Light-dependent nitration of bacteriorhodopsin.
Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.
D001436:Bacteriorhodopsins / Q000528:radiation effects*; D002338:Carotenoids / Q000528:radiation effects*; D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009584:Nitrogen; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013774:Tetranitromethane; D014443:Tyrosine
D002338:Carotenoids; D014443:Tyrosine; D001436:Bacteriorhodopsins; D013774:Tetranitromethane; D009584:Nitrogen