Abstract
The bacterial reaction center (RC) is a membrane protein complex that performs photosynthetic electron transfer from a bacteriochlorophyll dimer to quinone acceptors Q(A) and Q(B). Q(B) accepts electrons from the primary quinone, Q(A), in two sequential electron transfer reactions coupled to uptake of a proton from solution. It has been suggested that water molecules along the proton uptake pathway are protonated upon quinone reduction on the basis of FTIR difference spectra [Breton, J., and Nabedryk, E. (1998) Photosynth. Res. 55, 301-307]. We examined the possible involvement of water molecules in the photoreaction processes by studying (18)O water isotope effects on FTIR difference spectra resulting from formation of Q(A)(-) and Q(B)(-). Continuum bands in D(2)O due to Q(B)(-) formation in the 2300-1800 cm(-1) region did not show spectral shifts by (18)O water in the wild-type (WT) RC, suggesting that these bands do not originate from (protonated) water. In contrast, the Q(B)(-)/Q(B) spectrum of the EQ-L212 mutant RC showed a spectral shift of a band near 2100 cm(-1) due to (18)O water substitution, consistent with protonation of internal water. FTIR shifts due to (18)O water were also observed following formation of Q(A)(-) and Q(B)(-) in the spectral region of 3700-3500 cm(-1) characteristic of weakly hydrogen bonded water. The water responsible for the Q(B)(-) change was localized near Glu-L212 by spectral shifts in mutant RCs. The weakly hydrogen bonded water perturbed by quinone reduction may play a role in stabilizing the charge-separated state.