Effects of Tryptophan Oxidation on Bacteriorhodopsin Structure and Function

N‐bromosuccinimide at low molar ratios specifically oxidizes tryptophan residues in purple membranes (bR). Loss of 1 mol tryptophan per mol bR (which corresponds to oxidation of Trp10 or Trp12 or part of both) produces slight changes in the absorption and CD spectra and in the photobleaching kinetics. The efficiency of reconstituting the retinylidene protein after bleaching and heptane extraction of these membranes was comparable to control values(–80%). Photocycling yield (M412) with 265 nm or 530 nm excitation (15 ns pulse) was only slightly decreased. Another mol of tryptophan was reacted at higher NBS:bR molar ratio (10:1). Partial oxidation of several residues rather than titration of a specific residue occurred: HPLC indicated no reaction with the chromophore. A considerable loss of extinction at 570 nm including enhanced red and blue shifts of LDmax at low and high pH respectively, was observed. Also the photobleaching rate was faster and functional retinylidene membranes could not be reconstituted from heptane‐extracted, apomembranes. Exogenous retinal could still locate the attachment site, based on formation of the fluorescent NaBH4‐reduced retinoyl adduct. Perturbation in the near UV and visible CD infer changes in helical conformation, trimer dissociation and decreased asymmetry of the chromophore. Photocycling efficiency was greatly decreased. The relative decrease was greater for 265 nm rather than 530 nm excitation. These results are consistent with co‐operative destabilization of the protein conformation by oxidized tryptophan residues, which leads to a decrease in the hydrophobicity of chromophoric site.