Raman Spectroelectrochemical Investigations of Immobilized Redox Proteins

Redox proteins adsorbed on electrodes have gained increasing attention of researchers in both fundamental and applied science. Surface-enhanced resonance Raman (SERR) spectroelectrochemistry is a particularly suitable technique for studying heterogeneous electron transfer reactions as it selectively probes the redox sites of the adsorbed proteins. SERR spectroscopy combines the advantages of the surface and resonance enhancement by exciting a heme protein immobilized on rough silver electrodes in the Soret absorption band region. Protein immobilization methods involve coating the electrodes with biocompatible lms in order to prevent protein denaturation. This work focuses on the elucidation of heterogeneous electron transfer mechanism and dynamics of the soluble cytochrome c (Cyt c) and the membranebound cytochrome c oxidase (CcO) attached to electrodes modi ed with di erent functional groups. First, Cyt c coordinatively attached to self-assembled monolayers of pyridine-terminated alkanethiols on electrodes was employed as a model system to study basic aspects of heterogeneous electron transfer. Electron transfer at short spacer lengths was monitored by time-resolved SERR spectroscopy. The electron transfer rate constant increased with increasing driving force indicating a change of mechanism from a nonadiabatic, tunneling controlled reaction at long distances to the electrode, to an increasingly solvent/protein friction controlled mechanism at shorter spacers. Second, polyelectrolyte-Cyt c multilayers were investigated, which are of interest in the fabrication of biosensors. An anionic polyelectrolyte, the poly(aniline sulfonic acid) (PASA), was used to intercalate the cationic redox protein. The PASA-Cyt c multilayer assemblies were, however, not stable on rough Ag electrodes coated with a mixed carboxyl/hydroxyl-terminated lm resulting in a one PASA-Cyt c layer system. As indicated by SERR measurements, Cyt c within the PASA layer retains its native properties. Redox potentials and standard rate constants of the immobilized Cyt c determined by SERR spectroelectrochemistry display an excellent agreement with the values obtained by cyclic voltammetry. Stationary and time-resolved SERR experiments reveal reversible spectral changes of PASA that provide evidence for electron exchange between Cyt c and PASA pointing to a tight coupling of the two species in the multilayer assemblies. Finally, a novel immobilization technique was employed to attach CcO to an electrode and subsequently reconstitute the lipid environment. The immobilization procedure is based on the a nity of a metal-chelating surface modi cation to a histidine-tag, engineered to di erent sides of the enzyme, and hence is applicable to a large variety of (membrane) proteins. Upon avoiding the very facile photoreduction, SERR spectra of the immobilized CcO reveal preservation of the native structures of the heme a and heme a3 sites. The electron transfer reaction was independent of enzyme orientation and the applied driving force suggesting that it involves multi-step electron tunneling or hopping over a distance of ca. 50 Å. Moreover, electrochemical reduction was restricted to heme a pointing to a perturbation of the native network of cooperativities between electron transfer and proton translocation in the immobilized enzyme.