Properties of the Anion-Binding Site of pharaonis Halorhodopsin Studied by Ultrafast Pump−Probe Spectroscopy and Low-Temperature FTIR Spectroscopy

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• 作者：Keisuke Nakashima ; Takumi Nakamura ; Satoshi Takeuchi ; Mikihiro Shibata ; Makoto Demura ; Tahei Tahara ; Hideki Kandori
• 刊名：Journal of Physical Chemistry B
• 出版年：2009
• 出版时间：June 18, 2009
• 年：2009
• 卷：113
• 期：24
• 页码：8429-8434
• 全文大小：236K
• 年卷期：v.113,no.24(June 18, 2009)
• ISSN：1520-5207

文摘

Halorhodopsin (HR) is a light-driven chloride pump. Cl⁻ is bound in the Schiff base region of the retinal chromophore, and unidirectional Cl⁻ transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. It is known that HR from Natronobacterium pharaonis (pHR) also pumps NO₃⁻ with similar efficiency, suggesting that NO₃⁻ binds to the Cl⁻-binding site. In the present study, we investigated the properties of the anion-binding site by means of ultrafast pump−probe spectroscopy and low-temperature FTIR spectroscopy. The obtained data were surprisingly similar between pHR−NO₃⁻ and pHR−Cl⁻, even though the shapes and sizes of the two anions are quite different. Femtosecond pump−probe spectroscopy showed very similar excited-state dynamics between pHR−NO₃⁻ and pHR−Cl⁻. Low-temperature FTIR spectroscopy of unlabeled and [ζ-¹⁵N]Lys-labeled pHR revealed almost identical hydrogen-bonding strengths of the protonated retinal Schiff base between pHR−NO₃⁻ and pHR−Cl⁻, which is similarly strengthened after retinal isomerization. There were spectral variations for water stretching vibrations between pHR−NO₃⁻ and pHR−Cl⁻, suggesting that the water molecules hydrate each anion. Nevertheless, the overall spectral features were similar for the two species. These observations strongly suggest that the anion-binding site has a flexible structure and that the interaction between retinal and the anions is weak, despite the presence of an electrostatic interaction. Such a flexible hydrogen-bonding network in the Schiff base region in HR appears to be in remarkable contrast to that in light-driven proton-pumping proteins.