PATHWAY OF PROTON-TRANSFER IN BACTERIAL REACTION CENTERS - ROLE OF ASPARTATE-L213 IN PROTON TRANSFERS ASSOCIATED WITH REDUCTION OF QUINONE TO DIHYDROQUINONE

The role of Asp-L213 in proton transfer to reduced quinone Q(B) in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [k(AB)(2)] and proton uptake rates associated with the second electron reduction of Q(B) (Q(A)-Q(B)- + 2H+ --> Q(A)Q(B)H2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that k(AB)(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced k(AB)(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pK(a) (pK(a) greater-than-or-equal-to 7) than Asp-L213 (pKa less-than-or-equal-to 4), as indicated by the slower rate of charge recombination (D+Q(A)Q(B)- --> DQ(A)Q(B)) in the mutant RCs. The importance of the pK(a) and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried Q(B) molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near Q(B).