Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, Q(A) and Q(B), play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from Q(A) to Q(B) takes place in the time range of 5-200 mu s. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which Q(B)(-) formation precedes Q(A)(-) oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between Q(A) and Q(B) has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the Q(A) to Q(B) electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, Q(A), and Q(A)(-) [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either Q(A) or Q(B) measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the Q(A)(-)/Q(A) and Q(B)(-)/Q(B) difference spectra. The calculated Q(A)(-)Q(B)/Q(A)Q(B)(-) double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported Q(A)(-)X(+)/Q(A)X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from Q(B) and Q(B)(-), respectively. Further comparison of the Q(A)(-)Q(B)/Q(A)Q(B)(-) double-difference spectra in native and DN(L210) RCs also allows the possibility that Q(B)(-) formation precedes Q(A)(-) reoxidation to be ruled out for native RCs.