FTIR Spectroscopy of the O Photointermediate in pharaonis Phoborhodopsin

详细信息    查看全文

• 作者：Yuji Furutani ; Masayuki Iwamoto ; Kazumi Shimono ; Akimori Wada ; Masayoshi Ito ; Naoki Kamo ; and Hideki Kandori
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：May 11, 2004
• 年：2004
• 卷：43
• 期：18
• 页码：5204 - 5212
• 全文大小：184K
• 年卷期：v.43,no.18(May 11, 2004)
• ISSN：1520-4995

文摘

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is aphotoreceptor protein for negative phototaxis in Natronobacterium pharaonis. During the photocycle ofppR, the retinal chromophore is thermally isomerized from the 13-cis to all-trans form. We employedFTIR spectroscopy of ppR at 260 K and pH 5 to reveal that this isomerization occurs upon formation ofthe O intermediate (ppR_O) by using ppR samples reconstituted with 12,14-D₂-labeled retinal. In ppR_O,C=O stretching vibrations of protonated carboxylates newly appear at 1757 (+)/1722 (-) cm^-1 in H₂Oand at 1747 (+)/1718 (-) cm^-1 in D₂O in addition to the 1765 (+) cm^-1 band of Asp75. Amide I vibrationsare basically similar between ppR_M and ppR_O, whereas unique bands of ppR_O are also observed such asthe negative 1656 cm^-1 band in D₂O and intense bands at 1686 (-)/1674 (+) cm^-1. In addition, O-Dstretching vibrations of water molecules in the entire mid-infrared region are assigned for ppR_M andppR_O, the latter being unique for ppR, since it can be detected at low temperature (260 K). The ppR_Mminus ppR difference spectra lack the lowest frequency water band (2215 cm^-1) observed in the ppR_Kminus ppR spectra, which is probably associated with water that interacts with the negative charges in theSchiff base region. It is likely that the proton transfer from the Schiff base to Asp75 in ppR_M can beexplained by a hydration switch of a water from Asp75 to Asp201, as was proposed for the light-drivenproton-pump bacteriorhodopsin (hydration switch model) [Tanimoto, T., Furutani, Y., and Kandori, H.(2003) Biochemistry 42, 2300-2306]. In the transition from ppR_M to ppR_O, a hydrogen-bonding alterationtakes place for another water molecule that forms a strong hydrogen bond.