J. Phys. Chem. B 2001, 105, 9887-9895
9887
Spectral Tuning of Photoactive Yellow Protein. Theoretical and Experimental Analysis of Medium Effects on the Absorption Spectrum of the Chromophore Masaki Yoda, Hirohiko Houjou, Yoshio Inoue, and Minoru Sakurai* Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan ReceiVed: May 7, 2001; In Final Form: July 25, 2001
We apply a SCRF-PCM-CI calculation to elucidate the mechanism of spectral tuning in photoactive yellow protein (PYP). It is shown that the calculation well reproduces solvatochromic shifts observed for some model compounds of the PYP chromophore. By regression analysis, we obtain an empirical equation to predict solvatochromic shifts of these compounds for a given set of dielectric constant and refractive index. Next, using a classical electrostatic theory and the crystal structure of PYP, the value of refractive index is calculated for the chromophore-binding pocket. The value of the dielectric constant is estimated from the fact that the binding pocket is highly hydrophobic. On the basis of these results we predict the absorption maximum of PYP. In addition, the spectral tuning mechanism in PYP is divided into three factors, that is, counterion effect, hydrogen-bonding effect, medium effect of the protein matrix, and each contribution is quantitatively evaluated. It is shown that the electronic polarization effects of the protein matrix plays a nonnegligible role in tuning the absorption maximum of PYP as similar to the case of bacteriorhodopsin.
Introduction Photoactive Yellow Protein (PYP) isolated from Ectothiorhodospira halophila is a small water-soluble protein1 and thought to be a photoreceptor for negative phototaxis.2 In response to blue light, PYP enters a photocycle, resembling the one observed in the sensory rhodopsins from archaebacteria.3 Under physiological conditions, this photocycle involves at least two intermediates, which exhibit a red-shifted and blue-shifted absorption maximum relative to that of the ground state, respectively.4,5 The chromophore of PYP has been identified as deprotonated p-coumaric acid (trans-4-hydroxycinnamic acid) covalently bound via thiol ester linkage to Cys 69.6-8 In the dark state, a central, 6-stranded, antiparallel β-sheet flanked by five R-helices is in an R/β fold and the chromophore is buried in the hydrophobic core.9 The hydroxy group of the chromophore is deprotonated and participates in a hydrogen-bonding network with the side chains of surrounding residues, Tyr 42, Glu 46, and Thr 50. The primary event of the photocycle of PYP has been shown to be the transfcis photoisomerization of the vinyl double bond of the chromophore.10-12 Much evidence has been accumulated that large protein conformational changes occur during the photocycle in aqueous solution.13-19 It has been proposed that these conformational changes result in the formation of a signaling state of PYP. PYP shows an absorption maximum at 446 nm, resulting in its bright yellow color.1 On the other hand, free p-coumaric acid absorbs maximally at 284 nm in water. Thus, a large red shift of 13000 cm-1 is induced on binding of the chromophore to the apoprotein. The occurrence of such a red shift is relevant to the biological function of PYP. Elucidation of the physical mechanism of this spectral tuning has attracted much attention. Kroon et al. have investigated the effects of the following three factors on the absorption maximum of the chromophore:20 (i) formation of thiol ester bond, (ii) deprotonation of the chro* Corresponding author. Fax: 81-45-924-5827. E-mail: msakurai@bio. titech.ac.jp.
mophore, and (iii) specific protein-chromophore interactions. According to their results, the contributions of the factors (i), (ii), and (iii) are ∼6000, 4700, and 2300 cm-1, respectively. For convenience, the factor (iii) is hereinafter called PYP shift, which corresponds to the so-called opsin shift observed in retinal proteins such as rhodopsin and bacteriorhodopsin.21 Possible origins of the PYP shift are as follows:20 (i) weakening of the electrostatic interaction between the deprotonated chromophore and its counterion (Arg 52), (ii) conformational change around the C-C single bond of the -CdC-C(-S-)dO fragment of the chromophore, (iii) protein-induced torsional strain on the trans CdC bond in the chromophore. In addition, a forth factor, namely the stabilization of the excited state of the chromophore by polar or polarizable side chains in the binding pocket, should be considered, because recent studies of the opsin shift in bacteriorhodopsin have elucidated a significant contribution of this factor.22,23 It is however difficult to separately quantify these factors by experiment alone. Quantum chemical calculation is expected to provide a good insight into the elucidation of the PYP shift.24,25 Recently, we have developed a new self-consistent reaction field polarizable continuum model (SCRF-PCM), capable of evaluating medium effects with taking into account both orientational and electronic polarization effects of solvent.26 Namely, the excitation energy of a solute molecule is given as a function of dielectric constant and refractive index. Combining the SCRF-PCM theory with configuration interaction calculation (SCRF-PCM-CI method), we can calculate the excitation energy of a solute molecule in solvent.26 With the aid of this SCRF-PCM-CI method, we have succeeded in quantitatively reproducing the opsin shift of bacteriorhodopsin and shown that the polarizable medium effect of the protein matrix, the forth factor mentioned above, is an important factor causing the opsin shift.22 In this study, our SCRF-PCM-CI method is applied to the problem of the PYP shift. It is shown that the calculation well reproduces solvatochromic shifts observed for some model
10.1021/jp011722v CCC: $20.00 © 2001 American Chemical Society Published on Web 09/14/2001
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Figure 1. Molecular structures of chromophore models used in this study.
compounds of the PYP chromophore. By regression analysis, we obtain an empirical equation to predict solvatochromic shifts of these compounds for a given set of dielectric constant and refractive index. Next, using a classical electrostatic theory and the crystal structure of PYP, the value of refractive index is calculated for the chromophore-binding pocket. The value of the dielectric constant is estimated from the fact that the binding pocket is highly hydrophobic. The absorption maximum of PYP is calculated by using the above empirical equation with these medium parameters. In addition, the PYP shift is divided into three factors, that is, counterion effect, hydrogen-bonding effect, medium effect of the protein matrix and each contribution is quantified. It is shown that the electronic polarization effects of the protein matrix plays a nonnegligible role in tuning the absorption maximum of PYP as similar to the case of bacteriorhodopsin.22,23 Methods Modeling of Chromophores. The structures of model compounds studied here are shown in Figures 1 and 2. For propyl p-coumarate (1), S-propyl thio-p-coumarate (2), sodium salt of propyl p-coumarate (3), sodium salt of S-propyl thio-pcoumarate (4), the geometrical parameters were optimized by the HF/6-31+G (d, p) level of theory, using the Gaussian 94 program.27 In 3a-d and 4a-d, the sodium ion, the phenolate oxygen, and the carbon atom covalently bound to that oxygen are placed in a straight line. As a result of the full geometry optimization of 3a and 4a, the optimal distance between the sodium ion and the phenolate oxygen was determined to be 2.0 Å. In the optimization of 3b-d and 4b-d, the cation-anion distances were fixed to be 2.5, 3.0, and 3.5 Å for b, c, and d, respectively. Figure 3 shows a schematic representation of the hydrogen-bonding network formed between the phenolate oxygen of the chromophore and the side chains of neighboring residues, Tyr42, Glu46, and Thr50 and protonated Arg52 in the crystalline state. Compound 5 shown in Figure 2 is a model for
the complex of the chromophore with its counterion, protonated Arg52,28 where the quanidium cation is replaced by a sodium ion. A model for the hydrogen-bonding network shown in Figure 3 is designated as 6 in Figure 2, where the hydroxy groups in Tyr42, Glu46, and Thr50 were replaced by those of methanol molecules and the guanidium cation of Arg52 was again replaced by a sodium ion. The spatial arrangements of the methanol molecules and the sodium ion were determined by reference to the crystalline structure of PYP9 (PDP entry code: 2PHY). In 5 and 6, the sodium ion was placed at the Cζ position of the side chain of Arg52. In 6, the hydroxy oxygen atoms of the three methanol molecules were placed at the positions of the hydroxy oxygen atoms of Tyr42, Glu46, and Thr50, respectively. Then, the methyl carbon atoms were placed at the positions of the carbon atoms directly bound to the hydroxy oxygen atoms in these residues, respectively. Since the crystal structure lacks the coordinates of hydrogen atoms, the positions of the hydrogen atoms in 5 and 6 were determined by the HF/ 6-31+G (d, p) level of calculation. Computational Details. The computational scheme based on the SCRF-PCM-CI method described in ref 26 was incorporated into an INDO/S molecular orbital program,29 which can handle a single-excitation configuration interaction (CI). We took into account the configurations whose zeroth order excitation energies were lower than 12 eV. This threshold was confirmed to be sufficient in reproducing the experimental absorption maxima of p-coumaric acid derivatives. For each molecule, the lowest π-π* excitation energy was regarded as the calculated absorption maximum. The medium surrounding a solute is characterized using two nonspecific parameters, i.e., static dielectric constant and refractive index n. The calculation of excitation energy was performed for several sets of and n values. The value of was taken to be 2.0, 4.0, 10.0, and 80.0, and that of n to be 1.0, 1.2, 1.4, and 1.6.
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J. Phys. Chem. B, Vol. 105, No. 40, 2001 9889
Figure 3. Hydrogen-bonding network existing in the chromophore binding pocket and the counterion against the phenolate oxygen of the chromophore in PYP.
TABLE 1: The List of Aprotic Solvents Used for the UV-Vis Measurements
Figure 2. Molecular structures of the chromophore and its neighboring residues taken from the crystal structure of PYP.
Basically, the cavity in which the solute molecule was accommodated was prepared according to the following previously reported procedure:30 (i) a van der Waals (VDW) sphere was placed at each atomic center of the solute molecule; (ii) the surface of each sphere was divided into longitude-latitude grids with a dividing angle of 10; (iii) grid points placed in the overlapping region of the VDW spheres were deleted. Here we used the effective VDW radii defined as below rather than the standard ones, because the experimental absorption maxima of the model compounds studied were better reproduced by the use of them. The effective VDW radius for each atom of solute is defined as the sum of the standard VDW value for the atom and the standard VDW radius of a representative atom of solvent. This corresponds to the approximation that so-called solvent accessible surface is taken to be an envelope made by the centroid of that representative atom rolling on the VDW surface of the solute. We chose the carbon atom as the representative atom for all the solvents studied, and the resultant effective VDW radii are as follows: 2.77 Å for hydrogen, 3.16 Å for carbon, 2.97 Å for oxygen, 3.42 Å for sulfur, and 2.52 Å for sodium. Experimental. Propyl p-coumarate (abbreviated as propyl pCA) was synthesized by the Fischer esterification reaction and S-propyl thio-p-coumarate (abbreviated as S-propyl TpCA) was
no.
solvent
dielectric constanta
index of refractionb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
pentane hexane heptane benzene toluene diethyl ether diisopropyl ether tetrahydrofrane 1,4-dioxane ethyl acetate acetone 2-butanone dimethyl sulfoxide butyronitrile acetonitrile dimethylformamide pyridine carbon tetrachloride chloroform dichloromethane 1,2-dichloroethane
1.84 1.89 1.92 2.27 2.38 4.34 3.88 7.58 2.21 6.02 20.7 18.5 45.8 20.3 37.5 36.7 12.3 2.23 4.81 8.90 10.4
1.358 1.375 1.388 1.501 1.494 1.350 1.366 1.405 1.420 1.373 1.359 1.376 1.477 1.380 1.346 1.429 1.523 1.463 1.446 1.424 1.442
a
At 25 °C. b For D line at 25 °C.
synthesized according to ref 20. It was confirmed by 1H and 13C NMR experiments that the vinyl bonds of these compounds have trans configuration (data not shown). The solvent dependence of the absorption maximum of each compound was measured using 21 kinds of aprotic solvent listed in Table 1. On the other hand, the sodium salts of propyl pCA and S-propyl TpCA were difficult to be dissolved in low polar solvents. First, propyl pCA and an excess amount of sodium hydroxide were dissolved in ethanol. The concentration of the ethanol solution was adjusted so that the UV/vis spectrum of the chromophore was definitely measurable when a 2-µL aliquot of the solution was added to 3 mL of a solvent listed in Table 1. The UV/vis spectra of the sodium salt of S-propyl TpCA were measured in a similar manner. All the UV/vis spectra were recorded on a Shimadzu UV-2100 spectrometer at room temperature.
9890 J. Phys. Chem. B, Vol. 105, No. 40, 2001
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Results Propyl pCA and S-Propyl TpCA. To assess the reliability of the SCRF-PCM-CI method, we first compared the calculated results for the model compounds 1 and 2 with the corresponding experimental results. According to ref 26, the excitation energy of a chromophore depends on the two medium parameters, namely, the static dielectric constant stat and the optical dielectric constant opt. Hereafter, the static dielectric constant stat is simply denoted as , and the optical dielectric constant opt as n2. The reaction field potential changes as a function of or n2, and thus the absorption maximum λmax (given in cm-1) of the solute is approximately expressed as follows:
νmax ) Af() + Bf(n2) + C
(1)
where f(x) (x ) or n2) is given by
f(x) )
x-1 x+1
(2)
and the coefficients A, B, and C are the parameters intrinsic to the given solute molecule, including its geometry and shape of the cavity. In eq 1, coefficients A and B are regarded as measures of sensitivity to and n2, respectively, while C is the extrapolated value of the excitation energy toward the gaseous state. The first term in eq 1 involves both orientational and electronic polarization effects of solvents, while the second term does only the electronic effect. A way of assessing the accuracy of the calculation is to compare the values of these coefficients with the corresponding experimental values. The absorption spectrum of propyl pCA exhibited a main band split into two peaks due to the presence of a vibrational structure (data not shown). In low dielectric media, the intensity of the peak on the shorter wavelength side (peak 1) was larger than that of the other peak. With increasing dielectric constant, the longer wavelength peak (peak 2) became dominant, with nearly equal intensities in ether ( ) 4). Thus, the data for the low and high dielectric media were separately subjected to regression analysis. In the absorption spectrum of S-propyl TpCA, such vibrational peaks were not observed, and thereby the data in the whole range of dielectric constant were subjected to single regression analysis. In both cases of propyl pCA and S-propyl TpCA, several data points deviated from the regression lines (solid symbols in Figure 4); in the case of propyl pCA the data for chloroform, dichloromethane, water, and ethanol, and in the case of S-propyl TpCA those for dichloromethane, 1,2-dichloromethane, 1,4-dioxane, water, and ethanol. Any specific interactions might work in these solvents, and thereby those points were excluded in the final regression analysis. As can be seen from Figures 4A and 4B, there is good correlation between observed and regression values based on eq 1. Thus the solvatochromic shifts of the absorption maxima are explained well in terms of the continuum model, in other words, in terms of nonspecific interactions between the solute and solvent molecules. The resultant values of coefficients A, B, and C are summarized in Table 2. The values of A and B are negative, indicating that the effects depending on and n2 both cause red shifts. It should be noted that the absolute value of B is considerably larger than that of A, which indicates that the electronic polarization of solvent more strongly affects the absorption maximum of the solute than does the orientational polarization. Regression analysis was also carried out for processing primary data from the calculation because usually these are scattering due to numerical errors occurring in the solvent effect
Figure 4. (A) Correlation between the observed absorption maxima of propyl p-coumarate and their regression values based on eq 1. Numbers represent the solvents listed in Table 1. Solid symbols indicate the data excluded from the regression analysis. (B) Correlation between the observed absorption maxima of S-propyl thio-p-coumarate and their regression values based on eq 1. Numbers represent the solvents listed in Table 1. Solid symbols indicate the data excluded from the regression analysis.
calculation, especially in the numerical evaluation of the reaction field. Those results are also summarized in Table 2. For both of propyl pCA and S-propyl TpCA, the calculated values fairly agree with the corresponding experimental ones, although the calculated value of the coefficient A is smaller than the experimental value in the case of S-propyl TpCA. Therefore, the SCRF-PCM-CI method used is sufficiently reliable for the purpose of the present study. Sodium Salts of Propyl pCA and S-Propyl TpCA. Figure 5, parts A and B, show the correlation between the observed absorption maxima of the sodium salts of propyl pCA and S-propyl TpCA and the results for their regression analyses. The absorption maxima of both salts shift according to eq 1 in low dielectric solvents ( < 10). However, in both cases, there were found systematic derivations from eq 1 in high dielectric solvents ( > 10). Thus, the regression analysis was applied only to the data for the low dielectric solvents (open symbols in Figure 5). In addition, because of low solubility, the
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J. Phys. Chem. B, Vol. 105, No. 40, 2001 9891
TABLE 2: Comparison between the Experimental and Calculated Results for Regression Analysis of the Absorption Maxima of Chromophore Models coefficients/103 cm-1 propyl p-coumarate expt. (peak 1) expt. (peak 2) calcd.a S-propyl thio-p-coumarate expt. calcd.b sodium salt of S-propyl thio-p-coumarate expt. calcd.c sodium salt of S-propyl thio-p-coumarate expt. calcd.d chromophore models in PYP calcd.e calcd.f
C
r2
g
A
B
-0.46 -0.62 -0.18
-6.05 -9.73 -7.34
36.7 0.970 35.8 0.955 36.0 0.966
-2.10 -0.29
-7.72 -7.83
34.7 0.901 34.6 0.929
-1.72 -0.31
-5.24 -6.31
31.0 0.911 30.8 0.949
-1.97 -0.31
-6.76 -7.13
29.8 0.953 29.7 0.957
0.24 0.08
-8.15 -8.32
24.6 0.987 26.3 0.956
a Calculated value for compound 1. b Calculated value for compound 2. c Calculated value for compound 3. d Calculated value for compound 4. e Calculated value for compound 5. f Calculated value for compound 6. g The square of the correlation coefficient.
absorption maximum of sodium salt of propyl pCA could not be determined in diisopropyl ether, dichloromethane, and 1,2dichloroethane, and similarly that of the sodium salt of S-propyl TpCA in benzene, toluene, diisopropyl ether, carbon tetrachloride, dichloromethane, and 1,2-dichloroethane. The observed and calculated values of the coefficients A, B, and C for both salts are summarized in Table 2, where the regression analysis for the calculated values was applied to the data for the absorption maxima of 3a and 4a. The observed solvatochromic shift depends on n2 larger than on , the trend of which is reproduced well by the calculation. Thus, the electronic polarization effect of solvent is an important factor causing the solvatochromic shifts of the absorption maxima of both salts in low dielectric media. In high dielectric media (solid symbols in Figure 5) except water and ethanol, the absorption maxima of both salts appear to systematically deviate from the regression line: namely, the deviation becomes large with increasing dielectric constant. Obviously, a main origin of such deviation is ascribed to electrostatic effects. The dielectric effect of solvent should weaken the salt bridge between the sodium ion and the chromophore. As a result, their distance may be increased, resulting in an increased π-electron delocalization causing a red shift of the chromophore absorption maximum. On the other hand, in low dielectric media, the anion and cation form a tight salt bridge and thereby the solvatochromic shifts are dominated by the nonspecific interactions described by eq 1. To support this interpretation, we obtained the absorption maxima of 3a-d and 4a-d with fixing the medium parameters at ( ) 36.7, n ) 1.429). Here, the four molecules 3a-d (or 4a-d) are different only in the location of the counterion. In Figure 6, the absorption maxima of 3a-d and 4a-d are plotted against 1/R2, where R is the distance between the sodium ion and the phenolate oxygen in the chromophore. As described in the section of “modeling of chromophores”, the R-values of 3a and 4a both are 2.0 Å, those of 3b and 4b 2.5 Å, those of 3c and 4c 3.0 Å, and those of 3d and 4d 3.5 Å. Figure 6 shows that the absorption maxima of both sodium salts of propyl pCA 3 and S-propyl TpCA 4 shift to red with an increase in the R value. In addition, the changes in the absorption maxima are proportional to that in 1/R2 and the slopes for 3 and 4 are nearly equal to each other (2.1 and 1.8) × 103 cm-1 Å2, respectively). These findings
Figure 5. (A) Correlation between the observed absorption maxima of sodium salt of propyl p-coumarate and their regression values based on eq 1. Numbers represent the solvents listed in Table 1. Solid symbols indicate the data excluded from the regression analysis. (B) Correlation between the observed absorption maxima of the sodium salt of S-propyl thio-p-coumarate and their regression values based on eq 1. Numbers represent the solvents listed in Table 1. Solid symbols indicate the data excluded from the regression analysis.
indicate that the R-dependence of the absorption maxima is hardly affected by the difference of the terminal structure, namely ester or thiol ester linkage. This is consistent with the results shown in Figure 5, where the way of deviating from the regression lines is similar in both cases of propyl pCA and S-propyl TpCA. The absorption maxima of 3d and 4d shift to red more than 3000 cm-1 relative to those of 3a and 4a, respectively. This implies that the distance between the counterion and the chromophore is a decisive factor governing the change of the absorption maxima of these compounds. The medium parameters ( ) 36.7, n ) 1.429), used in the calculation of Figure 6, correspond to those of DMF, which has the highest dielectric constant among the solvents used here (Table 1). As can be seen from Figure 5, the deviation from the regression line observed for DMF (data point 16) is more than 3000 cm-1 in both salts. According to Figure 6, such an amount of deviation can be accounted for on the assumption that the anion-cation distances in the solute molecules are elongated, in this case, R g 3.5 Å. The data for the counterion
9892 J. Phys. Chem. B, Vol. 105, No. 40, 2001
Yoda et al. TABLE 3: List of Molar Refractivity (Rm) for Sodium (D) Light C H O N S
2.42 1.10 1.53b 2.45d 7.97h
3.29a 2.21c 2.65e
2.27f
2.71g
Aliphatic carbon. b O-H. c OdC. d Aliphatic primary amine. Aliphatic secondary amine. f Secondary amide. g Tertiary amide. h RS-R. a
e
Figure 6. Calculated absorption maxima of 3a-d (]) and 4a-d ([). In calculation the values of the medium parameters were assumed to be that of DMF, namely ) 36.7, n ) 1.429. R is the distance between the phenolic oxygen of the deprotonated chromophore and sodium ion.
effect obtained here are useful for interpreting the PYP shift as described below. Discussion Spectral Tuning Mechanism in PYP. In the case of propyl pCA, the nonspecific solvent effects cause a red shift of about 3300 cm-1, on going from the absorption maximum (34800 cm-1) in pentane to that (31500 cm-1) in pyridine (Figure 4A). This shift includes a contribution of about 1000 cm-1 that originates from the alternation of the vibrational peak top, namely from peak 1 to peak 2. Figure 4B indicates that the absorption maximum of S-propyl TpCA undergoes a red shift of about 2200 cm-1 from the value (31800 cm-1) in pentane to the value (29600 cm-1) in pyridine. Similarly, from Figures 5A and 5B, the absorption maxima of the sodium salts of propyl pCA and S-propyl TpCA undergo red shifts of about 1100 cm-1 and 1200 cm-1 in the low dielectric media (open circles), respectively. Furthermore, in the absorption spectra of these salts in high dielectric media, the additional red shifts might arise from the change in location of the counterion, the amount of which reaches maximally 24000 and 21700 cm-1 for the sodium salts of propyl pCA and S-propyl TpCA, respectively. Therefore, the experimental results show that the maximal spectral shifts, being observable in solution, reach totally 5000 and 5500 cm-1 for the sodium salts of propyl pCA and S-propyl TpCA, respectively. It is of interest to compare the absorption maximum of the chromophore having ester linkage with that of the corresponding thiol ester. As can be understood from the physical meaning of eq 1, the coefficient C shown in Table 2 indicates the absorption maximum (given in cm-1) of each compound in vacuo. Irrespective of whether the phenolic OH of the chromophore is protonated or deprotonated, the thiol ester compound absorbs at a longer wavelength by about 1000 cm-1 than does the ester. This is consistent with results from the orbital analysis based on ab inito and INDO/S calculations (data not shown), which indicated that by substituting the C-S-CS group for the C-O-C the energies of HOMO and LUMO become higher and lower, respectively, resulting in the decrease in the HOMOLUMO gap. Therefore, the natural selection of thiol ester linkage rather than ester one significantly contributes to a function of PYP as a blue light sensor. In addition, the observed absorption maximum of sodium salt of S-propyl TpCA was longer than
450 nm in both DMSO and DMF (Figure 5B), the value of which shifts to red relative to that (446 nm) of PYP. This implies that the absorption maximum of PYP in the native state can be explained only by the combined effect of the nonspecific interaction considered here and the positional change of the counterion. To support this statement, it may be necessary to estimate the effective values of the medium parameters (dielectric constant, refractive index) of the environment surrounding the chromophore in PYP. In particular, the estimation of the refractive index is more important than that of dielectric constant, because the absolute value of B is considerably larger than that of A (Table 2). In our previous report on the opsin shift of bacteriorhodopsin,22 it was shown that aromatic residues significantly contribute to the increase in electronic polarizability of the chromophorebinding pocket. In that report, we evaluated the effective refractive index of the chromophore-binding pocket with the aid of a theory of electrostatics. Here, the same method is applied to the case of PYP. First, the chromophore-binding pocket is deined as a region made by interlocking spheres, with a radius of 4 Å, centered on each atom of the chromophore. As a result, Phe96 was found to be located in this region. This residue seems to contribute most significantly to the electronic polarizability of the chromophore-binding pocket. It is worth noting that Phe96 is conserved in PYP isolated not only from E. halophila but also from Chromatium salexigens and Rhodospirillum salexigens.31 To estimate the average refractive index of the chromophorebinding pocket in PYP, we used the Lorentz-Lorenz equation given by
n2 - 1 M ‚ ) n2 + 2 F
∑i (Rm)i
(3)
where Rm, M, and F are the molar refractivity of atom i, the sum of atomic weights of all atoms involved in the chromophore-binding pocket, and the density (g/cm-3) of this region, respectively. The value of Rm is assigned to each atom according to Table 3.32 The volume of the binding pocket, the values of M, F, and the right-hand side of eq 3 were evaluated to be 960 Å3, 674, 1.17 g/cm-1, and 175.9, respectively. Consequently, the average refractive index of the chromophore-binding pocket in PYP was determined to be 1.53. Interestingly, this value is close to that (1.51) for bacteriorhodopsin.22 In bacteriorhodopsin, the origin of the high refractive index was ascribed to the fact that several aromatic residues exist around the chromophore.22 In PYP, however, there is only one aromatic residue, that is, Phe96, in the binding pocket. The reason the binding pocket has a high refractive index is ascribed to a high atomic packing of 1.17 g/cm-3, the value of which is fairly higher than those of usual organic solvents, e.g., hexane: 0.66 g/cm-3, benzene: 0.88 g/cm-3. The dielectric constant of the interior of PYP may be inferred from the pKa values of ionizable residues located at the active site. It has been reported that in the dark state of PYP the pKa
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J. Phys. Chem. B, Vol. 105, No. 40, 2001 9893
TABLE 4: Absorption Maxima Observed for Mutants of PYP ∆νb/cm-1
λmax/nm wild type Y42Fa E46Qa T50Va R52Qa
446 458 460 457 447
-587 -682 -539 -50
a
Taken from ref 35. b The shift from the absorption maximum of the wild type. The minus sign indicates red shift.
TABLE 5: Comparison between the Calculated and Experimental PYP Shifts this work ∆λdeprot ∆λdeprot (PYP shift) a
TABLE 6: Calculated Absorption Maxima (/103 cm-1) of the Chromophore Modelsa
calcd.
expt.
Kroon et al.a
4700 4400
4500 4700
4310 2960
Taken from ref 20.
of the OH group of the chromophore is 3< and that of the carboxyl side chain of Glu46 is 7
