Asp⁹⁶ deprotonation and transmembrane α-helical structural changes in bacteriorhodopsin

The M→N transition in the photocycle of bacteriorhodopsin involves the transfer of a proton from Asp⁹⁶ to the retinylidene Schiff base, possibly through a network of hydrogen-bonded amino acid residues and water molecules (Rothschild, K. J., He, Y. W., Sonar, S., Marti, T., and Khorana, H. G. (1992) J. Biol. Chem. 267, 1615-1622). A conformational change of the protein backbone is also observed during this transition. In this work, we have investigated the effects of replacing the residue Thr⁴⁶, which might be part of this chain, with an aspartic acid. Both Fourier transform infrared and resonance Raman spectroscopy show that the chromophore structure of this mutant (T46D) is normal. However, N formation is accelerated and N decay is significantly slowed compared to wild-type bacteriorhodopsin. This effect causes the N intermediate to accumulate under steady-state illumination thereby facilitating spectroscopic studies under normal pH conditions. Fourier transform infrared difference spectroscopy reveals that like native bacteriorhodopsin, N formation in T46D involves deprotonation of Asp⁹⁸, reprotonation of the Schiff base, and a change in the backbone secondary structure. However, in contrast to bacteriorhodopsin, bands assigned to the C=O stretch mode of the carboxylic acid group of Asp⁹⁶ are upshifted by 10 cm^-1 reflecting a change in the Asp⁹⁶ environment and a drop in its effective pK_a throughout the photocycle. This change in the pK_a can directly account for changes in the photocycle kinetics and indicates that Asp⁹⁶ deprotonation/protonation are the rate limiting steps in the formation and decay of the N intermediate. By studying the effects of H/D exchange, evidence is found that the backbone structural changes involve transmembrane α-helices. It is proposed that these structural changes serve to modulate the local environment and protonation state of Asp⁹⁸ during the photocycle and are also essential for formation of the proton conducting hydrogen bonded network which functions during Schiff base reprotonation.