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.