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 (lambdamax = 570 nm) to the blue form (lambdamax = 605 nm) of bR (pKa = 3.5 in 20 mM NaCl). The Purple <=> 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 <=> 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 A1 and A2. These findings bear on the mechanism of the vectorial proton translocation associated with the photocycle of bR.
Friedman
N
N
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Rousso
I
I
Sheves
M
M
Fu
X
X
Bressler
S
S
Druckmann
S
S
Ottolenghi
M
M
D001224:Aspartic Acid / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D002412:Cations; D003116:Color; D003624:Darkness; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000737:chemistry* / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D011522:Protons; D013053:Spectrophotometry; D017186:Titrimetry
D002412:Cations; D007473:Ion Channels; D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins