Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin.
The light-driven chloride pump halorhodopsin (HR), a halobacterial retinal protein, was studied by comparing wild type with specific mutants. Changes of conserved arginine and threonine residues in the transmembrane regions could be classified in two categories: in the extracellular half of the molecule, mutations influence anion uptake and binding. R108 mutations abolish all anion effects previously attributed to two distinct binding sites and change the characteristic photochemistry. Neutral residues at position 108 completely inactivate the pump. T111 increases the affinity of this anion binding site without being essentially important. In the photochemical cycles of the mutants T111V and Q105E, a red-shifted absorbing intermediate is enriched indicating retarded anion uptake. On the cytoplasmic side, mutations do not change anion binding properties of the unphotolyzed protein, but slow down anion release thereby reducing the chloride transport activity and the photocycling rate. The lowest activity is found for T203V, while R200 mutations have weaker effects. Thus, in the symmetrically arranged pairs R108/T111 and T203/R200, threonine and arginine play different roles, reflecting high affinity anion uptake by the former and effective anion release catalyzed by the latter residues. A model for the anion transport mechanism in HR is suggested comprising the specific functions of channel-lining residues.
Rüdiger
M
M
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Oesterhelt
D
D
D000838:Anions / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D018118:Chloride Channels / Q000378:metabolism*; D015870:Gene Expression; D025602:Halorhodopsins; D008027:Light; D013912:Threonine / Q000378:metabolism*
D000838:Anions; D018118:Chloride Channels; D025602:Halorhodopsins; D013912:Threonine; D001436:Bacteriorhodopsins; D001120:Arginine