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 modulated excitation by periodic potential pulses with frequencies between 20 Hz and 500 Hz, time-resolved (tr) 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. Overlapping structural bands in the amide I region were deconvoluted by application of Phase Sensitive Detection (PSD). Analyzing
the phase lags of single components acquired at different stimulation frequencies allowed the validation of deconvoluted structural bands. Identified vibrational components were used to fit the band intensities in tr-IR spectra. Obtained band absorbances versus time were then fitted to a computational model for the sequential ET, yielding parameters such as the electrochemical rate constant from the electrode to CuA, ke, as well as E0 and pKr and pKo of the four centers in the CcO.
|Konferenz||57 th Annual Meeting Biophysical Society, Philadelphia, Pennsylvania|
|Zeitraum||2/02/13 → 6/02/13|