Probing Redox Reactions of Immobilized Cytochrome c Using Evanescent Wave Cavity Ring‐Down Spectroscopy in a Thin‐Layer Electrochemical Cell

Abstract

We report the use of evanescent wave cavity ring-down spectroscopy (EW-CRDS) to monitor the reduction by ethylenediaminetetraacetic acid iron(II) complex, [FeEDTA](2-), of an adsorbed layer of oxidized cytochrome c immobilized on fused silica. The adsorption of cytochrome c at the silica-water interface was also probed using EW-CRDS and found to be in qualitative agreement with previous studies. The reduction of the adsorbed cytochrome c was achieved by using a strategically positioned electrode to electrogenerate FeEDTA(2-), which diffused to the silica surface and reduced the cytochrome c. The difference in the absorption spectra of the reduced and oxidized forms of cytochrome c at 400 nm allowed the direct monitoring of the electron transfer in real time. Using finite-element modelling, the rate constant of electron transfer (ET) between FeEDTA(2-) and cytochrome c was found to be 4.3(± 0.6) × 10(-9) cm s(-1) equivalent to 2.7(± 0.4) M(-1) s(-1). The latter value is considerably lower than previously reported ET rate constants between cytochrome c and FeEDTA(2-) in solution, which can be attributed to the confinement of the immobilized cytochrome c on the surface and possible effects from molecular crowding. This study highlights the importance of new methods which can be used to study ET at interfaces and opens up the possibility of studying ET to proteins in biologically relevant environments using EW-CRDS.

Electroactive Interface for Enabling Spectroelectrochemical Investigations in Evanescent-Wave Cavity-Ring-Down Spectroscopy

“…38 In a subsequent work, it was used to change the solution pH in a controlled manner via the potential-pulsed chronoamperometric oxidation of water. 39 In another study, 40 it was applied to investigate the adsorption process of Cyt-C protein at a silica−water interface. In this last report, a chronoamperometry technique was applied to provide soluble redox mediators, which after diffusion, could chemically control the redox state of an adsorbed layer of Cyt-C species.…”

“…In another embodiment, a liquid/liquid interface was studied by the EW-CRDS technique with electrochemical potential control located in the bulk phase of one of the liquids . However, in all of these studies, − the surface under optical interrogation by the EW-CRDS platform was not electrically active, so the redox process probed by the EW-CRDS optical signal was always limited by the mass diffusion of the redox agents, which certainly imposes limitations for many electrochemical studies, as for instance in investigations of time rates of charge transfer in redox events.…”

Spectroscopic analysis of immobilised redox enzymes under direct electrochemical control

“…29 An interesting recent application of evanescent wave cavity ring-down spectroscopy (EW-CRDS) to monitor the reduction of horse heart cytochrome c adsorbed at the silicawater interface in a thin-layer electrochemical cell has been demonstrated by Unwin and co-workers. 78 Although in this case the electron transfer was mediated by [Fe(EDTA)] 2À , and hence was diffusion limited, EW-CRDS is capable of following interfacial dynamics on sub-millisecond timescales 79 and is compatible with a variety of different interfaces. 80 Coupled with a suitable electrochemical cell that allows un-mediated direct electron transfer, therefore, EW-CRDS could present a useful addition to the suite of spectroelectrochemical methods employed to study the dynamics of electron transfer processes in redox proteins.…”

Evanescent wave cavity-based spectroscopic techniques as probes of interfacial processes

“…65 Our group recently used a thin layer electrochemical cell (similar to that described in Sections 2.2 and 2.3) to study the redox reaction between an adsorbed layer of cytochrome c immobilised on fused silica and the ethylenediaminetetraacetic acid iron(II) complex (FeEDTA 2À ). 66 This latter work showed that it is possible to directly monitor electron transfer between surface immobilised proteins and molecules using EW-CRDS, and obtain quantitative information on the kinetics.…”