FTIR studies of internal water molecules in the Schiff base region of bacteriorhodopsin.
In a light-driven proton pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. Previously, low-temperature Fourier-transform infrared (FTIR) difference spectra between BR and the K photointermediate in D(2)O revealed six O-D stretches of water in BR at 2690, 2636, 2599, 2323, 2292, and 2171 cm(-)(1), while five water bands were observed at 2684, 2675, 2662, 2359, and 2265 cm(-)(1) for the K intermediate. The frequencies are widely distributed over the possible range of stretching vibrations of water, and water molecules at <2400 cm(-)(1) were suggested to hydrate negative charges because of their extremely strong hydrogen bonds. In this paper, we aimed to reveal the origin of these water bands in the K minus BR spectra by use of various mutant proteins. The water bands were not affected by the mutations at the cytoplasmic side, such as T46V, D96N, and D115N, implying that the water molecules in the cytoplasmic domain do not change their hydrogen bonds in the BR to K transition. In contrast, significant modifications of the water bands were observed for the mutations in the Schiff base region and at the extracellular side, such as R82Q, D85N, T89A, Y185F, D212N, R82Q/D212N, and E204Q. From these results, we concluded that the six O-D stretches of BR originate from three water molecules, water401, -402, and -406, involved in the pentagonal cluster. Two stretching modes of each water molecule are highly separate (300-470 cm(-)(1) for O-D stretches and 500-770 cm(-)(1) for O-H stretches), which is consistent with the previous QM/MM calculation. The small amplitudes of vibrational coupling are presumably due to strong association of the waters to negative charges of Asp85 and Asp212. Among various mutant proteins, only D85N and D212N lack strongly hydrogen-bonded water molecules (<2400 cm(-)(1)) and proton pumpimg activity. We thus infer that the presence of a strong hydrogen bond of water is a prerequisite for proton pumping in BR. Internal water molecules in such a specific environment are discussed in terms of functional importance for rhodopsins.
Shibata
Mikihiro
M
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
Kandori
Hideki
H
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics; D019617:Halobacterium salinarum; D006860:Hydrogen Bonding; D009154:Mutation; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics; D012545:Schiff Bases; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics; D014867:Water / Q000737:chemistry*
D019843:Archaeal Proteins; D017494:Proton Pumps; D012545:Schiff Bases; D014867:Water; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; D001120:Arginine