Biophys J, April 1999, p. 1951-1958, Vol. 76, No. 4

Chloride Ion Binding to Bacteriorhodopsin at Low pH: An Infrared Spectroscopic Study

Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, H-6701 Hungary

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl transporter. There is strong evidence that at very low pH and in the presence of Cl, bR transports Cl ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl, Cl binds and Asp-212 also protonates. The binding of Cl and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.

Biophys J, April 1999, p. 1951-1958, Vol. 76, No. 4
© 1999 by the Biophysical Society   0006-3495/99/04/1951/08  $2.00