Existence of a proton transfer chain in bacteriorhodopsin: Participation of Glu-194 in the release of protons to the extracellular surface

被引:169
作者
Dioumaev, AK
Richter, HT
Brown, LS
Tanio, M
Tuzi, S
Saito, H
Kimura, Y
Needleman, R
Lanyi, JK [1 ]
机构
[1] Univ Calif Irvine, Dept Physiol & Biophys, Irvine, CA 92697 USA
[2] Himeji Inst Technol, Kamigori, Hyogo 67812, Japan
[3] Biomol Engn Res Inst, Dept Biol Struct, Osaka 565, Japan
[4] Wayne State Univ, Dept Biochem, Detroit, MI 48201 USA
关键词
D O I
10.1021/bi971842m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protanation of Asp-SS during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pK(a) of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement. of Glu-194 with aspartate raises the pK, for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm(-1). It can be assigned from [4-(13)C]aspartate and D(2)O isotope shifts to the C=O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pK(a) of about 9. We show that this pK(a) is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation: while (13)C NMR titration indicates that Asp-194 has an initial pK(a) of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pK(a)'s change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.
引用
收藏
页码:2496 / 2506
页数:11
相关论文
共 53 条
  • [51] Conformation and dynamics of [3-C-13]Ala-labeled bacteriorhodopsin and bacterioopsin, induced by interaction with retinal and its analogs, as studied by C-13 nuclear magnetic resonance
    Tuzi, S
    Yamaguchi, S
    Naito, A
    Needleman, R
    Lanyi, JK
    Saito, H
    [J]. BIOCHEMISTRY, 1996, 35 (23) : 7520 - 7527
  • [52] Temperature-dependent conformational change of bacteriorhodopsin as studied by solid-state C-13 NMR
    Tuzi, S
    Naito, A
    Saito, H
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1996, 239 (02): : 294 - 301
  • [53] PATHWAYS OF PROTON RELEASE IN THE BACTERIORHODOPSIN PHOTOCYCLE
    ZIMANYI, L
    VARO, G
    CHANG, M
    NI, BF
    NEEDLEMAN, R
    LANYI, JK
    [J]. BIOCHEMISTRY, 1992, 31 (36) : 8535 - 8543