Specific binding sites for cations in bacteriorhodopsin

The Asp-85 residue, located in the vicinity of the retinal chromophore, pla ys a key role in the function of bacteriorhodopsin (bR) as a light-driven p roton pump. In the unphotolyzed pigment the protonation of Asp-85 is respon sible for the transition from the purple form (lambda (max) = 570 nm) to th e blue form (lambda (max) = 605 nm) of bR. This transition can also be indu ced by deionization (cation removal). It was previously proposed that the c ations bind to the bR surface and raise the surface pH, or bind to a specif ic site in the protein, probably in the retinal vicinity. We have reexamine d these possibilities by evaluating the interaction between Mn2+ and a nitr oxyl radical probe covalently bound to several mutants in which protein res idues were substituted by cystein. We have found that Mn2+, which binds to the highest-affinity binding site, significantly affects the EPR spectrum o f 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 simi lar to9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn2+ bound to t hese binding sites affects spin labels attached to residues 103C and 163C l ocated in the cytoplasmic side of the protein. The results indicate specifi c binding sites for the color-controlling cations, and suggest that the bin ding sites involve negatively charged lipids located on the exterior of the bR trimer structure.