Light-induced FTIR Q(A)-/Q(A) difference spectra corresponding to the photoreduction of the primary quinone acceptor Q(A) have been obtained for Rhodobacter sphaeroides RCs reconstituted with chainless symmetrical quinones in order to study the influence of the side chain and of molecular asymmetry on the binding of natural quinones to the Q(A) site. The main vibrational modes of the quinones in vivo were obtained by analysis of the isotope effects induced by O-18 substitution on the carbonyls and by comparison with the IR absorption spectra of the isolated quinones. For isolated 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ(0)), 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-1,4-naphthoquinone (DMNQ), the IR spectra together with mass spectroscopy data of partially O-18 labeled quinones show that the labeling of one carbonyl leads to only a minor shift of the vibrational frequency of the opposite carbonyl. This observation demonstrates an essentially uncoupled behavior of the two C=O groups. Upon reconstitution of Q(A)-depleted RCs with these symmetrical quinones, the double-difference spectra calculated from the Q(A)-/Q(A) spectra of the O-18-labeled and unlabeled quinones reveal a splitting of the quinone C=O modes. This splitting and the frequency downshift of the C=O vibrations upon binding to the Q(A) Site are comparable to those previously reported for the C=O modes of quinones containing an isoprenoid (Q(8), Q(6), Q(1)) or a phytyl chain (vitamin K-1) [Breton, J., Burie, J.-R., Berthomieu, C., Berger, G., and Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. This observation demonstrates that the replacement of the side chain by a methyl group does not impair the asymmetrical bonding interactions of the two quinone carbonyls with the protein. This asymmetry is traceable to the two distinct amino acid residues which have been proposed, on the basis of X-ray structural studies, to form hydrogen bonds with the carbonyls of the quinone. The close analogy between the double-difference spectra calculated for RCs reconstituted either with vitamin K-1 or with DMNQ shows that the phytyl chain of vitamin K-1 imparts no specific constraint on the geometry of the menaquinone head group in its binding site for both the neutral and the semiquinone state. In contrast, the double-difference spectra calculated for RCs reconstituted either with MQ(0) or with Q(6) (Or Q(1)) exhibit significant differences in the relative amplitudes of the bands assigned to the mixed C=O and C=C modes of the neutral quinones. This observation shows that, within the first isoprene unit of the side chain, at least one of the carbon atoms beyond the one proximal to the quinone ring must play an important role in allowing the native geometrical anchoring of the ubiquinones to the Q(A) site. For all of the investigated quinones, the protein response upon Q(A) reduction is highly conserved and the unsplit C-O anion mode indicates symmetrical bonding interactions of the semiquinone with the protein.
