Binding and interaction of the primary and the secondary electron acceptor quinones in bacterial photosynthesis: An infrared spectroelectrochemical study of Rhodobacter sphaeroides reaction centers

Abstract

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) c 2, and electrochemical rereduction of cyt c 2; (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).