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)(-) elect ron transfer in photosynthetic reaction centers from Rhodobacter sphae roides 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) Spectro sc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lent, F., Nabedr yk, E., Breton, J., Kreutz, W., & Mantele, W., (1992) Biochemistry 31, 5799-5808]. In the midinfrared region between 1695 and 1780 cm(-1), t ransient signals associated with Q(A)-Q(B) --> Q(A)Q(B)(-) electron tr ansfer were observed and characterized. The dominant transient absorba nce 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)-s ignal disappears upon H-1 --> H-2 exchange as expected for an accessib le COOH group and is absent in Glu L212 Gln mutant reaction centers, O n this basis, we propose an assignment of this signal to the COOH grou p of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions fr om ester C=O groups of bacteriopheophytins cannot be ruled out. In nat ive 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 f ast and the slow components are different, Biphasic kinetics with comp arable half-times were also observed for the Glu L212 to Gin mutant, T he simplest model to explain these results is that the fast phase repr esents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The diffe rence spectra of the slow component from native reaction centers show that the 1725 cm(-1) band corresponds to an absorbance increase and no t 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 C OO- group. With increasing pH, the amplitude of the slow component inc reases while that of the fast component decreases slightly. Both compo nents 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 assu mes that Glu L212 is partly protonated upon single-electron reduction of Q(B). On the basis of integrated extinction coefficients of COOH re sidues, a proton uptake of 0.3-0.6 H+ at pH 7 per reaction center by G lu 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 g roups in the Q(B) site.