thick glass coverslips. Cyt c (Sigma) was prepared and adsorbed on ITO from pH 7 phosphate buffer, as described previously,11 to produce films of near monolayer surface coverage. The electroactive surface coverage, measured by cyclic voltammetry, was 7.9 pmol/cm2.13 The double layer capacitance (Cdl) and uncompensated solution resistance (Rs) were determined on independently prepared films using electrochemical impedance spectroscopy and found to be 9.1 μF/cm2 and 1.1 kΩ.cm2, respectively. The time constant of the electrochemical cell with a bare ITO electrode was 4.9 ms.13,17 Complete descriptions of PM-ATR theory and instrumentation are given elsewhere.17 Here a polarizer and a 417 nm bandpass filter (3 nm fwhm) were placed between the light source and ATR cell to control polarization and spectral bandwidth, respectively. Sinusoidally modulating the electrode potential over a small range near the midpoint between the oxidation and reduction potentials Figure 1. Distribution of heme plane tilt angles, relative to the electrode surface plane, in a cyt c monolayer adsorbed to ITO. Data are replotted from the polar coordinate data presented in Figure 3b2 in ref 11 and normalized to a total probability of unity. The insets show representations of molecules with heme tilt angles near 0°, 50°, and 90°. Published on Web 01/15/2008

DOI: 10.1021/ja710156d

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@article{Araci2008CorrelatingMO, title={Correlating molecular orientation distributions and electrochemical kinetics in subpopulations of an immobilized protein film.}, author={Zeynep Ozkan Araci and Anne F Runge and Walter J. Doherty and S Scott Saavedra}, journal={Journal of the American Chemical Society}, year={2008}, volume={130 5}, pages={1572-3} }