Vibrational spectroscopy favors a unique QB binding site at the proximal position in wild-type reaction centers and in the Pro-L209 --> Tyr mutant from Rhodobacter sphaeroides.

In the various X-ray structures of native reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides, two distinct main binding sites (distal and proximal) for the secondary quinone Q(B) have been described in the literature. The movement of Q(B) from its distal to proximal position has been proposed to account for the conformational gate limiting the rate of the first electron transfer from the primary quinone Q(A-) to Q(B). Recently, Q(B) was found to bind in the proximal binding site in the dark-adapted crystals of a mutant RC where Pro-L209 was changed to Tyr [Kuglstatter, A., Ermler, U., Michel, H., Baciou, L., and Fritzsch, G. (2001) Biochemistry 40, 4253-4260]. To test the structural and functional implications of the distal and proximal sites, a comparison of the FTIR vibrational properties of Q(B) in native RCs and in the Pro-L209 --> Tyr mutant was performed. Light-induced FTIR absorption changes associated with the reduction of Q(B) in Pro-L209 --> Tyr RCs reconstituted with 13C-labeled ubiquinone (Q3) at the 1 or 4 position show a highly specific IR fingerprint for the C=O and C=C modes of Q(B) upon selective labeling at C1 or C4. This IR fingerprint is very similar to that of native RCs, demonstrating that equivalent interactions occur between neutral Q(B) and the protein in native and mutant RCs. Consequently, Q(B) occupies the same binding site in all RCs. Since the FTIR data fit the description of Q(B) bonding interactions in the proximal site, it is therefore concluded that neutral Q(B) also binds to the proximal site in native functional RCs. The implication of these new results for the conformational gate of the first electron transfer to Q(B) is outlined.