Time-resolved step-scan FTIR difference spectroscopy for the study of photosystem I with different benzoquinones incorporated into the A1 binding site

Abstract Time-resolved step-scan FTIR difference spectroscopy has been used to study photosystem I (PSI) with plastoquinone-9 (PQ) and two other benzoquinones (2,6-dimethyl-1,4-benzoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone) incorporated into the A 1 binding site. By subtracting a (P700 + A 1 −  − P700A 1 ) FTIR difference spectrum for PSI with the native phylloquinone (PhQ) incorporated from corresponding spectra for PSI with different benzoquinones (BQs) incorporated, FTIR double difference spectra are produced, that display bands associated with vibrational modes of the quinones, without interference from features associated with protein vibrational modes. Molecular models for BQs involved in asymmetric hydrogen bonding were constructed and used in vibrational mode frequency calculations. The calculated data were used to aid in the interpretation and assignment of bands in the experimental spectra. We show that the calculations capture the general trends found in the experimental spectra. By comparing four different FTIR double difference spectra we are able to verify unambiguously bands associated with phyllosemiquinone in PSI at 1495 and 1415 cm −1 . We also resolve a previously unrecognized band of phyllosemiquinone at 1476 cm −1 that calculations suggest is due in part to a C 4 − O stretching mode. For PSI with PQ incorporated, calculations and experiment taken together indicate that the C 1 − O and C 4 − O vibrational modes of the semiquinone give rise to bands at 1487 and 1444 cm −1 , respectively. This is very distinct compared to PSI with PhQ incorporated. From the calculated and experimental spectra, we show that it is possible to distinguish between two possible orientations of PQ in the A 1 protein binding site.