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.

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS

“…2B) similar to the ones obtained under anaerobic conditions. Differences were revealed, however, when the spectra were subjected to the socalled phase sensitive detection (PSD), described in [23] and in more detail in [22]. Although changes were noticeable also in the fingerprint region, we focused on the much more pronounced amide I region.…”

“…In this elevated state, the enzyme can be expected to undergo reversble conformational changes of secondary structures as a consequence of ET different from the ones before the transformation. This assumption will be tested in this study comparing our previous tr-SEIRAS studies of the CcO conducted under anaerobic conditions [22,23] with those carried out under aerobic conditions.…”

“…SEIRAS studies in an anaerobic environment [13,[19][20][21][22] had shown that the CcO could be converted reversibly into the fully reduced state. This state is necessary as a starting point for measurements under aerobic conditions, thus enabling the enzyme to undergo enzymatic oxidation.…”

“…The preparation was done as described in [22,38,39]. Briefly, after modification with MPTES, the ATR crystal was coated with a 25 nm gold film by electrothermal evaporation (HHV Edwards Auto 306, Crawley, UK).…”

“…CcO was immobilized on the ATR crystal as described in detail by Schwaighofer et al [22]. CcO from Rhodobacter sphaeroides with a His-tag engineered to the C terminus of subunit II was expressed and purified according to Hiser et al [40].…”