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 ligh t-driven proton pump. In the unphotolyzed pigment the protonation of A sp-85 is responsible for the transition from the purple form (lambda(m ax) = 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 chan ges offer a unique opportunity for time resolving the titration of a p rotein 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 o f the inducing concentration jump ([H+] or [cation]), the kinetics are found to exhibit analogous multicomponent features. Analysis of the d ata over a range of cation concentrations and pH values leads to the c onclusion that the rate-determining step in the overall titration of A sp-85 is proton translocation through a specific proton channel. The m ulticomponent kinetics, extending over a wide time range (10(-2)-10(4) s), are accounted for in terms of a pH-dependent heterogeneity of pro ton channels. A model is presented in which the relative weight of fou r proton channels is determined by the state of protonation of two int eracting, channel-controlling, protein residues A(1) and A(2). These f indings bear on the mechanism of the vectorial proton translocation as sociated with the photocycle of bR.