Reversible inhibition of the proton pump bacteriorhodopsin by modification of tyrosine 64.
Five mol of lysine per mol of bacteriorhodopsin were modified with methylacetimidate. This treatment did not inactivate bacteriorhodopsin but prevented all lysines from subsequent reaction with diazotized sulfanilic acid. This reaction predominantly modified tyrosine 64 and light-induced proton translocation was abolished. Reduction of the mono(p-azobenzene sulfonic acid) tyrosine 64 to the corresponding 3-amino derivative with sodium dithionite led to complete reactivation of the proton translocation activity of bacteriorhodopsin. The relative location of tyrosines 26 and 64 and the COOH terminus on the two surfaces of the purple membrane was determined by incorporation into phospholipid vesicles, subsequent modification, and proteolytic treatment. The results obtained support the models proposed by Engelmann et al. (Engelman, D. M., Henderson, R. McLauchlan, A. D., and Wallace, B. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2023-2027) and by Ovchinnikov et al. (Ovchinnikov, Yu. A., Abdulaev, N. G., Feigina M. Yu., Kiselev A. V., and Lobanov, N. A. (1979) FEBS Lett. 100, 219-224). Tyrosine 64 is located on the extracellular side of the membrane, whereas tyrosine 26 and the COOH terminus are located on the cytoplasmic side. Because specific nitration of tyrosine 26 also leads to inactivation of bacteriorhodopsin (Lemke, H. D., and Oesterhelt, D. (1981) Eur. J. Biochem. 115, 595-604), the results obtained demonstrate that amino acid residues located on both surfaces of the purple membrane are involved in proton translocation.
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002338:Carotenoids / Q000037:antagonists & inhibitors*; D055598:Chemical Phenomena; D002621:Chemistry; D003488:Cyanogen Bromide; D003979:Diazonium Compounds / Q000494:pharmacology; D006217:Halobacterium / Q000032:analysis*; D007096:Imidoesters / Q000494:pharmacology; D008081:Liposomes; D008239:Lysine; D010446:Peptide Fragments / Q000378:metabolism; D011522:Protons; D013329:Structure-Activity Relationship; D013425:Sulfanilic Acids / Q000031:analogs & derivatives / Q000494:pharmacology; D014443:Tyrosine
D003979:Diazonium Compounds; D007096:Imidoesters; D008081:Liposomes; D010446:Peptide Fragments; D011522:Protons; D013425:Sulfanilic Acids; D002338:Carotenoids; D014443:Tyrosine; D001436:Bacteriorhodopsins; C010222:methyl acetimidate; D008239:Lysine; D003488:Cyanogen Bromide; C000469:diazobenzenesulfonic acid