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 ² Hideki Kandori, ‡ Yoichi Yamazaki, ‡ Minoru Hatanaka, ‡ Richard Needleman, § Leonid S. Brown, | Hans-Thomas Richter, | Janos K. Lanyi, | and Akio Maeda* ,‡ Department of Biophysics, Graduate School of Science, Kyoto UniVersity, Sakyo-ku, Kyoto 606-01, Japan, Department of Biochemistry, Wayne State UniVersity School of Medicine, Detroit, Michigan 48201, and Department of Physiology and Biophysics, UniVersity of California, IrVine, California 92717 ReceiVed December 4, 1996; ReVised Manuscript ReceiVed February 18, 1997 X ABSTRACT: The last intermediate in the photocycle of the light-driven proton pump bacteriorhodopsin is the red-shifted O state. The structure and dynamics of the last step in the photocycle were characterized with time-resolved Fourier transform infrared spectroscopy of the mutants of Glu-204 and Leu-93, which accumulate this intermediate in much larger amounts than the wild type. The results show that E204Q and E204D give distorted all-trans-retinal chromophore like the O intermediate of the wild 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 have 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 intermediate but not to the N intermediate of the wild type with respect to structural changes in the peptide carbonyls. The structural changes around Asp-96 in the N intermediate are completely restored also in the red-shifted intermediates of the Leu-93 mutants like in the O intermediate. These results imply that the protein structural changes in the last step proceed regardless of thermal isomerization of the chromophore. Time-resolved Fourier transform infrared spectroscopy with the Glu- 204 mutants suggests that the response of Asp-204 (Glu-204 in the wild type) to the protonation of Asp- 85 during formation of the M intermediate, which results in proton release, is slow and may occur through structural changes. Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The retinal chromophore is covalently bound to Lys-216 through a protonated Schiff base and can assume both all-trans and 13-cis configurations. Light adaptation changes the 13-cis,15-syn form to all- trans,15-anti. Additionally, photoisomerization of the all- trans-bacteriorhodopsin (BR) 1 to 13-cis,15-anti triggers a cyclic reaction that comprises a series of intermediates. Each intermediate was originally identified by visible absorption spectroscopy as reflected in their subscripts with the absorp- tion maxima (K 600 ,L 550 ,M 412 ,N 565 , and O 640 ). 2 This means that the chromophore-protein interaction, reflected in the visible spectrum, is distinct in each of these photointerme- diates. Extensive spectroscopic studies have identified (i) the chromophore structure, (ii) the protonation state of the residues that contribute to proton pumping, and (iii) the protein structure of the intermediate states [for reviews see Mathies et al. (1991), Oesterhelt et al. (1992), Rothschild (1992), Lanyi (1993), Krebs and Khorana (1993), Ebrey (1993), and Maeda (1996)]. The K-to-L transition precedes the primary proton transfer from the Schiff base to Asp-85 with accompanying structural changes in the protein and internal bound water. The proton transfer in the L-to-M process causes proton release from Glu-204 to the extracel- lular aqueous phase (Brown et al., 1995). The protonated Asp-85 and the unprotonated Glu-204 thus formed appear to persist until the final step in the photocycle. The M-to-N process is accompanied by proton transfer from Asp-96 to the Schiff base. The greatest structural changes of the protein in the photocycle are observed in the M N and N intermediates (Sasaki et al., 1992; Kamikubo et al., 1996). Proton uptake from cytoplasmic aqueous phase to Asp-96 and thermal reisomerization from 13-cis to all-trans occurs in the step subsequent to the formation of the N intermediate, and the final proton transfer from Asp-85 to Glu-204 resets the original structure of BR. Unlike the case of visual rhodopsin, the initial state recovers thermally in the photocycle of BR, which allows it to turn over rapidly as required for an energy converter rather than a signal transduction device. Therefore, it is important to elucidate the molecular mechanism for how bacterior- hodopsin resets the system. However, in comparison with the earlier events, the structural changes in the late processes ² J.K.L. acknowledges a grant from the National Institutes of Health (GM 29498). This work is also supported by grants from the Japanese Ministry of Education, Culture, Sports, and Science to A.M. (06404082, 06044123, and 08268225) and H.K. (07839003 and 08238231) and by JSPS Research Fellowships for Young Scientists to Y.Y. * To whom correspondence should be addressed. ‡ Kyoto University. § Wayne State University. | University of California, Irvine. X Abstract published in AdVance ACS Abstracts, April 1, 1997. 1 Abbreviations: BR, bacteriorhodopsin (or its mutants) with all- trans chromophore; HOOP, hydrogen out-of-plane; TR-FTIR, time- resolved Fourier transform infrared. 2 These subscripts originate from Va ´ro ´ and Lanyi (1991). 5134 Biochemistry 1997, 36, 5134-5141 S0006-2960(96)02978-9 CCC: $14.00 © 1997 American Chemical Society