The titrations of Asp-85 and of the cation binding residues in bacteriorhodopsin are not coupled

An outstanding problem relating to the structure and function of bacteriorh odopsin (bR), which is the only protein in the purple membrane of the photo synthetic microorganism Halobacterium salinarium, is the relation between t he titration of Asp-85 and the binding/unbinding of metal cations, An exten sively accepted working hypothesis has been that the two titrations are cou pled, 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 a blue equilibrium and the bind ing 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 obta ined for native bR, bR mutants, artificial bR and chemically modified bR, W e find that in the native pigment the two titrations are separated by more than a pK(a) unit [Delta pK(a) = pK(a)(P/B)-pK(a)(Mn2+) = (4.2-2.8) = 1.4], In the non-native systems, Delta pK(a) values as high as 5 units, as well as negative Delta pK(a)s, are observed. We conclude that the pH titration o f 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 cat ion sites in bR and to their role in the photosynthetic function of the pig ment. (C) 1999 Federation of European Biochemical Societies.