Electron-Transfer Kinetics of Covalently Attached Cytochrome c/SAM/Au Electrode Assemblies

The rotational motion of cytochrome c has been restricted by cross-linking it to mixed self-assembled monolayers (SAMs) with the compositions S−(CH₂)_mCOOH/S−(CH₂)_nOH on gold electrodes via the formation of amide bonds between lysine residues on the protein and terminal carboxylate groups of the SAM. The effect of SAM thickness on the electron-transfer rate has been studied, and two main observations are drawn. First, the electron-transfer rate displays the same qualitative dependence on SAM thickness that was previously reported for electrostatically adsorbed and pyridine-ligated assemblies, suggesting a tunneling mechanism at long distance and some other rate-limiting process at short distance. Second, a significant effect on the rate is observed for mixed SAMs having a hydroxyl-terminated alkanethiol diluent when the diluent is more than one methylene group shorter than the carboxylic acid alkanethiol. These conclusions suggest that large-amplitude protein motion (i.e., gating) is not rate-limiting at short distance, though smaller-amplitude motions cannot be ruled out.