Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases protonuptake by Glu-L212 upon first electron transfer to Q(B) in reaction centers from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, th e first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located similar to5 Angstrom from the cent er of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 pl ay an important role in the proton transfer to the Q(B) site [Paddock, M. L ., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. ( 2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the ampl itude of the proton uptake by Glu-L212 at pH 8 is increased in the single a nd double mutant RCs, as is evident from the larger intensity (by 35-55%) o f the carboxylic acid band at 1727 cm(-1) in the Q(B)-/Q(B) difference spec tra Of Mutant RCs, compared to that at 1728 cm(-1) in native RCs. This impl ies that the extent of ionization of Glu-L212 in the Q(B) ground state is g reater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no c hanges in the protonation state or the environment of these two residues ar e detected upon Q(B) reduction. The absence of the 1727 cm-1 signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 c m(-1) probes the protonation of Glu-L212 in native and mutant RCs.