The role of tyrosine residues in the function of bacteriorhodopsin. Specific nitration of tyrosine 26.
Treatment of the purple membrane with tetranitromethane under controlled conditions leads to the nitration of 3 mol tyrosine/mol bacteriorhodopsin. The combination of subtilisin digestion and cyanogen bromide cleavage with subsequent analysis of the resulting peptide mixture by high-performance liquid chromatography, allows identification of the positions modified in the polypeptide chain. Tyrosines 26 and 64 are fully nitrated, whereas tyrosines 131 and 133 are nitrated to about 60% and 40%, respectively. Reduction of the nitrated membranes with the water-soluble ionic agent dithionite leaves only tyrosine 26 nitrated indicating that the residues 64, 131 and 133 are located on the membrane surface. As a result of nitration, the purple complex shifts its absorption maximum from 568 nm to 532 nm. Dithionite reduction of the nitrated membrane does not reverse this effect. Removal of the retinal and reconstitution maintains the blue-shifted absorption of the chromophore. A pH-dependent equilibrium of the chromophore with a further red-shifted form is observed. The pK of this transition is at about pH 9. Because nitration of tyrosine leads to a drastic decrease of its pK a participation of tyrosine 26 in the chromophoric structure via a hydrogen bridge is suggested. This finding is consistent with a model of chromophore structure published earlier [U. Fischer and D. Oesterhelt (1979) Biophys. J. 31, 139--146].
D000596:Amino Acids / Q000032:analysis; D001436:Bacteriorhodopsins; D002338:Carotenoids; D004227:Dithionite; D006217:Halobacterium / Q000032:analysis; D007700:Kinetics; D008697:Methane / Q000031:analogs & derivatives; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013053:Spectrophotometry; D013381:Subtilisins; D013774:Tetranitromethane; D014443:Tyrosine
D000596:Amino Acids; D010446:Peptide Fragments; D004227:Dithionite; D002338:Carotenoids; D014443:Tyrosine; D001436:Bacteriorhodopsins; D013381:Subtilisins; D013774:Tetranitromethane; D008697:Methane