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 ligh t-driven proton pump bacteriorhodopsin (BR) although a prerequisite fo r efficient vectorial. proton translocation is the efficient capture o f protons from the alkaline cytoplasm of the cell. To identify residue s 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 res idue by a neutral or positive one at the pH of investigation). The eff ect of these replacements on the photo- and transport cycle was examin ed by time-resolved visible and infrared spectroscopy, biochemical mod ification studies, and activity assays in intact eels. Of the five CP amino acids studied, only the replacement of D38 resulted in severe al terations of the reaction steps in BR during the second half of the ph otocycle. Our data show that D38, which seemed to be a freely accessib le CP surface residue lacking functional importance, is an essential p art of the CP proton uptake pathway connecting the membrane surface wi th the Schiff base of BR, probably as the first amino acid residue at the CP entrance. D38 influences the late steps in the functional cycle , such as the occurrence of the intermediates N and O, the modulation of the hydrogen-network, the conformational changes in the protein moi ety, and the deprotonation/reprotonation of D96. Opposed to this funct ion, the surface-exposed amino acids D36, D102, D104, and E161 seem to efficiently collect protons from the aqueous bulk phase and funnel th em to the entrance of the CP proton pathway.