In the light-driven proton pump bacteriorhodopsin, proton transfer from the retinal Schiff base to aspartate-85 is the crucial reaction of the transport cycle. In halorhodopsin, a light-driven chloride ion pump, the equivalent of residue 85 is threonine. When aspartate-85 was replaced with threonine, the mutated bacteriorhodopsin became a chloride ion pump when expressed in Halobacterium salinarium and, like halorhodopsin, actively transported chloride ions in the direction opposite from the proton pump. Chloride was bound to it, as revealed by large shifts of the absorption maximum of the chromophore, and its photointermediates included a red-shifted state in the millisecond time domain, with its amplitude and decay rate dependent on chloride concentration. Bacteriorhodopsin and halorhodopsin thus share a common transport mechanism, and the interaction of residue 85 with the retinal Schiff base determines the ionic specificity.

The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide binds near the Schiff base. In D85T this occurs near neutral pH, but in D85N only at pH < 4. The structures of the L and the unphotolyzed states of these proteins were examined with Fourier transform infrared spectroscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the wild-type protein. Shift of the ethylenic band toward lower frequency upon replacing chloride by bromide confirmed the contribution of the negative charge of the anions to the Schiff base counterion. These anions restored the change of water, which is bound near the protonated Schiff base but is absent in the blue form of the D85N mutant, though with stronger H-bonding than in the wild type. The C = N stretching vibration of the Schiff base in H2O and 2H2O was detected by Fourier transform Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to the mutants than with Asp85 as the counterion in the wild type. Thus, although the geometry of the environment is different, there is at least one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.