title: The titrations of Asp-85 and of the cation binding residues in bacteriorhodopsin are not coupled.
abstract: An outstanding problem relating to the structure and function of bacteriorhodopsin (bR), which is the only protein in the purple membrane of the photosynthetic microorganism Halobacterium salinarium, is the relation between the titration of Asp-85 and the binding/unbinding of metal cations. An extensively accepted working hypothesis has been that the two titrations are coupled, namely, protonation of Asp-85 (located in the vicinity of the retinal chromophore) and cation unbinding occur concurrently. We have carried out a series of experiments in which the purple blue equilibrium and the binding of Mn2+ ions (monitored by electron spin resonance) were followed as a function of pH for several (1-4) R = [Mn2+]/[bR] molar ratios. Data were obtained for native bR, bR mutants, artificial bR and chemically modified bR. We find that in the native pigment the two titrations are separated by more than a pKa unit [delta pKa = pKa(P/B)-pKa(Mn2+) = (4.2-2.8) = 1.4]. In the non-native systems, delta pKa values as high as 5 units, as well as negative delta pKas, are observed. We conclude that the pH titration of cation binding residues in bR is not directly related to the titration of Asp-85. This conclusion is relevant to the nature of the high affinity cation sites in bR and to their role in the photosynthetic function of the pigment.
authors.affiliation: Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: T
authors.initials: T
authors.lastname: Eliash
authors.forename: M
authors.initials: M
authors.lastname: Ottolenghi
authors.forename: M
authors.initials: M
authors.lastname: Sheves
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002412:Cations; D004578:Electron Spin Resonance Spectroscopy; D019617:Halobacterium salinarum / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009154:Mutation
chemical_list: D002412:Cations; D001224:Aspartic Acid; D008345:Manganese; D001436:Bacteriorhodopsins