Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3- [(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate miceiles at pH 7.3. In the Asp-85 -Ala and Asp-85 --Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85 -* Ala and Asp-85 -* Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but dearly precedes the rise of M. The amplitude of the early (<0.2 ps) reversed photovoltage component in the Asp-85 -* Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212 -* Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 Ls, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82 -Gln mutant, no net transient proton release was observed, whereas, in the Arg-82 Ala mutant,, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85 --Glu, Arg-82-Ala, and Arg-82 --Gin mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85 -Ala, Asp-85 --Asn, and Asp-212 --Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.Site-directed mutagenesis has shown and Asp-212 to be essential for proton translocation by bacteriorhodopsin (bR) (1). The very low activity in mutants Asp-96 -+ Ala (D96A) and Asp-96 -* Asn (D96N) at pH 7 is due to a markedly slowed-down decay of the photocycle intermediate M and the associated charge movement (2-5). was concluded to be the internal proton donor for the reprotonation of the Schiff base (SB) leading to the decay of M (3, 4). Fourier-transform infrared spectroscopy has revealed the protonation states of Asp-85, Asp-96, Asp-115, and Asp-212 in the K, L, and M intermediates and provided clues to the time course of proton transfer (6, 7). The data suggest that Asp-85 and Asp-212 are deprotonated in the ground state and become protonated in the L -* M transition (6). A...
