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 threonine but not be asparagine creates at pH<7 an anion-binding site in the molecular similar to that in chloride pump halorhodopsin. Binding of various anions to BR-D85T causes a blue shift of the absorption maximum by maximally 57 nm. Connected to this color change is a change in the absorption difference spectrum of the initial state and the longest living photo intermediate from a positive difference maximum at 460 nm in the absence of transported anions to one at 630 nm in their presence. Increasing anion concentration cause decreasing decay times of this intermediate. At physiological pH, BR-D85T but not BR-D85N transports chloride ions inward in green light, protons outward in blue or green light and protons inward in white light (directions refer to the intact cell). The proton movements are observable also in BR-D85N. Thus, creation of an anion-binding site in BR is responsible for chloride transport 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 light-induced isomerization of the retinal an ion transfer step and an accessibility change of the active site follow. The latter two steps occur independently. In order to complete the cyclic event, the accessibility change, ion transfer and isomerization state have to be reversed. The relative rates of accessibility changes and ion transfer steps define ultimately the vectoriality of ion transfers. All transport modes described here for the same molecule can satisfactorily be described in the framework of this general concept.
Tittor
J
J
Max-Planck-Institut für Biochemie, Martinsried, D82152, Germany.
Haupts
U
U
Haupts
C
C
Oesterhelt
D
D
Becker
A
A
Bamberg
E
E
D000838:Anions; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D006217:Halobacterium; D017136:Ion Transport; D008027:Light; D017354:Point Mutation; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013057:Spectrum Analysis
D000838:Anions; D002712:Chlorides; D011522:Protons; D001436:Bacteriorhodopsins; D012172:Retinaldehyde