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

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 (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR (pK(a) = 3.5 in 20 mM NaCl). The Purple double left right arrow 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 double left right arrow 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)-10(4) 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 A(1) and A(2). These findings bear on the mechanism of the vectorial proton translocation associated with the photocycle of bR.