D38 IS AN ESSENTIAL PART OF THE PROTON TRANSLOCATION PATHWAY IN BACTERIORHODOPSIN

At present, almost no knowledge exists about the functional relevance of the amino acid residues at the cytoplasmic (CP) surface of the light-driven proton pump bacteriorhodopsin (BR) although a prerequisite for efficient vectorial proton translocation is the efficient capture of protons from the alkaline cytoplasm of the cell. To identify residues involved in the proton transfer reaction steps in the CP part of BR, the aspartic and glutamic amino acids D36, D38, D102, D104, and E161 were replaced by cysteine and arginine (i.e., a negatively charged residue by a neutral or positive one at the pH of investigation). The effect of these replacements on the photo- and transport cycle was examined by time-resolved visible and infrared spectroscopy, biochemical modification studies, and activity assays in intact cells. Of the five CP amino acids studied, only the replacement of D38 resulted in severe alterations of the reaction steps in BR during the second half of the photocycle. Our data show that D38, which ...