BACTERIORHODOPSIN CAN FUNCTION WITHOUT A COVALENT LINKAGE BETWEEN RETINAL AND PROTEIN

Light energy is transferred from retinal to the protein in bacteriorho dopsin after absorption of a photon resulting in changes of protein co nformation. To examine whether the covalent bond, formed by the carbon yl group of retinal and the epsilon-amino group of lysine 216, is esse ntial for this process, a mutant with lysine 216 replaced by alanine w as expressed in Halobacterium salinarium L33 (BO-, retinal+). Reconsti tution of the chromoprotein with varying retinylidene-n-alkylamines wa s possible in isolated membranes as well as in whole cells. When the p rotein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein wa s formed with retinylideneethylamine. The absorbance maximum was at 47 5 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 l ed 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 lig ht saturation could be reached in these measurements in contrast to wi ld-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved. Action spec tra from flash photolysis experiments revealed that only the 568-nm fo rm led to a M-intermediate with a half-time of decay of 17 ms. In summ ary, it could be shown that the covalent linkage between retinal and t he protein is basically not required for the function of bacteriorhodo psin as a light-driven proton pump.