THE BINDING-SITES OF QUINONES IN PHOTOSYNTHETIC BACTERIAL REACTION CENTERS INVESTIGATED BY LIGHT-INDUCED FTIR DIFFERENCE SPECTROSCOPY - ASSIGNMENT OF THE Q(A) VIBRATIONS IN RHODOBACTER-SPHAEROIDES USING O-18-LABELED OR C-13-LABELED UBIQUINONE AND VITAMIN-K-1

Light-induced FTIR difference spectra of the photoreduction of the primary quinone acceptor Q(A) have been obtained for Rhodobacter sphaeroides RCs reconstituted with a series of isotopically labeled quinones in order to separate the contributions of the quinone from those of the protein. The isotopic shifts observed in the Q(A)(-)/Q(A) spectra of RCs reconstituted with ubiquinones (Q(1), Q(6)) or vitamin K-1 O-18-labeled on their carbonyl oxygens and with fully C-13-labeled Q(8) lead to a clear identification of the quinone bands from both the neutral and anion forms. Double-difference spectra from pairs of Q(A)(-)/Q(A) spectra obtained from O-18/O-16 Q(6), O-18/O-16 Q(1), C-13/C-12 Q(8), (CO)-C-13-O-18/(CO)-C-12-O-16 Q(8), and O-18/O-16 vitamin K-1 allow the C=O modes of Q(A) in vivo to be identified unambiguously for the first time. For all the investigated unlabeled quinones, two carbonyl bands are demasked, at 1660 and 1628 cm(-1) for neutral ubiquinones and at 1651 and 1640 cm(-1) for vitamin K-1, while C=C bands are found at 1608 and 1588 cm(-1) for vitamin K-1 and at 1601 cm(-1) for ubiquinones. Compared with the spectra of the isolated quinones, the generally smaller width observed for the C=O and C=C bands in vivo suggests precise interactions between the quinone and the contours of the protein at a single, well-defined Q(A) site. The different frequency downshifts of the two C=O bands upon binding to the Q(A) site underscore the inequivalence of the two carbonyls in providing asymmetrical banding interactions with the protein. The comparison of the isotopic shifts observed for the various quinone C=O and C=C bands in vitro and in vivo demonstrates that the admixture of C=O and C=C characters in these modes is strongly affected by the binding of Q(A) to its anchoring site. In particular, the bands at 1628 and 1601 cm(-1) of Q(6) in vivo exhibit highly mixed C=O and C=C characters. In contrast, the methoxy groups of the ubiquinones do not appear to suffer large strain upon binding. The closeness of the Q(A)(-)/Q(A) spectra for Q(1) and Q(6) indicates that a possible role of the chain in providing the proper positioning of the quinone ring in the site for both the oxidized and reduced states of Q(A) cannot extend significantly beyond the first isoprene unit. The comparison of the frequency of the anion bands of vitamin K-1 in vitro and in vivo indicates strong bonding of the carbonyls of Q(A)(-) to the protein. The absence of a splitting of the C-O mode in vivo is indicative of symmetrical bonding of the two carbonyls after photoreduction. The near identity of the protein signals in the Q(A)(-)/Q(A) spectra for vitamin K-1 and for Q(6) in the Q(A) Site of Rb. sphaeroides shows that the protein-quinone interactions are very similar for these two quinones. Furthermore, comparison of the Q(A)(-)/Q(A) spectra of Rb. sphaeroides RCs reconstituted with vitamin K-1 and of Rhodopseudomonas viridis containing the native menaquinone-9 demonstrates that the protein contours at the Q(A) Site of the two species offer similar interactions to these two closely related naphthoquinones. Accompanying the photoreduction of Q(A), specific microconformational changes of amino acid side chains and/or of the polypeptide backbone are detected. These localized structural changes probably occur in the close vicinity of Q(A), although electrostatic effects at more distant sites should also be considered.