2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer

Abstract

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.

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

“…Time constants = 400 ms were found for the bands at 1524 and 1739 cm -1 , indicating the A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 F-O H transition and Glu 278 protonation, respectively. The band around 1739 cm -1 , which we had found in our titration data of CcO [21] could not be discriminated from noise in the present tr-SEIRA spectra ( Fig. 2A and B).…”

“…1B). From titration studies we have indications that this treatment elicits a conformational change in the enzyme, which we explain in terms of an activated state of the enzyme mentioned above [13,15,21]. Subsequently, SEIRA measurements in the step-scan mode were conducted, under the same conditions, e.g.…”

“…Our ptBLM system has the further advantage that a thin gold layer to be used as an electrode can be placed on top of the ATR crystal of an IR spectrometer thus providing the means for coupled EC/FTIR studies. When the gold electrode is further provided with a nano-particulate surface, surface-enhanced infrared-absorption spectroscopy (SEIRAS) can be employed controlled by EC [13,[19][20][21]. This methodology had proved very useful in the past in our titration studies of the CcO as well as time-resolved (tr-) SEIRAS studies of CcO and the photosynthetic reaction center (RC) of R. sphaeroides.…”

“…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.…”

Vibrational spectroscopy for probing molecular-level interactions in organic films mimicking biointerfaces

“…Two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode was used to monitor the catalytic activity of Cytochrome c oxidase (CcO) immobilized on a tethered bilayer lipid membrane deposited on a gold support [173]. With potentiometric titrations of CcO, differences could be noted between the non-activated and the activated states of the enzyme, as indicated by changes in the 2D SEIRA spectra as the electric potential was varied.…”