Altered Hydrogen Bonding of Arg82 during the Proton Pump Cycle of Bacteriorhodopsin: A Low-Temperature Polarized FTIR Spectroscopic Study
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- 作者：Taro Tanimoto ; Mikihiro Shibata ; Marina Belenky ; Judith Herzfeld ; and Hideki Kandori
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：July 27, 2004
- 年：2004
- 卷：43
- 期：29
- 页码：9439 - 9447
- 全文大小：207K
- 年卷期：v.43,no.29(July 27, 2004)
- ISSN：1520-4995
文摘
Light-driven proton transport in bacteriorhodopsin (BR) is achieved by dynamic rearrangementof the hydrogen-bonding network inside the membrane protein. Arg82 is located between the Schiff baseregion and proton release group, and has a major influence on the pKa values of these groups. It is believedthat Arg82 changes its hydrogen-bonding acceptors during the pump cycle of BR, stages of which arecorrelated with proton movement along the transport pathway. In this study, we compare low-temperaturepolarized FTIR spectra of [
1,2-15N]arginine-labeled BR in the 2750-2000 cm-1 region with those ofunlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, 15N-shiftedmodes were found at 2292 (-)/2266 (+) cm-1 and at 2579 (-)/2567 (+) cm-1. The former correspondsto strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-Dstretches probably originate from Arg82, the former oriented toward water 406 and the latter toward theextracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization.This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no 15N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is notsignificantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolarorientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular toparallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 sidechain from the Schiff base region to the extracellular proton release group. Additional bands correspondingto weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes inhydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.
1,2-15N]arginine-labeled BR in the 2750-2000 cm-1 region with those ofunlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, 15N-shiftedmodes were found at 2292 (-)/2266 (+) cm-1 and at 2579 (-)/2567 (+) cm-1. The former correspondsto strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-Dstretches probably originate from Arg82, the former oriented toward water 406 and the latter toward theextracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization.This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no 15N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is notsignificantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolarorientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular toparallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 sidechain from the Schiff base region to the extracellular proton release group. Additional bands correspondingto weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes inhydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.