Electron Transfer Kinetics of Cytochrome c Probed by Time-Resolved Surface-Enhanced Resonance Raman Spectroscopy

Abstract

An improved setup including a measuring cell was designed for time-resolved surface-enhanced resonance Raman (SERR) spectroscopy. The cell is based on a rotating disk electrode (RDE) made from electrochemically roughened Ag. Cytochrome c (cc) adsorbed on a monolayer of mercaptoethanol is investigated with respect to heterogeneous electron transfer. Cyclic voltammograms and potential-dependent static SERR spectra indicate cc to be electroactive on the Ag electrode. The standard redox potential was found to be 234 mV. Time-resolved SERR spectra were then measured triggered by periodic potential pulses changing the protein between the oxidized and reduced state at a frequency of 10 Hz. Monoexponential functions obtained from the intensity of the band at 1361 cm-1 plotted versus time yielded the rate constants of heterogeneous electron transfer to be k(ox) = 46 +/- 7 s(-1) and k(red) = 84 +/- 20 s(-1). These relatively low rates are in line with the orientation of cc on the mercaptoethanol-modified Ag electrode. In this case the heme cleft pointed away from the surface thus hampering electron transfer.

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

“…The setup used for Raman experiments was described in detail in a previous publication [7]. All Raman experiments were performed using the 413 nm line of a Kr + laser (Innova 90C, Coherent).…”

“…The top planes of 12 mm thick silver rods were polished as described before [7] down to a RMS roughness of 3-5 nm. Self assembled monolayers (SAMs) were formed by immersion of the silver rods into an aqueous solution of cysteamine (CA) (10 mM/l) for 1 h. After thorough rinsing with water the modified silver rods were immersed into the nanoparticle suspension.…”

“…Electron transfer kinetics of heme proteins adsorbed on metal surfaces can be investigated by spectroelectrochemistry [3,7]. A combination of surface-enhanced Raman scattering (SERS) and resonance Raman scattering (RRS) is particularly advantageous in this context.…”

“…Rotating disc electrodes (RDE) proved to be a promising approach. We designed a new Raman measuring cell using this concept [7]. Furthermore a SER(R)Sactive silver modified silver substrate was developed and optimized for the excitation with the 413 nm line of a Kr + laser, offering ideal properties for studying heme proteins [8].…”

Sum Frequency Generation Spectroscopy in Biosensors Technology

“…In such cases, adapted characterisation techniques were used. For example, the orientation of heme complex in Cytochrome C p r o t e i n s w e r e d e t e r m i n e d b y R a m a n spectroscopy, therefore deducing the overall protein orientation (Mc Donald et al, 1996;Edminston et al, 1997;Keating et al, 1998;Yu et al, 2007;Sonois et al, 2009;Grosserueschkamp et al, 2009). Unfortunately, such investigations were limited to heme groups containing proteins.…”