Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82 → Ala and Asp-85 → Glu: The blue form is inactive in proton translocation

Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85 → Glu and Arg-82 → Ala mutants exist in a pH-dependent equilibrium between purple (λ_max ≈ 550/540 nm) and blue (λ_max ≈ 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85 → Asn mutant displays a blue chromophore (λ_max ≈ 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212 → Asn mutant is purple (λ_max ≈ 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pK_a of the purple-to-blue transition is directly influenced by the pK_a of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.