BINDING-SITES OF QUINONES IN PHOTOSYNTHETIC BACTERIAL REACTION CENTERS INVESTIGATED BY LIGHT-INDUCED FTIR DIFFERENCE SPECTROSCOPY - SYMMETRY OF THE CARBONYL INTERACTIONS AND CLOSE EQUIVALENCE OF THE Q(B) VIBRATIONS IN RHODOBACTER-SPHAEROIDES AND RHODOPSEUDOMONAS-VIRIDIS PROBED BY ISOTOPE LABELING

The photoreduction of the secondary quinone acceptor Q(B) in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis has been investigated by light-induced FTIR difference spectroscopy of RCs reconstituted with several isotopically labeled ubiquinones. The labels used were O-18 On both carbonyls and C-13 either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The Q(B)(-)/Q(B) spectra of RCs reconstituted with the isotopically labeled and unlabeled quinones as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for a number of bands attributed to vibrations of Q(B) and Q(B)(-) The vibrational modes of the quinone in the Q(B) Site are compared to those of ubiquinone in vitro, leading to band assignments for the C=O and C=C vibrations of the neutral Q(B) and for the C - O and C - C of the semiquinone. The C=O frequency of each of the carbonyls of the unlabeled quinone is revealed at 1641 cm(-1) for both species, This demonstrates symmetrical and weak hydrogen bonding of the two C=O groups to the protein at the Q(B) site, In contrast, the C=C vibrations are not equivalent for selective labeling at C-1 or at C-4, although they both contribute to the similar to 1617-cm(-1) band in the Q(B)(-)/Q(B) spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C=C mode of Q(B) does not involve displacement of the C-4 carbon atom, while the motion of C-1 is not hindered. Further analysis of the spectra suggests that the protein at the binding site imposes a specific constraint on the methoxy and/or the methyl group proximal to the C-4 carbonyl. The FTIR observations provide compelling evidence for almost identical conformation and identical interactions of the ubiquinone in the Q(B) binding site of Rb, sphaeroides and Rp. viridis in contrast to the X-ray structures, which yield different descriptions for the hydrogen-bonding pattern of Q(B) binding. In the semiquinone state, the bonding interactions of the C - O groups are also symmetrical and the C - C are inequivalent at C-1 and C-4. However, the interactions are almost the same in the RCs of both species.