Low-temperature FTIR study of Gloeobacter rhodopsin: presence of strongly hydrogen-bonded water and long-range structural protein perturbation upon retinal photoisomerization.
Gloeobacter rhodopsin (GR) is a light-driven proton-pump protein similar to bacteriorhodopsin (BR), found in Gloeobacter violaceus PCC 7421, a primitive cyanobacterium. In this paper, structural changes of GR following retinal photoisomerization are studied by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy. The initial motivation was to test our hypothesis that proton-pumping rhodopsins possess strongly hydrogen-bonded water molecules in the active center. Water O-D stretching vibrations at <2400 cm(-1) in D(2)O have been regarded as coming from such strongly hydrogen-bonded water, and there is a strong correlation between the proton-pumping activity and the presence of such water molecule. Since GR pumps protons, we expected that GR also possesses strongly hydrogen-bonded water molecule(s), and the FTIR results clearly show that this is indeed the case. In addition, another unexpected finding was gained from the frequency region of protonated carboxylic acids in the GR(K) minus GR spectra at 77 K, where we observed the unique bands of a protonated carboxylic acid at 1735 (+)/1730 (-) cm(-1). Comprehensive mutation study revealed that the vibrational bands originate from the carboxylic C=O stretch of Glu132 at the position corresponding to Asp96 in BR. Glu132 presumably functions as an internal proton donor for the retinal Schiff base, but they may be located far apart (ca. 12 A in BR). The present study demonstrates the long-range structural changes of GR along the proton pathway, even though the protein matrix is frozen at 77 K.
Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Kyohei
K
Hashimoto
Ah Reum
AR
Choi
Yuji
Y
Furutani
Kwang-Hwan
KH
Jung
Hideki
H
Kandori
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002264:Carboxylic Acids / Q000032:analysis; D000458:Cyanobacteria / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006860:Hydrogen Bonding; D007536:Isomerism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010777:Photochemistry; D011487:Protein Conformation; D012172:Retinaldehyde / Q000737:chemistry*; D025604:Rhodopsins, Microbial / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water / Q000737:chemistry
D000596:Amino Acids; D002264:Carboxylic Acids; D025604:Rhodopsins, Microbial; D014867:Water; D018698:Glutamic Acid; D012172:Retinaldehyde