title: Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.
abstract: Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.
authors.affiliation: Lehrstuhl für Biophysik, Fakultät Biologie, Ruhr-Universität-Bochum, Federal Republic of Germany.
authors.forename: J
authors.initials: J
authors.lastname: Le Coutre
authors.forename: J
authors.initials: J
authors.lastname: Tittor
authors.forename: D
authors.initials: D
authors.lastname: Oesterhelt
authors.forename: K
authors.initials: K
authors.lastname: Gerwert
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001386:Azides; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D006217:Halobacterium / Q000378:metabolism*; D006860:Hydrogen Bonding; D016296:Mutagenesis; D017354:Point Mutation; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D001386:Azides; D011522:Protons; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001216:Asparagine