Protein dynamics of Cytochrome c Oxidase: Analysis of vibrational components by Phase Sensitive Detection applied to Modulated Excitation-SEIRAS

Cytochrome c oxidase (CcO) from R. sphaeroides was investigated by Modulated Excitation Surface-Enhanced IR-Absorption Spectroscopy (ME-SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During the modulated excitation by periodic potential pulses with frequencies between 20 Hz and 500 Hz, time-resolved IR spectra were measured by the Step-Scan technique with time-resolution in the millisecond time range. Conformational changes of the protein structure as a result of ET lead to rather complex SEIRA spectra with many overlapping bands embedded in a broad background signal. Phase sensitive detection (PSD) was used to separate single components within the broad band of overlapping structural bands in the amide region. PSD is able to extract the periodic response of single components with the same frequency as the excitation from noise or from static background and therefore enhances the signal-to-noise ratio. Moreover, PSD enables the validation of the fit model utilized for the deconvolution of overlapping bands by analyzing the phase lags of single components acquired at different stimulation frequencies. The phase lags between the evaluated vibrational components and the modulated excitation increase with increasing excitation frequencies, which is an inherent prerequisite of the evaluation method.