Time-resolved titrations of ASP-85 in bacteriorhodopsin: The multicomponent kinetic mechanism

N. Friedman, I. Rousso, M. Sheves, X. Fu, S. Bressler, S. Druckmann, M. Ottolenghi

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (λ(max) = 570 nm) to the blue form (λ(max) = 605 nm) of bR (pK(a) = 3.5 in 20 mM NaCl). The Purple mutually implies Blue transition can also be induced by deionization (cation removal). These color changes offer a unique opportunity for time resolving the titration of a protein residue using conventional stopped-flow methodologies. We have studied the Purple mutually implies Blue equilibration kinetics in bR by exposing the system to pH and to cation jumps. Independently of the equilibration direction (Purple→Blue or Blue→Purple) and of the inducing concentration jump ([H+] or [cation]), the kinetics are found to exhibit analogous multicomponent features. Analysis of the data over a range of cation concentrations and pH values leads to the conclusion that the rate-determining step in the overall titration of Asp-85 is proton translocation through a specific proton channel. The multicomponent kinetics, extending over a wide time range (10-2 -104 s), are accounted for in terms of a pH-dependent heterogeneity of proton channels. A model is presented in which the relative weight of four proton channels is determined by the state of protonation of two interacting, channel-controlling, protein residues A1 and A2. These findings bear on the mechanism of the vectorial proton translocation associated with the photocycle of bR.

Original languageEnglish
Pages (from-to)11369-11380
Number of pages12
JournalBiochemistry
Volume36
Issue number38
DOIs
StatePublished - 23 Sep 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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