Cyclic voltammetry, square wave voltammetry or electrochemical impedance spectroscopy? Interrogating electrochemical approaches for the determination of electron transfer rates of immobilized redox proteins.

In this article, we cross-examine three well-established electrochemical approaches, namely cyclic voltammetry (CV), cyclic square-wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) to dissect the electron transfer (ET) rate of electrostatically immobilized cytochrome c on Ag electrodes. A detailed analysis supported by simulations of redox transition provided three distinct values for the heterogeneous electron transfer (HET) rate constant of cyt c interfaced on COOH-terminated C10-long alkanethiol, i.e., kHET= 47.8 (±2,91) s-1 in CV, kHET= 64.8 (±1,27) s-1 in SWV, and kHET= 26.5 s-1 in EIS. We discuss the obtained discrepancies obtained from electrochemical methods and compare them with the data from spectro-electrochemical experiments. A comprehensive selection list is created from which the most applicable approach can be chosen for studying proteins of interest. CV is most applicable to study the interfaced proteins exhibiting kHET of ca. 0.5 - 70 s-1, SWV is suitable for a broader range of kHET of 5 - 120 s-1 and EIS for kHET of 0.5 to 5 s-1 if alkanethiols are used as immobilization strategy.

Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

HYPOTHESIS: Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. EXPERIMENTS: Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). FINDINGS: The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his6-tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his6-tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode.