CHLORIDE AND PROTON TRANSPORT IN BACTERIORHODOPSIN MUTANT D85T - DIFFERENT MODES OF ION TRANSLOCATION IN A RETINAL PROTEIN

Replacement of aspartate 85 (D85) in bacteriorhodopsin (BR) by threoni ne but not be asparagine creates at pH < 7 an anion-binding site in th e molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maxim um by maximally 57 nm. Connected to this color change is a change in t he absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 n m in the absence of transported anions to one at 630 nm in their prese nce. Increasing anion concentration cause decreasing decay times of th is intermediate. At physiological pH, BR-D85T but not BR-D85N transpor ts chloride ions inward in green light, protons outward in blue or gre en light and protons inward in white Light (directions refer to the in tact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride t ransport and introduction of anion-dependent spectroscopic properties at physiological pH. The different transport modes are explained with the help of the recently proposed IST model, which states that after l ight-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps o ccur independently. Ln order to complete the cyclic event, the accessi bility change, ion transfer and isomerization state have to be reverse d. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport mod es described here for the same molecule can satisfactorily be describe d in the framework of this general concept. (C) 1997 Academic Press Li mited.