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 seemed to be a freely accessible CP surface residue lacking functional importance, is an essential part of the CP proton uptake pathway connecting the membrane surface with 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 moiety, and the deprotonation/reprotonation of D96. Opposed to this function, the surface-exposed amino acids D36, D102, D104, and E161 seem to efficiently collect protons from the aqueous bulk phase and funnel them to the entrance of the CP proton pathway.
Riesle
J
J
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Oesterhelt
D
D
Dencher
N A
NA
Heberle
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine; D018698:Glutamic Acid; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D003545:Cysteine