Monitoring molecular events during photo-driven ubiquinone pool reduction in PufX and PufX membranes from Rhobobacter capsulatus by time-resolved FTIR difference spectroscopy.

The PufX protein is found in the photosynthetic membranes of several purple bacteria and is involved in ubiquinol-ubiquinone exchange at the Q site of the reaction center. We have studied quinone pool reduction in chromatophores from PufX and PufX strains of Rhodobacter capsulatus by time-resolved FTIR difference spectroscopy under and after continuous illumination. To our knowledge, it is the first time that quinone pool reduction has been directly followed in real time in Rba. capsulatus membranes. Thanks to the availability in the literature of IR marker bands for protein conformational changes, ubiquinone consumption, ubiquinol production, Q---QH quinhydrone complex formation, as well as for RC-bound Q and Q semiquinone species, it is possible to follow all the molecular events associated with light-induced quinone pool reduction. In Rba. capsulatus PufX  chromatophores, these events resemble the ones found in Rba. sphaeroides wild-type membranes. In PufX chromatophores the situation is different. Spectra recorded during 22.7 s of illumination showed a much smaller amount of photoreduced quinol, consistent with previous observations that PufX is required for efficient QHQ exchange at the Q site of the RC. Q consumption and QH formation are rapidly associated with Q formation, showing that the structure of the RC-LH1 complex in PufX membranes does not provide efficient access to the Q site of the RC to a large fraction of the quinone pool, evidently because the LH1 ring increases in size to impair access to the RC. The presence of a positive band at 1560 cm suggests also the transient formation, in a fraction of chromatophores or of RC-LH1 complexes, of a Q---QH quinhydrone complex. Experiments carried out after 2-flash and 10-flash sequences make it possible to estimate that the size of the quinone pool with access to the Q site in PufX membranes is ≥ 5 ubiquinone molecules per RC. The results are discussed in the framework of the current knowledge of protein organization and quinone pool reduction in bacterial photosynthetic membranes.