TITRATION KINETICS OF ASP-85 IN BACTERIORHODOPSIN - EXCLUSION OF THE RETINAL POCKET AS THE COLOR-CONTROLLING CATION-BINDING SITE

The spectrum (the purple<->blue transition) and function of the light- driven proton pump bacteriorhodopsin are determined by the state of pr otonation of the Asp-85 residue located in the vicinity of the retinal chromophore. The titration of Asp-85 is controlled by the binding/unb inding of one or two divalent metal cations (Ca2+ or Mg2+), The locati on of such metal binding site(s) is approached by studying the kinetic s of the cation-induced titration of Asp-85 using metal ions and large molecular cations, such as quaternary ammonium ions, R4N+ CR Et, Pr, a divalent 'bolaform ion' [Et3N+-(CH2)(4)-N+Et3] and the 1:3 molecular complex formed between Fe2+ and 1,10-phenanthroline (OP). The basic m ulti-component kinetic features of the titration, extending from 10(-2 ) to 10(4) s, are unaffected by the charge and size of the cation, Thi s indicates that cation binding to bR triggers the blue-->purple titra tion in a fast step, which is not rate-determining, In view of the siz e of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface, This excludes previous models, which placed the color-control ling Ca2+ ion in the retinal binding pocket. (C) 1997 Federation of Eu ropean Biochemical Societies.