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 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)
Breton
J
J
Section de Bioénergétique, CEA-Saclay, Gif-sur-Yvette, France.
Burie
J R
JR
Boullais
C
C
Berger
G
G
Nabedryk
E
E
D001665:Binding Sites; D006860:Hydrogen Bonding; D009285:Naphthoquinones / Q000737:chemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000737:chemistry*; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D010837:Vitamin K 1 / Q000737:chemistry
D009285:Naphthoquinones; D045322:Photosynthetic Reaction Center Complex Proteins; D011809:Quinones; C063002:2,3-dimethoxy-1,4-naphthoquinone; D010837:Vitamin K 1