Factors that differentiate the H-bond strengths of water near the Schiff bases in bacteriorhodopsin and Anabaena sensory rhodopsin

Abstract

Bacteriorhodopsin (BR) functions as a light-driven proton pump, whereas Anabaena sensory rhodopsin (ASR) is believed to function as a photosensor despite the high similarity in their protein sequences. In Fourier transform infrared (FTIR) spectroscopic studies, the lowest O-D stretch for D(2)O was observed at ∼2200 cm(-1) in BR but was significantly higher in ASR (>2500 cm(-1)), which was previously attributed to a water molecule near the Schiff base (W402) that is H-bonded to Asp-85 in BR and Asp-75 in ASR. We investigated the factors that differentiate the lowest O-D stretches of W402 in BR and ASR. Quantum mechanical/molecular mechanical calculations reproduced the H-bond geometries of the crystal structures, and the calculated O-D stretching frequencies were corroborated by the FTIR band assignments. The potential energy profiles indicate that the smaller O-D stretching frequency in BR originates from the significantly higher pK(a)(Asp-85) in BR relative to the pK(a)(Asp-75) in ASR, which were calculated to be 1.5 and -5.1, respectively. The difference is mostly due to the influences of Ala-53, Arg-82, Glu-194-Glu-204, and Asp-212 on pK(a)(Asp-85) in BR and the corresponding residues Ser-47, Arg-72, Ser-188-Asp-198, and Pro-206 on pK(a)(Asp-75) in ASR. Because these residues participate in proton transfer pathways in BR but not in ASR, the presence of a strongly H-bonded water molecule near the Schiff base ultimately results from the proton-pumping activity in BR.

FIGURE 1.

Overview of the BR (PDB code 1C3W) and ASR (PDB code 1XIO) structures. Red spheres indicate O atoms of water molecules. Yellow arrows indicate proton transfer pathways.

FIGURE 2.

Residues and groups included in the QM regions for BR (a) and ASR (b). Dotted lines indicate hydrogen bonds. c, geometry of moieties near Lys-210 in ASR before (magenta) and after (yellow) the QM/MM optimization is shown.

FIGURE 3.

The geometric correlation of the O-D stretching frequencies with the O_acceptor…H-O_donor bond of H₂O (correlation with the O_donor–O_acceptor distance). Each open square (□) represents the O_donor–O_acceptor distance of the QM/MM optimized geometries (Tables 1 and 4) and the O-D stretching frequency calculated by a QM/MM numerical differentiation method (Tables 2, 3, and 5). The solid curve was determined using an empirical equation (Equation 1) proposed by Mikenda (34).

FIGURE 4.

H-bond geometries of water molecules near the Schiff base in BR and ASR. Pink arrows in model 1 indicate mode couplings of the two O-D bonds. Red dotted lines indicate “strong H-bonds” reported in FTIR studies (7).

FIGURE 5.

Potential energy profiles along the proton transfer coordinates of O_W402–H…O_Asp-85 in BR (black solid line) and O_W402–H…O_Asp-75 in ASR (blue solid line). ΔE describes the difference in energy relative to the energy minimum. Boxed arrows indicate the shifts in the potential energy curve accompanied by the pK_a(Asp) change from ASR to BR. The marginal energy drop near 1.74 Å in O_W402–H of ASR was due to an alteration of the H-bond pattern; below 1.74 Å, Ser-47 donates an H-bond to the carboxyl O atom of Asp-75, which is simultaneously H-bonded by W402. At 1.74 Å, the hydroxyl H atom is oriented toward another carboxyl O atom of Asp-75 due to the unusual proximity of the H atom of W402 to Asp-75.

FIGURE 6.

Residues that contribute to differences between pK_a(Asp) of BR (a) and ASR (b).