Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin - Presence of specific cation-binding sites

Single and multiple mutants of extracellular Glu side chains of bacteriorho dopsin were analyzed by acid and calcium titration, differential scanning c alorimetry, and thermal difference spectrophotometry. Acid titration spectr a show that the second group protonating with Asps' is revealed in E204Q in the absence of Cl- but is not observed in the triple mutant E9Q/E194Q/E204 Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu e as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(u) of Asp(85) protonation in water and in the deionized fo rms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type b acteriorhodopsin, differential scanning calorimetry reveals a lack of the p retransition in the multiple mutants, whereas in E9Q it appears at lower te mperature and with lower cooperativity. Additionally, at neutral pH the ban d at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsi n having as ligands Glu(9), Glu(194), Glu(204), and water molecules.