title: Deprotonation of the Schiff base of bacteriorhodopsin is obligate in light-induced proton pumping.
abstract: Bacteriorhodopsin (bR) in purple membranes was permethylated with formaldehyde and pyridine-borane with the incorporation of approximately 12 methyl groups. This new pigment, PMbR, absorbed light in the dark-adapted state with a lambda max at 558 nm, virtually the same as that of bR. Light adaptation of PMbR produced a lambda max of 564 nm with a slightly elevated epsilon. Similar changes occurred with bR. When incorporated into asolectin vesicles, PMbR was able to pump protons in the light with an efficiency similar to that of bR itself. Bleaching of PMbR exposed its active site lysine residue, which was monomethylated to form active site methylated bR (AMbR) after regeneration with all-trans-retinal. This blue pigment, which is a cyanopsin rather than a rhodopsin, showed an extraordinary red shift, absorbing light with a lambda max of 620 nm in the dark-adapted state. Light adaptation of AMbR resulted in a spectral shift to 616 nm with a decrease in epsilon. This change was completely reversible in the dark. This shift was interpreted to mean that an L-like intermediate was accumulating, as would be expected if deprotonation of the protonated Schiff base could not occur to produce the M intermediate. Furthermore, when incorporated into asolectin vesicles, AMbR proved incapable of pumping protons in the light. It was concluded from these experiments that deprotonation of the Schiff base of bR is obligate for light-induced proton pumping.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: C
authors.initials: C
authors.lastname: Longstaff
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D001436:Bacteriorhodopsins / Q000378:metabolism* / Q000528:radiation effects; D003624:Darkness; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008745:Methylation; D011522:Protons; D012545:Schiff Bases
chemical_list: D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins