title: Phase-sensitive detection in modulation excitation spectroscopy applied to potential induced electron transfer in cytochrome c oxidase.
abstract: Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides was investigated by modulated excitation surface-enhanced infrared-absorption spectroscopy (SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During modulated excitation by periodic potential pulses with frequencies between 20 and 500 Hz, time-resolved infrared spectra were measured by the step-scan technique, with time resolution in the millisecond 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 I 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 validation of the fit model used for the deconvolution of overlapping bands by analyzing phase lags of single components acquired at different stimulation frequencies. Phase lags between the evaluated vibrational components and the modulated excitation increase with increasing excitation frequencies, an inherent prerequisite of this evaluation method.
authors.affiliation: AIT Austrian Institute of Technology GmbH, Donau-City Str. 1, 1220 Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwaighofer
authors.forename: Shelagh
authors.initials: S
authors.lastname: Ferguson-Miller
authors.forename: Renate L C
authors.initials: RL
authors.lastname: Naumann
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Knoll
authors.forename: Christoph
authors.initials: C
authors.lastname: Nowak
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D015203:Reproducibility of Results; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013055:Spectrophotometry, Infrared / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D006418:Heme; D003576:Electron Transport Complex IV