Carboxyl groups and the proton pump of bacteriorhodopsin.

The purple membrane isolated from Halobacterium halobium contains only a single protein, bacteriorhodopsin, which functions as a light-driven proton pump. Substantial structural information has been obtained which has led to specific models of protein structure in the membrane (Engelman et al., 1982; Huang et al., 1982; Agard & Stroud, 1982). The retinal chromophore of bacteriorhodopsin is bound to the &-amino group of lysine-216 by a Schiff-base linkage (Katre et a/ . , 1981; Bayley et al., 1981). Light absorption by the chromophore initiates a photochemical reaction cycle which involves configurational changes of the retinal and conformational changes of the protein (for a review, see Stoeckenius et al., 1979). The role of the Schiffbase linkage in the reaction mechanism is well established, but the contribution of the protein moiety to chromophore structure, the photochemical reaction cycle, and to the proton-translocation mechanism is largely unknown. The carboxyl residues of aspartic and glutamic acid have been suggested to have the following roles in bacteriorhodopsin. (i) Structural: by forming ion-pairs within the membrane with the positively charged groups of lysine and/or arginine (Packer et al., 1979; Engelman et al., 1980). (ii) Chromophoric: by interacting with retinal and the Schiffbase nitrogen to modulate absorption properties (Fisher & Osterhelt, 1980). (iii) Catalytic: by functioning as protontranslocating groups during the photoreaction cycle. Chemical modification studies of carboxyl groups have indicated structural involvement in the photocycle (Herz & Packer, 1981). Recently, Fourier transform and kinetic i.r. spectroscopy studies (Rothschild et al., 1981 ; Siebert et a / . , 1982) have demonstrated changes in the protonation state of carboxyl groups correlated with the photocycle. It was suggested that these groups reside in a hydrophobic membrane environment. In addition, current models of bacteriorhodopsin structure place several carboxyl residues within hydrophobic membrane-protein domains. Thus knowledge of the topography of carboxyl residues is central to the understanding of the structure and function of the light-driven proton pump. We have used chemical modification techniques to attach reporter groups to the carboxyl residues of bacteriorhodopsin. The spin-label and chromophoric groups employed display characteristic spectra which are sensitive to changes in their micro-environment. This approach has allowed the correlation of functional properties of carboxyl groups with their local topography and mobility in distinct membraneprotein domains.