Specific binding sites for cations in bacteriorhodopsin.
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 (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.
Eliash
T
T
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Weiner
L
L
Ottolenghi
M
M
Sheves
M
M
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013113:Spin Labels
D002413:Cations, Divalent; D013113:Spin Labels; D008345:Manganese; D001436:Bacteriorhodopsins