Bacteriorhodopsin can function without a covalent linkage between retinal and protein.
Light energy is transferred from retinal to the protein in bacteriorhodopsin after absorption of a photon resulting in changes of protein conformation. To examine whether the covalent bond, formed by the carbonyl group of retinal and the epsilon-amino group of lysine 216, is essential for this process, a mutant with lysine 216 replaced by alanine was expressed in Halobacterium salinarium L33 (BO-, retinal+). Reconstitution of the chromoprotein with varying retinylidene-n-alkylamines was possible in isolated membranes as well as in whole cells. When the protein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein was formed with retinylideneethylamine. The absorbance maximum was at 475 nm in alkaline solution and 620 nm in acidic solution. At neutral pH values both species equilibrate with a third one absorbing maximally at 568 nm. Reconstitution of whole cells with retinylideneethylamine led to a specific proton pump activity of 30 mol of protons per mol of BR per minute. This value indicates a lower limit of transport; no light saturation could be reached in these measurements in contrast to wild-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved. Action spectra from flash photolysis experiments revealed that only the 568-nm form led to a M-intermediate with a half-time of decay of 17 ms. In summary, it could be shown that the covalent linkage between retinal and the protein is basically not required for the function of bacteriorhodopsin as a light-driven proton pump.
Schweiger
U
U
Max-Planck-Institute for Biochemistry, Martinsried, FRG.
Tittor
J
J
Oesterhelt
D
D
D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006217:Halobacterium / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D010782:Photolysis; D017494:Proton Pumps / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism*; D012176:Retinoids / Q000378:metabolism; D012545:Schiff Bases
D017494:Proton Pumps; D012176:Retinoids; D012545:Schiff Bases; D001436:Bacteriorhodopsins; D008239:Lysine; D012172:Retinaldehyde