BINDING AND INTERACTION OF THE PRIMARY AND THE SECONDARY-ELECTRON ACCEPTOR QUINONES IN BACTERIAL PHOTOSYNTHESIS - AN INFRARED SPECTROELECTROCHEMICAL STUDY OF RHODOBACTER-SPHAEROIDES REACTION CENTERS

The vibrational modes of the primary and secondary electron acceptors Q(A) and Q(B), their semiquinone anions and their respective protein environment in Rhodobacter sphaeroides reaction centers have been characterized using combinations of electrochemically-induced and light-induced Fourier-transform infrared (IR) difference spectroscopy. Q(A)-/Q(A) and Q(B)-/Q(B) IR difference spectra without contributions of other cofactors were generated by three different methods: (1) electrochemically, by reduction of Q(A) to Q(A)-; (2) photochemically, with flash-induced formation of P+Q(A)- or P+Q(B)-, rereduction of P+ by cytochrome (cyt) c2, and electrochemical rereduction of cyt c2; (3) photochemically and electrochemically, by subtraction of redox-induced P+/P difference spectra from light-induced P+Q(A)-/PQ(A) and P+Q(B)-/PQ(B) difference spectra. Although Q(A)- was generated by completely different methods, and in one case (3) a charge-separated state is involved, almost identical Q(A)-/Q(A) and only slightly different Q(B)-/Q(B) difference spectra have been obtained. Bands at 1630 cm-1 and 1640 cm-1 are proposed as candidates for the C=O modes of Q(A) and Q(B), respectively. The C-O modes of Q(A)- and Q(B)- are assigned to bands at 1462 cm-1 and 1478-88 cm-1, respectively. Difference bands at 1668 cm-1 and 1652 cm-1 in Q(A)-/Q(A) difference spectra are more likely to arise from amide-I modes or side chain vibrations of amino acids to which Q(A) is hydrogen-bonded. A number of difference bands between 1520 cm-1 and 1560 cm-1 possibly arise from amide-II vibrations and aromatic amino-acid side chain residues in the vicinity Of Q(A) and Q(B). A differential feature at 1734 cm-1/1726 cm-1 in Q(A-)/Q(A) difference spectra probably arises from changes in the protonation state or environment of distant carboxyl groups. An alternative explanation in terms of changes in the environment of the 10a ester C=O group of bacteriopheophytin L, however, cannot be excluded. Bands between 1724 cm-1 and 1740 cm-1 in Q(B)-/Q(B) difference spectra are tentatively assigned to a protonation of ASP L213 and/or a change in the environment of GLU L212, both being located in the vicinity Of Q(B) and involved in the proton transfer to Q(B) (Okamura, M.Y. and Feher, G. (1992) Annu. Rev. Biochem. 61, 861-896).