PROTONATION OF GLU-L212 FOLLOWING Q(B)(-) FORMATION IN THE PHOTOSYNTHETIC REACTION-CENTER OF RHODOBACTER-SPHAEROIDES - EVIDENCE FROM TIME-RESOLVED INFRARED-SPECTROSCOPY

The protonation events that occur upon Q(A)-Q(B) --> Q(A)Q(B)(-) electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mantele, W., Hienerwadel, R., Lent, F., Riedel, E., J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lent, F., Nabedryk, E., Breton, J., Kreutz, W., & Mantele, W., (1992) Biochemistry 31, 5799-5808]. In the midinfrared region between 1695 and 1780 cm(-1), transient signals associated with Q(A)-Q(B) --> Q(A)Q(B)(-) electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm(-1) and two negative signals at 1716 and at 1698 cm(-1). The 1725 cm(-1)-signal disappears upon H-1 --> H-2 exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers, On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C=O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-l exhibits a fast kinetic component of t(1/2) = 0.18 ms (25% of the total amplitude) and a slow one of t(1/2) = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm(-1) revealed that the spectral distributions of the fast and the slow components are different, Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gin mutant, The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm(-1) band corresponds to an absorbance increase and not to a shift of an existing band, The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm(-1) signal varies distinctly with pH as expected fdr protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly. Both components slow down with increasing pH reaching half-times of 0.3 and 2.5 ms, respectively, at pH 10. These results can be explained if one assumes that Glu L212 is partly protonated upon single-electron reduction of Q(B). On the basis of integrated extinction coefficients of COOH residues, a proton uptake of 0.3-0.6 H+ at pH 7 per reaction center by Glu L212 is proposed. This proton uptake shows little variation from pH 5 to pH 9 but increases above pH 9. This behavior cannot be explained by the titration of a single isolated titrating residue. However, it can be interpreted by the interaction of Glu L212 with other charged groups in the Q(B) site.