TIME-RESOLVED FOURIER-TRANSFORM INFRARED STUDY OF STRUCTURAL-CHANGES IN THE LAST STEPS OF THE PHOTOCYCLES OF GLU-204 AND LEU-93 MUTANTS OF BACTERIORHODOPSIN

The last intermediate in the photocycle of the light-driven proton pum p bacteriorhodopsin is the red-shifted O state. The structure and dyna mics of the last step in the photocycle were characterized with time-r esolved Fourier transform infrared spectroscopy of the mutants of Glu- 204 and Leu-93, which accumulate this intermediate in much larger amou nts than the wild type. The results show that E204Q and E204D give dis torted all-trans-retinal chromophore like the O intermediate of the wi ld type. This is simply due to the perturbation of the proton acceptor function of Glu-204 in the O-to-BR transition in the Glu-204 mutants. The corresponding red-shifted intermediates of L93M, L93T, and L93S h ave a 13-cis chromophore like the N intermediate of the wild type, as reported from analysis of extracted retinal [Delaney, J. K., Schweiger , U., & Subramaniam, S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11120 -11124]. In spite of their different chromophore structures from the O intermediate, the red-shifted intermediates are similar to the O inte rmediate but not to the N intermediate of the wild type with respect t o structural changes in the peptide carbonyls. The structural changes around Asp-96 in the N intermediate are completely restored also in th e red-shifted intermediates of the Leu-93 mutants like in the O interm ediate. These results imply that the protein structural changes in the last step proceed regardless of thermal isomerization of the chromoph ore. Time-resolved Fourier transform infrared spectroscopy with the Gl u-204 mutants suggests that the response of Asp-204 (Glu-204 in the wi ld type) to the protonation of Asp-85 during formation of the M interm ediate, which results in proton release, is slow and may occur through structural changes.