Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: Binding of chainless symmetrical quinones to the QA site of Rhodobacter sphaeroides

Light-induced FTIR QA-/QA difference spectra corresponding to the photoreduction of the primary quinone acceptor QA 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 QA site. The main vibrational modes of the quinones in vivo were obtained by analysis of the isotope effects induced by 18O 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 (MQ0) 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 18O 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 QA-depleted RCs with these symmetrical quinones, the double-difference spectra calculated from the QA-/QA spectra of the 18 O-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 QA site are comparable to those previously reported for the C=O modes of quinones containing an isoprenoid (Q8, Q6, Q1) or a phytyl chain (vitamin K1) [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