Light-induced QA-/QA FTIR difference spectra of the photoreduction of the primary quinone (QA) have been obtained for Rhodobacter sphaeroides reaction centers (RCs) reconstituted with ubiquinone (Q3) labeled selectively with 13C 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 QA 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 QA-/QA spectra of RCs reconstituted with either 13C1- or 13C4-labeled Q3 and with unlabeled Q3 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 QA. For the unlabeled QA, 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., & Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. The 1660-cm-1 band is unaffected upon selective labeling at C4 but shifts to approximately 1623 cm-1 upon 13C1 labeling, demonstrating that this band arises from the C1 carbonyl, proximal to the isoprenoid chain. The band at 1628 cm-1 shifts by 11 and 16 cm-1 upon 13C1 and 13C4 labeling, respectively, and is assigned to a C = C mode coupled to both carbonyls.(ABSTRACT TRUNCATED AT 250 WORDS)

The photoreduction of the secondary quinone acceptor QB 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 18O on both carbonyls and 13C either uniformly or selectively at the 1- or the 4-position, i.e., on either one of the two carbonyls. The QB-/QB 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 QB and QB-. The vibrational modes of the quinone in the QB site are compared to those of ubiquinone in vitro, leading to band assignments for the C = O and C = C vibrations of the neutral QB 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 QB site. In contrast, the C = C vibrations are not equivalent for selective labeling at C1 or at C4, although they both contribute to the approximately 1617-cm-1 band in the QB-/QB spectra of the two species. Compared to the vibrations of isolated ubiquinone, the C = C mode of QB does not involve displacement of the C4 carbon atom, while the motion of C1 is not hindered. Further analysis of the 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 C4 carbonyl. The FTIR observations provide compelling evidence for almost identical conformation and identical interactions of the ubiquinone in the QB 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 QB binding. In the semiquinone state, the bonding interactions of the C***O groups are also symmetrical and the C***C are inequivalent at C1 and C4. However, the interactions are almost the same in the RCs of both species.

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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 18O-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., & 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 K1 or with DMNQ shows that the phytyl chain of vitamin K1 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 MQ0 or with Q6 (or Q1) exhibited significant differences in the relative amplitudes of the bands assigned to the mixed C = O and C = C modes of the neutral quinones.(ABSTRACT TRUNCATED AT 400 WORDS)

On the basis of semiempirical calculations, the present study proposes a comprehensive interpretation of the crystallographic, vibrational, and electrochemical data on methoxy-substituted quinones, and in particular for ubiquinones, in terms of the orientation of the methoxy groups relative to the quinone ring plane. “Hindered” and “free” methoxy groups are considered depending on the presence or absence on the quinone ring of a bulky group in ortho position of the considered methoxy group, respectively. The free methoxy groups have their O−CH3 bond in the quinone ring plane while the hindered methoxy groups cannot adopt this conformation and have their methyl group tilted out of the quinone ring plane. The electron donation of the methoxy is dependent on the orientation of the O−CH3 bond and is maximum for a free methoxy group. This effect is revealed by the analysis of both electrochemical and IR data. An assignment of the ν(CO) modes of the quinones bearing such groups is proposed. From electrochemical data in literature, a new coefficient σpara, used in the Hammett equation, is determined for a hindered methoxy group (σpara = −0.07 compared to −0.27 for a free methoxy group). In the specific and biologically important case of the bulky group being another methoxy group, such as in ubiquinones (2,3-dimethoxy-substituted 1,4-benzoquinones), two types of conformation have to be considered. In the first type (conformer A), one methoxy adopts the conformation of a free methoxy group and the second that of a hindered methoxy group. In the second type (conformer B), both methoxy groups adopt the conformation of a hindered methoxy group. Both conformers appear equiprobable within the precision of our semiempirical calculations and a low rotational barrier, compared to k B T at room temperature, is found between them. Only conformers A are encountered in crystals. Using specific 13C labeling, IR data show that conformers A are mostly encountered at room temperature in solution while a mixture of both conformers is present at low temperature. On the other hand, electrochemical data on these quinones are best interpreted as the reduction of conformers B. This is explained by the higher electron affinity of conformers B compared to conformers A and by the low rotational barrier between the two conformers. Taking into account IR data of ubiquinone in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides, the 70 mV difference found in the redox potential of ubiquinone in the two quinone binding sites can be explained by a difference of orientation of the methoxy groups imposed by the protein. By selecting a different orientation of the methoxy groups in the two sites, the protein would thus tune the redox potential of the quinone present in each site.

In the various X-ray structures of native reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides, two distinct main binding sites (distal and proximal) for the secondary quinone Q(B) have been described in the literature. The movement of Q(B) from its distal to proximal position has been proposed to account for the conformational gate limiting the rate of the first electron transfer from the primary quinone Q(A-) to Q(B). Recently, Q(B) was found to bind in the proximal binding site in the dark-adapted crystals of a mutant RC where Pro-L209 was changed to Tyr [Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G. (2001) Biochemistry 40, 4253-4260]. To test the structural and functional implications of the distal and proximal sites, a comparison of the FTIR vibrational properties of Q(B) in native RCs and in the Pro-L209 --> Tyr mutant was performed. Light-induced FTIR absorption changes associated with the reduction of Q(B) in Pro-L209 --> Tyr RCs reconstituted with 13C-labeled ubiquinone (Q3) at the 1 or 4 position show a highly specific IR fingerprint for the C=O and C=C modes of Q(B) upon selective labeling at C1 or C4. This IR fingerprint is very similar to that of native RCs, demonstrating that equivalent interactions occur between neutral Q(B) and the protein in native and mutant RCs. Consequently, Q(B) occupies the same binding site in all RCs. Since the FTIR data fit the description of Q(B) bonding interactions in the proximal site, it is therefore concluded that neutral Q(B) also binds to the proximal site in native functional RCs. The implication of these new results for the conformational gate of the first electron transfer to Q(B) is outlined.

Selectively 13 C-labeled ubiquinone anion radicals in protic and aprotic solvents are investigated by EPR and ENDOR spectroscopy, yielding information about the effect of hydrogen bonds on the electronic g-tensor and the carbonyl carbon 13 C-hf tensors. Formation of the hydrogen bonds alter the g-tensor significantly to lower values and increases the A. component of the 13 C-hf tensor. Both effects can be explained by electrostatic interactions between the positively charged hydrogen and the electrons at the carbonyl oxygen leading to a redistribution of charge and rr-spin density. Two different hydrogen bonds were obtained for UQo' which are in agreement with the results of DFT (density functional theory) calculations.

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