Electron Transfer Kinetics of Cytochrome C in the Submillisecond Time Regime Using Time-Resolved Surface-Enhanced Infrared Absorption Spectroscopy

Abstract

The two-layer gold surface developed before is used for time-resolved surface-enhanced infrared absorption spectroscopy (tr-SEIRAS). The electron transfer (eT) rate to cytochrome c adsorbed to a self-assembled monolayer of 2-mercaptoethanol is measured using SEIRAS in the step-scan mode in an ATR configuration. Electron transfer is triggered by periodic potential pulses applied to the gold surface which is used both as an electrode and a surface enhancing layer. Phase sensitive detection is used to separate and determine band parameters of the strongly overlapping absorption bands of the amide region. The surface enhancement effect of the two-layer gold surface was determined to be 128. Rate constants for eT under conditions of the experiment were determined by tr-SEIRAS to be kox ) 1810 ( 239 s -1 and kred ) 1880 ( 232 s -1 for oxidation and reduction, respectively. This was deduced from the time-dependent change of one particular amino acid (His18) directly ligated to the porphyrine ring of the heme center. Different groups of the peptide backbone were found to be excited at different excitation frequencies.