Abstract
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.