| Abstract: | Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR. |
