Novel Time-Resolved Surface-Enhanced (Resonance) Raman Spectroscopic Technique for Studying the Dynamics of Interfacial Processes: Application to the Electron Transfer Reaction of Cytochrome c at a Silver Electrode

Abstract

A novel approach for time-resolved (TR) surface-enhanced (resonance) Raman (SE(R)R) spectroscopy is presented for probing potential-dependent processes of molecules adsorbed on a silver electrode. TR SE(R)R spectroscopy offers the unique advantage of providing structural and kinetic data exclusively of the adsorbed species and their reactions. These processes are initiated by a rapid potential jump and monitored by SE(R)R spectroscopy after a delay time δ for the probe interval Δt. The synchronization is achieved by a mechanical chopper that triggers the potential jump via a photodiode and gates the exciting continuous-wave laser beam. After the probe event, the potential is reset to its initial value. Thus, the original equilibrium is restored to allow a continuous repetition of the sequence of potential jumps and probe events. During the entire experiment, the detection system, a liquid nitrogen-cooled charge-coupled device (CCD) detector, is active so that the signal-to-noise ratio (SNR) can be iteratively improved. This mode of detection does not limit the time resolution, so that the present approach allows TR SE(R)R experiments down to the microsecond time scale without lowering the SNR. The possibilities and limitations of this method are discussed. As an example we present preliminary results of a TR SERR study on yeast iso-1 cytochrome c (Cyt-c) adsorbed on a Ag electrode by applying a potential jump from −0.4 V to +0.05 V (vs. saturated calomel electrode). The experiments are carried out with a rotating electrode to avoid photoinduced degradation and desorption processes. The SERR spectra, which were measured with delay times between 45 to 175 ms, were analyzed quantitively in terms of the various states of the adsorbed Cyt-c that are formed in this potential range. The results show that under these conditions the relaxation processes include the electron transfer of the adsorbed Cyt-c to the electrode and a subsequent conformational transition. The analysis of the data reveals a heterogenous oxidation rate constant of 10.3 s−1 and rate constant for the conformational transition of 4.3 s−1, supporting the view that the biological electron transfer of Cyt-c is coupled with conformational transitions.

Redox and Conformational Equilibria and Dynamics of Cytochrome c at High Electric Fields

“…[19,26] In general, the distribution between the conformational states B1 and B2 follows a potential-dependent equilibrium within the electrode potential range between about 0.1 and À 0.55 V, with B1 (reduced) and B2 (oxidised) being the thermodynamically preferred states below and above approximately À 0.1 V, respectively. SERR spectra measured in this potential range can be consistently simulated by the component spectra solely of the seven B1 and B2 species.…”

“…We have chosen a mixture of sulfate and phosphate, which, albeit less tightly bound to the electrode than halide ions, [24] do not narrow the accessible potential range such that the oxidation potential of Ag remains sufficiently separated from the redox potential of Cyt-c. As shown previously, Cyt-c binds strongly to the layer of chemisorbed oxoanions and gives rise to intense SERR signals. [19] In continuation of these studies, we now report a detailed analysis of the structure, thermodynamics and kinetics of the adsorbed Cyt-c and its interfacial reaction under the influence of very strong electric fields.…”

“…[15] This conformational equilibrium that is observed for horse-heart and yeast iso-1 Cyt-c is induced quite generally upon electrostatic binding to a variety of anionic ™surfaces∫, which include phospholipid vesicles and the binding domain of the natural redox partner Cyt-c oxidase. [15,19,21,22] In these experiments, the variation of the thickness of the SAM coating from approximately 2.4 to 0.7 nm allows an increase in the electric-field strength by a factor of about 2. [15] A further increase requires an increase of the charge density in the binding domain.…”

“…[14,19] This technique selectively probes the vibrational spectrum of (chromophoric) active sites solely of the immobilised biomolecules and thus provides information about their molecular structures and the thermodynamics and kinetics of their interfacial processes under the influence of the electric field. [15] Furthermore, the electrode may serve as an electron donor or acceptor, such that it is specifically suited to studying electron-transfer (ET) reactions.…”

Time-resolved surface-enhanced resonance Raman spectro-electrochemistry of heme proteins

“…The surface enhancement effect as well as the resonance Raman scattering help to overcome the problems associated with the weak interaction cross-section of the Raman effect. However, photo-degradation of the protein by the incident laser beam is a serious problem in Raman scattering [12]. In order to overcome this problem a number of setups were designed to reduce the exposure time of the active area to the laser beam.…”