Proton and electron transfer to the secondary quinone (Q(B)) in bacterial reaction centers: The effect of changing the electrostatics in the vicinity of Q(B) by interchanging Asp and Glu at the L212 and L213 sites

The bacterial reaction center (RC) plays a central role in photosynthetic energy conversion by facilitating the light induced double reduction and protonation of a bound quinone molecule, QB. TWO carboxylic acid residues, Asp-L213 and Glu-L212, located near Q(B), were previously shown to be important for proton transfer to Q(B). In this work, the ability of Glu to substitute for Asp at L213 and Asp to substitute for Glu at L212 was tested by site-directed mutagenesis. Both single mutants end a double mutant in which Asp and Glu were exchanged between the two sites were constructed, The electron transfer rate constants k(BD) (D(+)Q(A)Q(B)(-)-->DQ(A)Q(B)), and k(AB)((2)) (DQ(A)-Q(B)(-) + H+ --> DQ(A)(Q(B)H)(-)), that are known to be sensitive to the energy of the Q(B)(-) state, were found to be altered by Asp/Glu substitutions. Both rates were fastest (similar to 10-fold) in RCs with Asp at both sites, slowest with Glu at both sites (similar to 50-fold) and relatively unchanged by the caboxylic acid exchange. These changes could be explained if Asp was predominantly ionized and Glu was predominantly protonated at both sites (pH 7.5), The charge recombination k(BD) suggests an observed similar to 5 pK(a), unit difference of Glu over Asp, Modeling of k(BD) by strong electrostatic interactions (similar to 3-4 pK(a) units) among negatively charged acids and Q(B)(-) indicated a lower intrinsic pK(a) for Asp compared to Glu at either site of similar to 2-3 units. The mechanism of the k(AB)(2) reaction was determined to be the same in all mutant RCs as for native RCs, A quantitative explanation of the effect of the electrostatic environment on k(AB)((2)) was Obtained using the two-step model proposed for native RCs [Graige. M. S., Paddock, hi. L., Bruce, J. M., Feher, G., & Okamura, M. Y. (1996) J. Am. Chem Soc. 118, 9005-9016] which involves fast protonation of the semiquinone followed by rate-limiting electron transfer. Using simple models for the quinone/quinol conversion rate, it is shown that the optimal electrostatic potential for the Q(B) Site is close to that found in native RCs.