TIME-RESOLVED INFRARED-SPECTROSCOPY OF ELECTRON-TRANSFER IN BACTERIAL PHOTOSYNTHETIC REACTION CENTERS - DYNAMICS OF BINDING AND INTERACTION UPON Q(A) AND Q(B) REDUCTION

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [Mantele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5-mu-s, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor Q(A) and its environment upon formation of Q(A)-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [Mantele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; Mantele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (I 988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., Mantele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., Mantele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120-mu-s were observed and attributed to Q(A) --> Q(B) electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes Of Q(A) and Q(B) and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C=O and C=C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-mu-s signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site. Since these conformational changes display kinetic parameters identical to those of the quinone modes, it appears that most of the protein relaxation processes closely follow the quinone reduction. A signal of 1617 cm-1, however, manifests itself with an additional absorbance decrease component exhibiting a half-time of slightly above 1 ms. This component, which is also present with a less pronounced contribution at other frequencies, is tentatively assigned to the antisymmetric C-O vibration of a carboxylate group disappearing upon formation of the carboxylic group, presumably that of Asp L213, and attributed to a (partial) protonation upon a pK shift following a single electron reduction Of Q(B).