Time-resolved FTIR difference spectroscopy for the study of quinones in the A1 binding site in photosystem I: Identification of neutral state quinone bands

Abstract

Infrared absorption bands associated with the neutral state of quinones in the A1 binding site in photosystem I (PSI) have been difficult to identify in the past. This problem is addressed here, where time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study PSI with six different quinones incorporated into the A1 binding site. (P700+A1− – P700A1) and (A1− – A1) FTIR difference spectra (DS) were obtained for PSI with the different quinones incorporated, and several double-difference spectra (DDS) were constructed from the DS. From analysis of the DS and DDS, in combination with density functional theory based vibrational frequency calculations of the quinones, the neutral state bands of the incorporated quinones are identified and assigned. For neutral PhQ in the A1 binding site, infrared absorption bands were identified near 1665 and 1635 cm−1, that are due to the C1O and C4O stretching vibrations of the incorporated PhQ, respectively. These assignments indicate a 30 cm−1 separation between the C1O and C4O modes, considerably less than the ~80 cm−1 found for similar modes of PhQ−. The C4O mode downshifts due to hydrogen bonding, so the suggestion is that hydrogen bonding is weaker for the neutral state compared to the anion state, indicating radical-induced proton dynamics associated with the quinone in the A1 binding site in PSI.