Factors That Differentiate the H-bond Strengths of Water Near the Schiff Bases in Bacteriorhodopsin and Anabaena Sensory Rhodopsin*

Background: Bacteriorhodopsin (BR) functions as a proton pump, whereas Anabaena sensory rhodopsin (ASR) functions as a photosensor. Results: pKa of the conserved Asp residues near the Schiff base significantly differ between BR and ASR. Conclusion: The O-D stretching frequencies for D2O are correlated with pKa(Asp). Significance: The presence of a strongly H-bonded water results from the proton-pumping activity in BR. 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 D2O 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 pKa(Asp-85) in BR relative to the pKa(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 pKa(Asp-85) in BR and the corresponding residues Ser-47, Arg-72, Ser-188-Asp-198, and Pro-206 on pKa(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.

Bacteriorhodopsin (BR) 2 functions as a light-driven proton pump, and the driving force for its action is provided by photoisomerization of the all-trans retinal chromophore, which is covalently attached to Lys-216 via the Schiff base, to 13-cis. This leads to proton transfer pathways that proceed toward the extracellular side via Asp-96, the Schiff base, Asp-85, and Glu-204 (1)(2)(3). Asp-85 is located near the Schiff base and serves as the counterion. The presence of water molecules in this region had been suggested by Fourier transform infrared (FTIR) studies (4,5), and these were later confirmed as W401, W402, and W406 in the high resolution crystal structure of the BR ground state (PDB code 1C3W; see Fig. 1) (6). The three water molecules form an H-bond network with Asp-85, Arg-82, Asp-212, and the Schiff base. Among the three water molecules, the chemical properties of W402 are of particular interest because the water molecule is located at H-bond distances of 2.63 and 2.87 Å from the carboxyl O atom of Asp-85 and the Schiff base N atom, respectively (see Table 1). From FTIR analysis of various mutants in D 2 O, the lowest O-D stretching frequency of 2171 cm Ϫ1 in BR was assigned to the O W402 -H…O Asp-85 bond, implying that this is the strongest H-bond in the network near the Schiff base (7). The band assignment presented in FTIR studies was also supported by previous computational studies by Hayashi et al. (8,9).
Water molecule W402 was also found in the crystal structure of Anabaena sensory rhodopsin (ASR) (PDB code 1XIO; see Fig. 1) (10). In contrast to the proton pumping of BR, ASR is believed to function as a photosensor. Despite the high similarity of their protein sequences, some key residues that are functionally important in the proton-pumping event in BR are not conserved in ASR; Asp-96 in BR, which serves as a proton donor to the Schiff base, is replaced with Ser-86 in ASR, and  and Glu-204 in BR, which is located in the terminal region of the proton transfer pathway, are replaced with Ser-188 and Asp-198, respectively. Although W402 is present in both BR (6) and ASR (10), Asp-212 in BR is replaced with Pro-206 in ASR (Fig.  1). The absence of W401 and W406 in the corresponding H-bond network of ASR may be associated with the absence of an acidic residue corresponding to Asp-212 in BR. FTIR studies have suggested that the O-D stretch in water molecules was only observed at Ͼ2500 cm Ϫ1 in ASR (11), which is ϳ300 cm Ϫ1 higher than the lowest O-D stretching frequency of 2171 cm Ϫ1 in BR (7). The significant difference in the lowest O-D stretching frequency implies that the H-bond properties of W402 are significantly different in BR and ASR. It has been established by FTIR that the lowest O-D stretching frequency of water can be found at less than 2400 cm Ϫ1 in a number of proton-pumping rhodopsins (12). The same tendency also holds true for BR and ASR.
In the BR and ASR crystal structures, the angle defined by the Schiff base N, W402 O, and Asp-85 O atoms (N Lys …O W402 …O Asp ) differs significantly; the angle is 106°in BR (6) and 83°in ASR (10). Thus, it was proposed that the angle difference may differentiate the H-bond strength of the water molecules in BR and ASR (11). On the other hand, it also appears that the O W402 -H…O Asp angle is more crucial than the N Lys …O W402 …O Asp angle to the energetics of the H-bond as it involves a H atom. In general, the O donor -H…O acceptor angle strongly depends on the O donor -O acceptor distance, i.e. the O donor -H…O acceptor angle is more linear when the O donor -O acceptor bond is shorter (13).
In addition, the N Lys …O W402 …O Asp angle in the crystal structure can be directly altered in response to changes in the O W402 -H…O Asp distance. Because of the nature of H-bonds (14 -17), the O W402 -H…O Asp-85 distance could also be predominantly determined by the pK a difference between the H-bond donor (W402) and acceptor (Asp) moieties. In general, a smaller pK a difference yields a short, symmetrical H-bond (14 -17), and the H atom of O W402 -H migrates toward the acceptor moiety, Asp. Unfortunately, the energetics of the O W402 -H…O Asp bond, particularly the pK a difference between W402 and the Asp residue, remain unclear. Thus, it is essential to clarify how the O W402 -H…O Asp distance is energetically determined in the BR and ASR crystal structures.
Here we present calculated O-D stretching frequencies of D 2 O near W402 in BR and ASR in the ground state using a large scale quantum mechanical/molecular mechanical (QM/MM) approach. We also present the potential energy profiles of the H-bond between W402 and Asp-85 in BR and between W402 and Asp-75 in ASR and demonstrate that the frequencies and H-bond properties are ultimately determined predominantly by the pK a values of H-bond acceptors Asp-85 and Asp-75. By calculating pK a (Asp-85) in BR and pK a (Asp-75) in ASR through solving the linear Poisson-Boltzmann equation with explicit consideration of the protonation states for all titratable residues, we are finally able to pinpoint the factors that cause a difference of ϳ300 cm Ϫ1 between the O-D stretching frequencies of W402 in BR and ASR.

EXPERIMENTAL PROCEDURES
As demonstrated in our previous work with photoactive yellow protein (18), we employed the following systematic modeling procedure.
First, we constructed initial molecular models of BR and ASR using their crystal structures and adding hydrogen atoms. Second, to gain better understanding of the electronic structure of W402 and the associated H-bond network, we performed large scale QM/MM calculations for the entire BR and ASR proteins. To gain insight into the QM/MM potential energy profiles of the H-bonds, O W402 -H…O Asp-85 in BR and O W402 -H…O Asp-75 in ASR, we calculated pK a (Asp-85) in BR and pK a (Asp-75) in ASR by simultaneously titrating all titratable residues in BR and ASR. Technical details of each modeling procedure are summarized below.
Atomic Coordinates and Charges-As a basis for the computations, the crystal structures of BR (PDB codes 1C3W (6) and 2NTU (19)) and ASR (PDB code 1XIO (10)) were used. To generate the initial geometries, the positions of the H atoms were energetically optimized with CHARMM (20) using the CHARMM22 force field. During this procedure, the positions of all non-H atoms were fixed, and the standard charge states of all the titratable groups were maintained (i.e. basic and acidic groups were considered to be protonated and deprotonated, respectively). The Schiff base was considered to be protonated. Atomic partial charges of the amino acids and the Schiff base were adopted from the all-atom CHARMM22 (20) parameter set.
Protonation Pattern and pK a -The present computation is based on the electrostatic continuum model created by solving the linear Poisson-Boltzmann equation with the MEAD program (21). To facilitate a direct comparison with previous computational results (e.g. see Refs. 22 and 23), identical computational conditions and parameters, such as atomic partial charges and dielectric constants, were used. To obtain absolute pK a values of target sites (e.g. pK a (Asp-85) of BR), we calculated the difference in electrostatic energy between the two protonation states, protonated and deprotonated, in a reference model system using a known experimentally measured pK a value (e.g. 4.0 for Asp (24)). The difference in the pK a value of the protein relative to the reference system was added to the known reference pK a value. The experimentally measured pK a values employed as references were 7.2 for the Schiff base (25,26), 12.0 for Arg, 4.0 for Asp, 9.5 for Cys, 4.4 for Glu, 10.4 for Lys, 9.6 for Tyr (24), and 7.0 and 6.6 for the N⑀ and N␦ atoms of His, respectively (27)(28)(29). All other titratable sites were fully equilibrated to the protonation state of the target site during the titration. The ensemble of the protonation patterns was sam-pled by a Monte Carlo method with Karlsberg (30). The dielectric constants were set to ⑀ p ϭ 4 inside the protein and ⑀ w ϭ 80 for water. All computations were performed at 300 K, pH 7.0, and an ionic strength of 100 mM. The linear Poisson-Boltzmann equation was solved using a three-step grid-focusing procedure at resolutions of 2.5, 1.0, and 0.3 Å. The Monte Carlo sampling yielded the probabilities (protonated and deprotonated) of the two protonation states of the molecule. The pK a value was evaluated using the Henderson-Hasselbalch equation. A bias potential was applied to obtain an equal amount of both protonation states (protonated ϭ deprotonated), yielding the pK a value as the resulting bias potential.
QM/MM Calculations-We employed an electrostatic embedding QM/MM scheme and used the Qsite (Version 5.6, Schrödinger, LLC, New York) program code as performed in previous studies (32). We employed the restricted density functional theory method with B3LYP functional and LACVP**ϩ basis sets. The geometries were refined using a constrained QM/MM optimization whereby the coordinates of the heavy atoms in the surrounding MM region were fixed to the original x-ray coordinates, whereas those of the H atoms in the MM region were optimized with the OPLS-2005 force field. To investigate the energetics of the entire H-bond network, the QM region was defined as follows (  Table S1 for BR and Table S2 for ASR. The potential energy profile of the H-bond was obtained in the following manner. First, we optimized the geometry without constraints using QM/MM and used the resulting geometry as the initial geometry for the subsequent steps. Next, we moved the H atom from the H-bond donor atom (O donor ) to the acceptor atom (O acceptor ) by 0.05 Å, optimized the geometry by constraining the O donor -H and O acceptor …H distances, and then calculated the energy of the resulting geometry. This procedure was repeated until the H atom reached the O acceptor atom. After obtaining the stable geometry of the QM fragment, we calculated the O-D stretching frequencies of W401, W402, and W406 in BR and of W402 in ASR. The frequency calculations were performed using a numerical differentiation method at the same level as the QM/MM geometry optimization. The calculated frequencies were scaled using a standard factor of 0.9614 for B3LYP (33).

Calculation of the O-D Stretching Frequencies of D 2 O from an Empirical Equation-
The O-D stretching resulting of D 2 O were also evaluated on the basis of the resultant QM/MM optimized geometries by using the correlation of stretching frequency with respect to bond distance of the O donor -D…O acceptor bond, as previously proposed by Mikenda (34). This correlation was empirically expressed with the O-D stretching frequency, O-D , and the O donor -O acceptor bond dis- where A ϭ 2.11 ϫ 10 6 , and B ϭ 3.23 for the O water -D water …O acceptor bond (34). Note that these parameters were derived from spectroscopic and x-ray diffraction data for 61 different solid deuterates, e.g. CaSO 4 ⅐2D 2 O and MnCl 2 ⅐6D 2 O (34).
To predict the frequencies, Equation 1 requires only the O donor -O acceptor distance, which can be preferentially taken from the QM/MM optimized geometry. Remarkably, the O-D stretching frequencies calculated by a QM/MM numerical differentiation method were reasonably well reproduced using Equation 1 solely from the optimized O donor -O acceptor distance, particularly for frequencies Ͻ2500 cm Ϫ1 (see Table 3).
Notably, Equation 1 indicates that the O-D stretching frequency of 2500 cm Ϫ1 corresponds to the O acceptor -O donor distance of ϳ2.8 Å. The deviation from the values calculated with the QM/MM numerical differentiation method appears to be more pronounced for frequencies Ͼ2500 cm Ϫ1 (Fig. 3). Because the original parameters were derived from solid hydrates (34), the larger deviation for weak H-bonds at these frequencies may be due to H-bond patterns or mode couplings specific to the protein environments.

H-bond Geometries Near W402 in BR
The O-D stretching frequencies of D 2 O predominantly depend on the H-bond geometry, particularly the H-bond donor-acceptor distance (O donor -O acceptor ) (34). If the assumed H-bond pattern is not correct, the resulting QM/MM optimized geometries significantly deviate from the original crystal structure. Thus, although the QM/MM optimized geometries (especially the heavy atom positions) are not always consistent with the crystal structures, it is necessary to carefully evaluate the H-bond pattern with respect to the atomic coordinates of the crystal structures before frequency calculations.
In BR, two H-bond patterns are geometrically possible for the H-bond network of W402 (Fig. 4). A model previously presented from FTIR studies (7) suggests that an H atom of W401 has no H-bond acceptor (model 1), whereas an alternative model suggests that an H atom of W406 has no H-bond acceptor (model 2). The QM/MM-optimized geometries of the two models appear to represent the actual H-bond patterns of the   Tables 2 and 3). In the reported QM/MM geometry, W402 forms a new H-bond with another carboxyl O atom of Asp-212 (Fig. 4). Hence, it is to be considered that the band assignment reported by Hayashi and Ohmine (8) (Table  1). However, the small discrepancies could also be due to being artifacts of the crystallization process. Because the resolutions of the two crystal structures are 1.53 and 1.55 Å (which are essentially the same), both of the H-bond tautomers are likely to contribute to the O-D stretching frequencies measured by FTIR. If both tautomers are present, this might corroborate the rapid H/D exchange observed in this region.

Calculated O-D Stretching Frequencies in BR
In FTIR studies (7), the three H-bonds O W401 -H…O Asp-85 , O W402 -H…O Asp-85 , and O W406 -H…O Asp-212 were classified as being strong H-bonds because of the assignment of low frequencies (Fig. 4).
Model 1-In FTIR studies (7) (Table 3). Furthermore, the lowest calculated frequency was observed for O W402 -H…O Asp-85 , which is also consistent with FTIR studies (7). Significantly high correlations of the frequencies predicted from Equation 1 suggest that assignment of the O-D stretching frequencies on the basis of model 2 is also justified by the H-bond geometry. Note that no remarkable mode couplings were observed for model 2 in the QM/MM calculations. 1 and 2, Fig. 4) Distances are in Å, and angles are in degrees. ND, not determined. An underlined number indicates a significant deviation from the distance in the crystal structure. See supplemental S1 for the atomic coordinates of the QM/MM geometries.

H-bond Strength of Water in BR and ASR
OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41

JOURNAL OF BIOLOGICAL CHEMISTRY 34013
In the following section further analysis of BR will be discussed on the basis of model 2 using the crystal structure by Luecke et al. (PDB code 1C3W) (6) unless otherwise specified.

H-bond Geometries near W402 and O-D Stretching Frequencies in ASR
In contrast to BR, the ASR crystal structure only possesses a single water molecule, W402, near the Schiff base (Fig. 4). The H-bond geometry of the crystal structure was reasonably reproduced by the QM/MM geometry optimization (Table 4). In the QM/MM calculations, the lowest O-D stretching frequency of 2376 cm Ϫ1 in ASR, which was found for O W402 -H…O Asp-75 (Table 5), was significantly larger (by ϳ300 cm Ϫ1 ) than that for the corresponding O W402 -H…O Asp-85 in BR (2078 cm Ϫ1 , Table  3). In FTIR studies, the O-D stretching of D 2 O was only observed in the Ͼ2500-cm Ϫ1 region for ASR (11), which was also ϳ300 cm Ϫ1 higher than the lowest frequency of 2171 cm Ϫ1 in BR (7). Because the main purpose of this study is to understand the reason for the significant discrepancy between the lowest frequencies of D 2 O (assumed as W402 (11)) of BR and ASR, these computational results are sufficiently accurate for our purpose to describe the chemical properties of W402.

Significant Differences between the H-bond Energy Profiles of BR and ASR
To identify the origin of the difference in O-D stretching frequencies of W402, we analyzed the potential energy profiles of H-bonds O W402 -H…O Asp-85 in BR and O W402 -H…O  in ASR by altering the H atom position along the O W402 -O Asp-85/75 bond (see Fig. 5). In both H-bonds, the energy minimum was found at the W402 moiety rather than at the Asp moiety, suggesting that in the ground state W402 exists as H 2 O in the presence of deprotonated Asp in BR and ASR.
On the other hand, the potential energy profiles of the Asp moiety were significantly different for BR and ASR. For an H atom along the O W402 -O Asp-85/75 bond, the energy of the Asp-85 moiety of BR is significantly lower than that of the Asp-75 moiety of ASR. Thus, it is more favorable for an H atom of O W402 -H to be at the Asp moiety in BR than in ASR, indicating that pK a (Asp) is significantly higher for BR than ASR (arrow 1 in Fig. 5). As clearly shown in Fig. 5, the lower energy near the Asp-85 moiety in BR leads to greater broadening of the potential-well width near W402 toward Asp-85, which corresponds to an elongation of O W402 -H. Thus, the H atom of O W402 -H migrates toward the H-bond acceptor Asp more in BR than in ASR (arrow 2 in Fig. 5). It should be noted that the longer O W402 -H corresponds to a smaller O-D stretching frequency. It is also obvious from the width of the entire well of the potential energy profiles that the O W402 -O Asp-85 length in BR (ϳ2.6 Å, Table 1), which is shorter than the O W402 -O Asp-75 length in ASR (ϳ2.7 Å, Table 4), is a result of the smaller pK a difference between W402 and the Asp residue in BR relative to the corresponding difference in ASR (arrow 3 in Fig. 5). Thus, from the potential energy profiles (Fig. 5), the difference of ϳ300 cm Ϫ1 in the O-D stretching frequency is mainly due to the difference between the pK a values for the Asp residues in BR and ASR.

Factors that Differentiate pK a (Asp) of BR and ASR
By solving the linear Poisson-Boltzmann equation, we calculated pK a (Asp-85) in BR and pK a (Asp-75) in ASR to be 1.5 and Ϫ5.1, respectively, in the presence of the protonated Schiff base ( Table 6). The calculated pK a (Asp-85) of 1.5 is consistent with an experimentally measured pK a (Asp-85) of 2.2-2.6 in the presence of the protonated Schiff base (2, 35). Although pK a (Asp-75) is not known, the significantly low pK a (Asp-85) in  a Excluding Lys-210 due to obvious displacement of the sidechain carbon atoms from the original crystal structure (Fig. 2c). BR relative to pK a (Asp-75) in ASR is consistent with the QM/MM-calculated potential energy profiles (Fig. 5). The significantly low pK a (Asp-75) in ASR appears to be consistent with the fact that Asp-75 remains deprotonated throughout the photocycle (36). Asp-212 in BR-The most significant difference observed between pK a (Asp-85) and pK a (Asp-75) was induced by the substitution of Asp-212 in BR for Pro-206 in ASR (Fig. 6), which is responsible for the pK a difference of ϳ6 (Table 6). Because Asp-212 is an H-bond acceptor for O W402 -H and is thus far the most proximal negative charge to Asp-85 (5.1 Å), Asp-212 upshifts pK a (Asp-85) by the largest amount among all residues in BR (Table 6).
Although the most significant difference between pK a (Asp-85) and pK a (Asp-75) induced by the substitution of Asp-212 in BR for Pro-206 in ASR is remarkable, this is not sufficient to entirely explain the difference between pK a (Asp-85) and pK a (Asp-75) (see below), which is in agreement with mutational studies of ASR mutant P206D (43).
Ser-47 in ASR-Ser-47 donates an H-bond to Asp-75 in ASR, decreasing pK a (Asp-75) by 3.2, whereas the corresponding residue in BR is the non-polar Ala-53 (Table 6). This was responsible for a pK a difference of 2.7 between Asp-85 and Asp-75 (Table 6).
Glu-194 -Glu-204 Pair in BR-Another remarkable pK a (Asp-85) upshift in BR originates from the Glu-194 -Glu-204 moiety near the terminal region of the proton transfer pathway (2, 3) (Fig. 6). It is likely that the two acidic residues share a proton. The corresponding acidic residue pair is absent in ASR, thereby contributing slightly to the larger pK a (Asp) in BR relative to that in ASR (ϳ0.5 pK a units, Table 6). In this study Glu-204 is protonated and Glu-194 is deprotonated when Ser-193 donates an H-bond to , which is consistent with the previously reported pK a (Glu-204) of ϳ9 (35,44,45).
Nevertheless, the geometry of the crystal structure also suggests that protonated Glu-194 and deprotonated Glu-204 are almost equally possible when Ser-193 donates an H-bond to Glu-204, as suggested by Gerwert and co-workers (46). Thus, it would be more plausible to consider the two acidic residues as a pair that possesses ϳ1 H ϩ as concluded in previous QM/MM simulations (47). This resembles a pair of acidic residues, Glu-L212 and Asp-L213, in the proton transfer pathway of photosynthetic reaction centers, which has been interpreted as sharing ϳ1 H ϩ (48 -50). Deprotonated Glu-194 contributes to an

TABLE 6
Contribution of residues to the pK a shifts of Asp-85 in BR and Asp-75 in ASR (relative to the bulk solvent) in the presence of the protonated Schiff base (in pK a units) For clarity, residue pairs that are responsible for differences of Ͻ0.7 between pK a (Asp-85) and pK a (Asp-75) are not listed except for the Glu-204/Asp-198 pair. Side, side chain; bb, backbone. increase in pK a (Asp-85) of ϳ1 (Table 6); this, therefore, corroborates the linkage between pK a (Asp-85) and pK a (Glu-204) previously reported in mutant BR studies (35) if we consider Glu-194 -Glu-204 as a pair of acidic residues sharing ϳ1 H ϩ .
Orientation of Arg-82 in BR and Arg-72 ASR-Arg-82 was found to contribute to an increase in pK a (Asp-85) of 3.1 in BR (Table 6), which is in agreement with a similar increase (ϳ4.5) reported for BR mutants R82Q and R82A (51,52). Irrespective of the conservation of Arg-82/Arg-72 in BR/ASR, their influence on pK a (Asp-85) and pK a (Asp-75) significantly differs by ϳ2 because the side chain of Arg-72 is oriented away from the Schiff base and toward the extracellular side in ASR (O Asp-75 -N Arg-72 ϭ 9.6 Å) (10), which is the opposite of the orientation of Arg-82 in BR (O Asp-85 -N Arg-82 ϭ 6.6 Å) (6) (Fig. 6). Thus, the differing orientations of the Arg side chains decrease the pK a (Asp) difference between BR and ASR (Table 7).
In summary, the significantly large pK a (Asp-85) in BR relative to pK a (Asp-75) in ASR is largely due to the presence of Asp-212 near Asp-85 in BR and the absence of the corresponding acidic residue in ASR. In addition, the donation of an H-bond from Ser-47 to Asp-75 lowers the pK a (Asp-75) in ASR by facilitating deprotonation. The corresponding polar residue is absent in BR; this upshifts pK a (Asp-85) in BR relative to pK a (Asp-75) in ASR. The Glu-194 -Glu-204 pair is located near the terminal region of the proton transfer pathway in BR, whereas only a single acidic residue is located near the corresponding position in ASR. This also upshifts pK a (Asp-85) in BR relative to pK a (Asp-75) in ASR.

Conclusions
QM/MM calculations revealed that the heavy atom positions in the BR crystal structures could be explained by one of two models. Model 2 can reasonably explain the strong H-bond (2292 cm Ϫ1 ) of O W406 -H…O Asp-212 and the weak H-bond (2636 cm Ϫ1 ) of O W406 -H…O W401 , as previously assigned by FTIR (Table 3). In model 2, the lowest calculated frequency was observed at O W402 -H…O Asp-85 , which is also consistent with FTIR studies. Essentially the same frequencies were also predicted solely from the O donor -O acceptor distances using Equation 1 proposed by Mikenda (34). This suggests that Equation 1 can be used to support the frequency assignments made from spectroscopic studies. In ASR, the lowest calculated O-D stretching frequency is observed at O W402 -H…O Asp-75 , which is ϳ300 cm Ϫ1 higher than that of BR (Tables 3 and 6), as previously reported in FTIR studies of BR (7) and ASR (11).
The potential energy profiles of two H-bonds, O W402 -H…O Asp-85 in BR and O W402 -H…O Asp-75 in ASR, demonstrate that the significant pK a difference between Asp-85 in BR and Asp-75 in ASR is ultimately responsible for the difference of ϳ300 cm Ϫ1 in the O-D stretching frequency of W402 between BR and ASR (Fig. 5). Electrostatic calculations resulted in a pK a (Asp-85) of 1.5 in BR and a pK a (Asp-75) of Ϫ5.1 in ASR ( Table 6). The pK a difference is mainly due to differences in the influences of Ala-53, Arg-82, Glu-194, and Asp-212 on Asp-85 in BR and the corresponding residues Ser-47, Arg-72, Ser-188, and Pro-206 on Asp-75 in ASR, indicating the presence of long FIGURE 6. Residues that contribute to differences between pK a (Asp) of BR (a) and ASR (b).

TABLE 7
Key residues that alter the pK a difference between Asp-85 and Asp-212 in the presence of the protonated Schiff base (in pK a units) Side, side chain; bb, backbone; ND ϭ not determined. range electrostatic interactions along the proton transfer pathways (31). A strongly H-bonded water near the Schiff base, which results from electrostatic interactions between Asp-85 and these acidic residues in BR, could play a key role in proton transfer.