BINDING-SITES OF QUINONES IN PHOTOSYNTHETIC BACTERIAL REACTION CENTERS INVESTIGATED BY LIGHT-INDUCED FTIR DIFFERENCE SPECTROSCOPY - ASSIGNMENT OF THE INTERACTIONS OF EACH CARBONYL OF Q(A) IN RHODOBACTER-SPHAEROIDES USING SITE-SPECIFIC C-13-LABELED UBIQUINONE

Light-induced Q(A)-/Q(A) FTIR difference spectra of the photoreduction of the primary quinone (Q(A)) have been obtained for Rhodobacter sphaeroides reaction centers (RCs) reconstituted with ubiquinone (Q(3)) labeled selectively with C-13 at the 1- or 4-position of the quinone ring, i.e., on either of the two carbonyls. The vibrational modes of the quinone in the Q(A) site are compared to those in vitro. IR absorption spectra of films of the labeled quinones show that the two carbonyls contribute equally to the split C=O band at 1663-1650 cm(-1). This splitting is assigned to the two different geometries of the methoxy group nearest to each carbonyl. The Q(A)(-)/Q(A) Spectra of RCs reconstituted With either C-13(1-) or C-13(4)-labeled Q(3) and with unlabeled Q(3) as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for the bands assigned to C=O and C=C vibrations of the neutral Q(A). For the unlabeled Q(A), these bands correspond to the bands at 1660, 1628, and 1601 cm(-1) previously detected upon nonselective isotopic labeling [Breton, J., Burie, J.-R., Berthomieu, C., Berger, G., and Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. The 1660-cm(-1) band is unaffected upon selective labeling at C-4 but shifts to similar to 1623 cm(-1) upon C-13(1) labeling, demonstrating that this band arises from the C-1 carbonyl, proximal to the isoprenoid chain. The band at 1628 cm(-1) shifts by 11 and 16 cm(-1) upon C-13(1) and C-13(4) labeling, respectively, and is assigned to a C=C mode coupled to both carbonyls. The band at 1601 cm(-1), which shifts to 1578 cm(-1) upon labeling at C-4 and is unaffected by labeling at C-1, corresponds to cm the C-4 carbonyl, proximal to the methyl group. Additional O-18 labeling on the carbonyls of the selectively labeled Q(3) confirms these assignments. The large difference in the IR frequencies of the two C=O modes of QA underscores the inequivalent interactions of the two carbonyls with the protein. The extreme downshift of the frequency of the C-4=O group in the QA binding site compared to that in vitro, together with the strongly mixed C=C and C=O characters of the 1628- and 1601-cm(-1) modes, points to a strong perturbation of the C-4 carbonyl. The large downshift of C-4=O probably is caused by hydrogen bonding with the imidazole ring of His M219, which is located close to this carbonyl group in the most recent X-ray structure of the RC [Ermler, U., Fritzsch, G., Buchanan, S., and Michel, H. (1992) in Research in Photosynthesis (Murata, N., Ed.) Vol. I, pp 341-347, Kluwer Academic Publishers, Dordrecht]. This hydrogen bond would be Stabilized by the linkage: of N epsilon 2 of the imidazole to the non-heme Fe2+,]However, the FTIR data do not support the suggestion based on the X-ray structure; that the C-1 carbonyl forms a hydrogen bond with the peptide NH of Ala M260; C-1=O appears not to interact significantly with the protein. In contrast to the uncoupled behavior of the C=O modes of the neutral QA, the two C - O modes of the semiquinone in the Q(A)(-)/Q(A) spectra are coupled. They are also coupled to the C --C modes and are both strongly downshifted compared to the C - O mode of the semiquinone in vitro. Double-difference spectra calculated from the P(+)Q(A)(-)/PQ(A) spectra at 100 K are very similar to the Q(A)(-)/Q(A) double-difference spectra at 278 K, showing that the center of mass of QA does not move appreciably upon reduction.