Interaction of proton and chloride transfer pathways in recombinant bacteriorhodopsin with chloride transport activity: implications for the chloride translocation mechanism.

When the protonated retinal Schiff base dissociates in the photocycle of the proton pump bacteriorhodopsin, asp-85 is the proton acceptor. Replacing this residue with threonine confers halorhodopsin-like properties on the protein, including chloride transport [Sasaki, J., Brown, L.S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., & Lanyi, J.K. (1995) Science 269, 73-75]. However, the electrostatic interaction between the vicinity of residue 85 and glu-204, a residue located about 10 A away near the extracellular surface, that is a part of the proton transport mechanism, should still exist. We find that in the D85T mutant glu-204 becomes protonated when chloride is added. This indicates that the binding of chloride at thr-85 must be equivalent to deprotonation of asp-85. The protonation state of glu-204 reports therefore on the presence or absence of chloride bound at thr-85. During the chloride-transport cycle of D85T, but not D85T/E204Q, fluorescein and pyranine detect the transient release of protons from the protein to the surface and the bulk. The release and the subsequent uptake of the protons occur during the rise and decay of a red-shifted photointermediate, respectively, and confirm the earlier suggestion that this state has the same role in the chloride transport as the M intermediate in the proton transport. Consistent with the red-shift of the absorption maximum, the chloride bound near the Schiff base had already moved away, presumably to be released at the cytoplasmic surface, but another chloride ion has not yet been taken up from the extracellular surface. The switch of the connectivity of the chloride binding site from the cytoplasmic to the extracellular membrane surface must occur therefore during the lifetime of this photointermediate.