HYDRATION OF THE COUNTERION OF THE SCHIFF-BASE IN THE CHLORIDE-TRANSPORTING MUTANT OF BACTERIORHODOPSIN - FTIR AND FT-RAMAN STUDIES OF THE EFFECTS OF ANION-BINDING WHEN ASP85 IS REPLACED WITH A NEUTRAL RESIDUE

The chromophores of the D85T and D85N mutants of bacteriorhodopsin are blue but become purple like the wild type when chloride or bromide bi nds 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 state s of these proteins were examined with Fourier transform infrared spec troscopy. The difference spectra of the purple forms, but not the blue forms in the absence of these anions, resembled the spectrum of the w ild-type protein. Shift of the ethylenic band toward lower frequency u pon replacing chloride by bromide confirmed the contribution of the ne gative charge of the anions to the Schiff base counterion. These anion s restored the change of water, which is bound near the protonated Sch iff base but is absent in the blue form of the D85N mutant, though wit h stronger H-bonding than in the wild type, The C=N stretching vibrati on of the Schiff base in H2O and 2H2O was detected by Fourier transfor m Raman spectroscopy. The H-bonding strength of the Schiff base in the unphotolyzed state was weaker when chloride or bromide was bound to t he mutants than with Asp85 as the counterion in the wild type. Thus, a lthough the geometry of the environment is different, there is at leas t one water molecule coordinated to the bound halide in these mutants, in a way similar to water bound to Asp85 in the wild type.