Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

• DOI: 10.1073/pnas.92.11.4962
• Publication Date: 2006-05-31T13:11:38Z
• Authors: J Le Coutre; J Tittor; D Oesterhelt; K Gerwert
• Keywords: Halobacterium; 0301 basic medicine; Aspartic Acid; Azides; Binding Sites; Protein Conformation; Hydrogen Bonding; Catalysis; Recombinant Proteins; 03 medical and health sciences; Mutagenesis; Bacteriorhodopsins; Spectroscopy, Fourier Transform Infrared; Point Mutation; Amino Acid Sequence; Asparagine; Protons

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.