J. Phys. Chem. 1987, 91, 804-819
804
structure and choice of model cluster.
Conclusions CHFPT calculations using large s,p bases and flexible d polarization functions semiquantitatively reproduce the experimentally observed high 31Pchemical shielding of P4. Although dependence of the total shielding on the choice of gauge origin is still substantial, the results clearly indicate that a center of mass choice of origin is to be preferred. In contrast to P,, the molecules P2 and PN have negative isotropic chemical shieldings and very large shift anisotropies. The large magnitude of upavin P2 can be related to mixing of P 3po P 3 p r * character. When the
-
P 3pr* orbital is filled in P2-, the magnitude of upav is greatly reduced. The high shielding of P4 arises from unexpectedly positive values of oPav,particularly for the contributions from the 2e and 6t2 orbitals. Calculations with an s,p basis only give much more negative values for these orbital contributions. It thus appears that presently feasible C H F P T calculations can describe some of the major trends in 31PN M R shifts and can relate them to their electronic origins.
Acknowledgment. This work was supported by the National Science Foundation, Grant No. EAR-82- 13115. Registry No. P,. 12185-10-3; P,, 12185-09-0; PN, 17739-47-8.
Vibrational Analysis of the 13-cis-Retinal Chromophore in Dark-Adapted Bacteriorhodopsin Steven 0. Smith,la,cJohannes A. Pardoen,lb Johan Lugtenburg,lb and Richard A. Mathies*'" Departments of Chemistry, University of California, Berkeley, California 94720, and Leiden Unioersity, 2300 RA Leiden, The Netherlands (Received: July 11, 1986)
We have obtained resonance Raman spectra of the 13-cis-retinal chromophore in dark-adapted bacteriorhodopsin (BR548) using purple membrane regenerated with isotopic retinal derivatives. 13C substitutions were at positions 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15, while the deuterium substitutions were at positions 7, 8, IO, 11, 12, 14, and 15 and on the Schiff base nitrogen. Raman spectra have also been obtained of the 13-cis-retinal Schiff base and protonated Schiff base model compounds. The major lines in the BR,,, spectrum are assigned to specific vibrations on the basis of their isotopic shifts and by comparison with the assignments in BRm. A modified Urey-Bradley force field was adapted from vibrational calculations on BR,,, and refined to reproduce the frequencies and isotopic shifts in the BR548 derivatives. The distinctive pattern of vibrational frequencies and intensities observed in the 1100-1 300-cm-' "fingerprint region" in BR548results in part from the reduced frequency of the 1167-cm-' CI4-Cl5stretching mode compared to BR,,,. The lower Cl4-CI5frequency is due primarily to decreased kinetic interaction between the c14-c]5stretch and the skeletal bends associated with the cis CI3=CI4 and C=N bonds. The intense 1183-cm-' fingerprint line is assigned to the Clo-CIIstretching mode, while the methyl-substituted Cs-C9 and CI2-Cl3stretches are at 1202 and 1234 cm-', respectively. Comparison of the C-C stretching vibrations of BR548 with the 1 3 4 s (C=N trans) protonated retinal Schiff base shows a general increase in the C-C frequencies in the pigment due to increased *-electron delocalization once the effects of C=N isomerization are taken into account. Evidence for the C=N cis configuration in BR,,, comes from the large (41 cm-') shift of the c14-c]5stretch upon N deuteriation as well as the high frequency of the CI5Drock (1047 cm-I). The CI4Hout-of-plane wagging vibration at 800 cm-' in native BR548 and the CI2D+ C14Din-plane rock combination at 944 cm-I in 12,14-dideuterio BR,,, provide reliable marker bands for the CI3=Cl4 cis configuration. Comparison of the BR548spectra with those of the all-trans and 1 3 4 s Schiff base model compounds and with BRS6,allows us to identify the spectral features and vibrational coupling patterns which are diagnostic of the C13=C14and C=N configurations in retinal pigments.
Bacteriorhodopsin (BR)2 is a photoreactive membrane protein ~ found in the purple membrane of Halobacterium h a l o b i ~ m . The light-sensitive chromophore in BR is the protonated Schiff base (PSB) of retinal bacteriorhodopsin is covalently bound to lysine 216 within the protein interior., In the light-adapted state (BR568) the retinal-protein complex exhibits a broad visible absorption band with a maximum at 568 nm which gives rise to its char(1) (a) University of California, Berkeley. (b) Leiden University. (c) Current address: Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA 021 39. (2) Abbreviations used: BR, bacteriorhodopsin; PSB, protonated Schiff base; RR, resonance Raman; FTIR, Fourier transform infrared; HOOP, hydrogen out-of-plane. (3) For bacteriorhodopsin reviews, see: (a) Birge, R. R. Annu. Reo. Eiophys. Bioeng. 1981, 10, 315. (b) Stoeckenius, W.; Bogomolni, R. A . Annu. Reti. Biochem. 1982, 51, 5 8 7 . (4) (a) Kouyama, T.;Kinosita, K.; Ikegami, A. J . Mol. Biol. 1983, 165, 91. (b) Rothschild, K. J.; Argade, P. V.; Earnest, T. N.; Huang, K.-S.; London, E.; Liao, M.-J.; Bayley, H.; Khorana, H. G.; Herzfeld, J. J . Biol. Chem. 1982, 257, 8592. (c) Huang, K.-S.; Liao, M.-J.; Gupta, C. M.; Royai, N.; Biemann, K.; Khorana, H . G. J . B i d . Chem. 1982, 257, 8596. (d) Katre, N. V.: Wolber, P. K.; Stoeckenius, W.; Stroud, R. M. Proc. Natl. Acad. Sci. CLS.A. 1981. 78, 4068.
acteristic purple color. Absorption of a photon by BR56Binitiates a cyclic photochemical reaction which drives the transport of protons across the bacterial cell membrane. The intermediates in this proton-pumping photocycle were first characterized by their absorption spectra and reaction kinetics., In the dark, bacteriorhodopsin converts to a dark-adapted state (BR,,,) which contains a mixture of BR,,, and BR,,,. Photoisomerization of the retinal chromophore in bacteriorhodopsin's photocycle has been studied extensively by resonance Raman (RR)6 and Fourier transform infrared (FTIR)' spec(5) Lozier, R. H.; Bogomolni, R. A,; Stoeckenius, W. Biophys. J . 1975, 15, 955. (6) (a) Smith, S. 0.; Lugtenburg, J.; Mathies: R. A . J . Memb. Biol. 1985, 85, 95. (b) Mathies, R. A,; Smith, S. 0.;Palings, 1. In Biological Applications ofRaman Spectroscopy, Spiro, T. G., Ed.; Wiley: in press. (c) Terner, J.: El-Sayed, M. A. Acc. Chem. Res. 1985, 18, 331. (d) Stockburger, M.; Alshuth, T.: Oesterhelt, D.; Gartner, W. Ado. Infrared Raman Spectrosc., in press. (7) (a) Rothschild, K. J.; Roepe, P.; Lugtenburg, J.; Pardoen, J . A,; Biochemistry 1984, 23, 6103. (b) Bagley, K.; Dollinger, G.; Eisenstein, L.; Singh, A . K.; Zimanyi, L. Proc. Natl. Acad. Sci. U.S.A. 1982, 7 9 , 4972. (c) Engelhard, M.: Gerwert, K.; Hess, B.; Kreutz, W.; Siebert, F. Biochemistry 1985. 24. 400.
0022-3654187I209 1-0804$0 1.5010 0 1987 American Chemical Society
The Journal of Physical Chemistry, Vol. 91, No. 4. 1987 805
Vibrational Analysis of BRs48 troscopy. In the resonance Raman experiments, laser excitation within the visible absorption band of bacteriorhodopsin selectively enhances Raman scattering from the chromophore. FTIR vibrational spectra of the retinal chromophore are obtained by difference techniques which eliminate protein vibrations that do not change during the photochemical reaction. With both methods, the frequencies and intensities of the lines observed in the vibrational spectra are sensitive to chromophore structure and protein environment. Qualitative comparison of BR vibrational spectra with spectra of geometric isomers of retinal and its Schiff bases has been used to argue that the retinal configuration is all-trans in BR568and 13-cis in the K, L, and M intermediates.*-l' A more quantitative approach can be built upon differences in geometry-sensitivevibrational coupling between particular internal coordinates of the chromophore. In this case, the observed spectral lines are assigned to specific vibrational normal modes, and model compound assignments and normal-mode calculations are used to determine the expected changes in mode character or frequency which arise from a change in chromophore structure. For example, specific assignments have recently been used to determine the C=N configuration and the CI4-Cl5conformation in BR568and many of its intermediate^.^^.'^ To confirm and extend these structural conclusions, and to address more subtle effects of protein environment, it is necessary to compare complete vibrational assignments for the retinal model compounds and bacteriorhodopsin's intermediates. The first step in this process was the vibrational analysis of the retinal isomers and their isotopic derivatives by Curry et al.14-16 Subsequent analysis of the all-trans protonated Schiff base explored the effect of Schiff base formation and protonation on the Raman spectrum.]' The effect of binding the all-trum-PSB to bacterio-opsin has recently been studied by performing a detailed vibrational analysis of BR568 and its isotopic derivatives.I8 However, the vibrational analysis of a cis-retinal isomer in a pigment has not yet been reported. In this paper we present a vibrational analysis of the 13-cisretinal chromophore in BR548(I). The analysis presented here
H 0 0
5
A-
strongly supports the conclusions that the C=N linkage is pro(8) (a) Braiman, M.; Mathies, R. Biochemistry 1980, 19, 5421. (b) Braiman, M.; Mathies, R. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 403. (9) Aton, B.; Doukas, A. G.; Callender, R. H.; Becher, B.; Ebrey, T. G. Biochemistry 1977, 16, 2995. (10) (a) Stockburger, M.; Klusmann, W.; Gatterman, H.; Massig, G.; Peters, R. Biochemistry 1979, 18, 4886. (b) Terner, J.; Hsieh, C.-L.; Burns, A. R.; El-Sayed, M. A. Biochemistry 1979, 18, 3629. (c) Argade, P. V.; Rothschild, K. J. Biochemistry 1983, 22, 3460: (11) Lewis, A,; Spoonhower, J.; Bogomolni, R. A.; Lozier, R. H.; Stoeckenius, W. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4462. (12) Smith, S. 0.;Myers, A. B.; Pardoen, J. A,; Winkel, C.; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2055. I (13) Smith, S.0.;Hornung, I.; van der Steen, R.; Pardoen, J. A.; Braiman, M. S.;Lugtenburg, J.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 967. (14) Curry, B.; Broek, A.; Lugtenburg, 3.; Mathies, R. J . Am. Chem. Soc. 1982, 104, 5214. (15) Curry, B.; Palings, I.; Broek, A.; Pardoen, J. A,; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R. J . Phys. Chem. 1984, 88, 688. (16) Curry, B.; Palings, I.; Broek, A,; Pardoen, J. A,; Lugtenburg, J.; Mathies, R. Adu. Infrared Raman Spectrosc. 1985, 12, 115. (17) Smith, S. 0.;Myers, A. B.; Mathies, R. A,; Pardoen, J. A,; Winkel, C.; van den Berg, E. M. M.; Lugtenburg, J. Biophys. J . 1985, 47, 653. (18) Smith, S. 0.;Braiman, M.; Myers, A. B.; Pardoen, J. A.; Courtin, J. M. L.; Winkel, C.; Lugtenburg, J.; Mathies, R. A. J . A m . Chem. SOC.,in press.
I
I
I
I
I
I
I
I
I
800 I000 1200 1400 160C Figure 1. Resonance Raman spectra of dark-adapted BRSm(A), lightadapted BR568(B), and BRSd8( C ) . The spectrum of BRsa was obtained by subtracting -40% of spectrum B from spectrum A.
tonated, that the C13=C14 bond is in the cis c o n f i g ~ r a t i o n , ~ - ~ ~ ~ ~ ~ and that the C=N Schiff base bond is cis (or syn).12,20 Comparison of the BR548spectra and assignments with those of BR568 and the 13-cis-retinal model compounds allows us to identify the effects of retinal isomerization on the vibrational spectrum of a protein-bound chromophore. This provides a more detailed understanding of the vibrational structure of BR548 and also forms a basis for the interpretation of the vibrational spectra of other 13-cis intermediates in bacteriorhodopsin, as well as in halorhodopsin and sensory rhodopsin.
Experimental Section The syntheses of the 13C-and 2H-labeled derivatives of retinal have been reviewed in ref 21. The isomeric purity was 298% as determined by high-performance liquid chromatography. The isotopic purity was 198% for each position deuterated and 292% for each position labeled with 13C based on mass spectrometric analysis. Bacterio-opsin was isolated and purified from a retinal deficient strain of H.halobium (JW5)and regenerated with isotopically labeled retinal according to procedures in ref 22. Raman spectra of dark-adapted bacteriorhodopsin were obtained with 514.5-nm excitation by using a recirculating rapid-flow system.22 The laser power was reduced to 5 mW and cylindrically focused (focal length = 3.7 cm) to keep the sample from becoming light-adapted during the course of the experiment. In general, = (3.824 X the bulk photoalteration parameter,23Fbulk P@eLTV',was kept below 0.1. In this equation, P is the incident laser power, L is the path length of the laser beam through the sample, Tis the total irradiation time, Vis the total sample volume, @ is the quantum yield for photoreaction, and t is the extinction coefficient. In cases where V was below 150 mL, it was necessary to wait 10-20 min between successive scans for the sample
-
(19) (a) Terner, J.; Hsieh, C.-L., El-Sayed, M. A. Biophys. J. 1979, 26, 527. (b) Alshuth, Th.; Stockburger, M. Ber. Bunsenges. Phys. Chem. 1981, 85, 484. (c) Marcus, M. A,; Lewis, A. Biochemistry 1978, 17, 4722. (20) Harbison, G. S.;Smith, S . 0.; Pardoen, J. A,; Winkel, C.; Lugtenburg, J.; Herzfeld, J.; Mathies, R.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1706. (21) (a) Lugtenburg, J. Pure Appl. Chem. 1985, 57, 753. (b) Lugtenburg, J. Spectroscopy of Biological Molecules, NATO AS1 Series C, 139; Reidel: Boston, 1984; pp 447-455. (22) Smith, S.0.;Pardoen, J. A,; Mulder, P.P. J.; Curry, B.; Lugtenburg, J.; Mathies, R. Biochemistry 1983, 22, 6141. (23) Mathies, R. Chem. Biochem. Appl. Lasers 1979, 4, 55.
The Journal of Physical Chemistry, Vol. 91, No. 4, 1987
Smith et al.
to become fully dark-adapted. Since the rate of dark adaptation increases with t e m p e r a t ~ r ethe , ~ ~spectra were obtained with the sample reservoir maintained at -28 OC. The purple membrane solutions were buffered in distilled water at pH 7 with 10 mM Hepes and had an absorbance of 1-2 at 570 nm (1-cm path length). Figure 1 illustrates the procedure for obtaining spectra of BRS4. The Raman spectrum of light-adapted bacteriorhodopsin (BR568) is subtracted from that of dark-adapted bacteriorhodopsin (BR560) to produce the BR548spectrum. This method is based on chemical extraction results which indicate that the 13-cis component (Le. BR548) represents 50-60% of the dark-adapted mixture.2s This procedure has been tested by showing that spectra of BR,,, obtained by using bacteria-opsin regenerated with 13-cis-retinal are identical with those obtained by subtraction.z6 Raman spectra of the 13-cis Schiff base and protonated Schiff base model compounds were obtained in melting point capillaries by using 40 mW of 676-nm excitation and 15-20 mW of 752-nm excitation, respectively. N o evidence of isomerization or sample degradation was observed for the Schiff bases. The 13-cis protonated Schiff bases isomerized rapidly to the all-trans configuration if the incident laser power or sample temperature was too high. This was most easily monitored by following the increase in the 1237-cm-' line which is characteristic of the all-trans isomer. To decrease the rate of conversion, spectra were obtained in a Harney-Miller cell at -0 "C. The Raman apparatus consists of a Spex 1401 double monochromator with a Spex 1419 illuminator and photon-counting detection (PAR 1105/1120). The monochromator was stepped in 2-cm-l increments and the spectral resolution was -4 cm-l. Digitized data were averaged, smoothed (three point sliding average), and corrected for detector sensitivity. Fluorescence backgrounds (simulated by a quartic polynomial) were subtracted with a PDP 11 /23 computer. Computational Methods. The normal-mode calculations were performed by using the Wilson FG method.27 The geometry for the 6-s-trans- 13,15-di-cis-PSB was obtained from a calculation using the QCFF-a method.28 The C6-C7 bond was placed in the s-trans conformation to be consistent with recent solid-state N M R and chemical analogue experiment^.^^ The methyl groups were made tetrahedral to allow the transfer of force constants developed for small molecules and were rotated to have reflection symmetry in the plane of the polyene chain. The chromophore was truncated by replacing carbons 1 , 4, and 18 of the ionone ring and the &carbon of the lysine group with "R" groups, which have a mass of 15, a valence of 1, and the default potential parameters of an sp3 carbon. The starting force field was taken from calculations on BR568.18Force field changes appropriate for a 13,15-di-cis chromophore were adapted from vibrational calculations on 13cis-retinalk5and polyene model compounds30 and are indicated ) by an asterisk in Table I. The (Cl,Cl,Cl,, C l 3 C I 4 C l 5and (C,,C,,N, Cl5NClys)interaction constants across the cis CI3=Cl4 and C=N bonds, respectively, were set equal to 0.17 mdyn A/rad2 in analogy with the values used for cis bend-bend interaction constants in 13-cis-retinal and cis-hexatriene. In analogy with 13-cis-retinal, the (C20C,3C14,C13C14H)interaction constant was set equal to -0.026 mdyn &'rad2 and a Urey-Bradley interaction constant F(HI2-Hl5) was introduced between the hydrogens o n C I 2and C I S . The (HC,,N, C,,NH) interaction constant for the
cis bends associated with the C=N bond is similar to the (HClICl2,CIICI2H)constant used in calculations on 11-cis-retinal. The force field was refined by fitting to the observed frequencies in native BR5@and 16 of its isotopic derivatives: N-D; 15-D; 14-D; 12-D; 1 I-D; 10,l l-Dz; 8-D;7-D; 14,15-13C; 13-I3C; l2-I3C; 10,l l-I3C; 9-I3C; 8-I3C; 7-I3C; and 6-I3C. These derivatives incorporate I3C and 2H labels at each position of interest along the retinal chain and were the minimum set necessary for successful iteration of the force constants. For the out-of-plane vibrations, only the sign and magnitude of the (15w,Nw) and (13CH3w,14w) interaction constants were changed from the BR568out-of-plane force field to fit the experimental frequencies of native BRS4,and its N-D, 15-D, 14-D, 12-D, and 12,14-D2derivatives. Curry et al.I5 have shown that the interaction constant between cis wags should be approximately -2/3 the value of trans wags. Changes to the out-of-plane field were minimized since most of the HOOP vibrations have little or no intensity in BR548. Experimental frequencies corresponding to vibrations localized on the methyl groups were not included in the force constant refinement. The final force field in Table I was iterated to fit 210 in-plane frequencies of native BR548and 16 isotopic derivatives with a rms error of 5.2 cm-' (maximum error 21 cm-'). In addition, the final force field fit 120 in-plane frequencies of an additional 10 isotopic derivatives with an rms error of 5.5 cm-' (maximum error 24 cm-I). The molecular geometry and the complete force field are available in the supplementary material.
806
(24) Sperling, W.; Carl, P.; Rafferty, Ch. N.; Dencher, N. A. Biophys. Struct. Mech. 1977, 3, 79. ( 2 5 ) Pettei, M . J.; Yudd, A. P.; Nakanishi, K.; Henselman, R.; Stoeckenius, W.Biochemistry 1977, 16, 1955. (26) Braiman, M. S . Ph.D. Dissertation, University of California, Berkeley, 1983. (27) Wilson, E. B.; Decius, J. C.; Cross, P. C. Molecular Vibrations; McGraw-Hill: New York, 1955. (28) Warshel, A.; Karplus, M . J . Am. Chem. SOC.1974, 96, 5677. (29) (a) Harbison, G. S.; Smith, S. 0.;Pardoen, J. A,; Courtin, J. M. L.; Lugtenburg, J . ; Herzfeld, J.; Mathies, R. A,; Griffin, R. G. Biochemistry 1985, 24, 6955. (b) van der Steen, R.; Biesheuvel, P. L.; Mathies, R. A,; Lugtenburg, J. J . Am. Chem. SOC.1986, 108, 6410. (30) Curry, B. Ph.D. Dissertation, University of California, Berkeley, 1982.
Results
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The strong vibrational lines observed in the Raman spectrum a* of BR548 (Figure 1C) are enhanced by the resonant a electronic transition of the protein-bound retinal chromophore. Those vibrational modes associated with the nonconjugated 0ionone ring and lysine side chain are generally weak or absent in the Raman spectrum. Since the chromophore is nearly planar, the BR548vibrational spectrum can be roughly divided between in-plane and out-of-plane normal modes. We will first discuss the in-plane vibrations involving the C=C stretches (1 500-1600 cm-'), the C C H rocks (1250-1450 cm-I), and the C-C stretches (1 160-1 250 cm-I), and then the out-of-plane vibrations involving the chain vinyl hydrogens (700-1000 cm-I). Finally, we present the assignments of the C - C stretching modes in the 13-cis Schiff base model compounds. ( A ) In-Plane Chain Vibrations. C=C Stretches. The Raman spectra of BRS4, and its '3C-isotopic derivatives are presented in Figure 2. The calculated normal modes and assignments for ethylenic stretching native BR54 are given in Table 11. The C==€ region from 1500 to 1600 cm-' in native BR548(Figure 2A) exhibits only two resolved bands at 1536 and 1599 cm-I. Deconvolution of the intense 1536-cm-' band reveals three less intense lines at 1515, 1550, and 1570 cm-I (Table 111). The 1536-cm-' ethylenic mode is 9 cm-' higher in frequency than the 1527-cm-' ethylenic mode in BR568. A similar shift in the main ethylenic frequency was observed between all-trans- and 13-cis-retinal. This was attributed to a decreased potential interaction between the C=C stretching coordinates due to the cis bend which shortens the effective region of conjugation and decreases a-electron del o ~ a l i z a t i o n .This ~ ~ ~shift ~ ~ is correlated with the blue shift in A, between all-trans- (380 nm) and 13-cis- (374 nm) retinal as well as between and BR548. To reproduce the higher C=C frequency in the BR,,, calculation, the magnitude of the k(C=C, C=C) coupling constant between the conjugated stretches was lowered from its value of -0.28 mdyn/A in BR,,, to -0.24 mdyn/A;. This change is similar to that needed to reproduce the C=C frequencies in calculations on all-trans- and 13-cis-retinal (-0.3 1 -0.25 mdyn/A). The I3C shifts of the observed vibrational lines provide an estimate of the contribution of the individual C=C stretching internal coordinates to the ethylenic normal modes. For example, a pure C=C stretching coordinate should shift -30 cm-I upon a single I3C substitution. The main ethylenic line is calculated to be an in-phase combination of the five C=C stretches (Table 11). We observe shifts of 12 cm-I in both the 11- and 12-[I3C]
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The Journal of Physical Chemistry, Vol. 91, No. 4 , 1987 807
Vibrational Analysis of BRS48 TABLE I: Modified Urey-Bradley Force Field for BR," coordinate
force constant
coordinate
Chain Stretches K(5=6) K(7=8) K(9=10) K(11=12) K(13=14) K(C=N) K(6-7) K(8-9) K(10-11) K( 12-1 3) K( 14-1 5) K(C-R) K(C1,s-R) K(C-H) K(C15-H) K(N-H) K(C1,s-H) K(N-Ci,,)
force constant non-Urey-Bradley
6.98 6.50 5.85 5.59 5.70 7.40 2.44 3.47 4.15 4.72 4.43 2.55 2.23 4.83 4.00 4.83 4.71 2.74 Chain Bends
H(CCC) H(CCC) cisb H(C=CH) H(C-CH) H(RCR) H(CC=N) H(C=NC) * H~N-CR)' H(N=CH) H(C=NH) H(C-NH) H(R-CH) H(N-CH) H(HCI,sH) H(C6C7H) H(C9C8H)
0.570 0.760 0.300 0.330 0.700 0.570 0.570 0.690 0.194 0.293 0.345 0.310 0.389 0.506 0.450 0.423
Urey-Bradley * F(H12***H15)C F(C=CH) F(C-CH) F(CCC) F(RCR) or (RCC) F(RCH) F(N=CH) F(C=NH) F(C-NH) F(N-CH) F(CC=N) F(C=NC) F(N-CR) F(HCI,SH)
0.06 0.52 0.49 0.35 0.59 0.55 1.20 0.50 0.55 0.48 0.80 0.59 0.32 0.13
k(C=C,C=C) k(C=C,C=N) k(C=C,C-C) k(C-c,c-C) k(CC,CR) k(CC,CC)" m(CC,bend)e h (bend,bend)' h(bend,bend)g h(CCC,CCC) trans h(CCC,CCR) cis ~(Cl2Cl3C,4~Cl3Cl4H)* ~(Cl2Cl3Cl4~CI3Cl4CI5), ~(C2oCl3Cl4~Cl3Cl4CI5~* ~(C2OCl,Cl4,CI3C14H)* ~(HCI&"CI~NH)* h(Cl'lCl,N,Cl5NCI,~)*
-0.240 -0.673 0.237 -0.060 0.103 -0.130 0.046 0.034 0.070 0.1 12 0.338 0.06 0.17 0.04 -0.026 0.10 0.17
CHI Group K(CW K(C-CH3) H(CCH) H(HCH) F(CCH) F(HCH) h(HCH,HCH) h(CCH.CCH)
4.709 2.550 0.395 0.506 0.480 0.130 -0.020 0.034 Out-of-Plane
H(7w) H(8w) H(l0w) H(l1w) H ( 12w) H(14w) H(15w) HWw) H(CH3) t (C-C) t(C=C) t(C-CH3) t(N-CHJ ( 6 ~ ~ 7 ~ ) (8~,9CH3w) (lOw,l lw) (12w,13CH3w) (14w, 15w) ( 5 ~ ~ 6 ~ ) (7 w ,8w ) (9CH3w,low) (1 lw,12w) ( 13CH3w,14w)* (15w,Nw)* (14w,Nw) (bW)d (t*w), (t(C-CH,),w) I(t,CCH) I(w,CCH) methyl I(w,CCH) adjacent
0.520 0.494 0.475 0.476 0.480 0.450 0.553 0.386 0.570 0.197 0.545 0.081 0.081 -0.130 0.088 -0.063 0.073 -0.055 -0.172 -0.178 0.174 -0.168 -0.094 0.045 -0.014 0.292 0.116 -0.052 0.056 0.080 -0.018
'Modified Urey-Bradley force field adapted from ref 18. Asterisks indicate force constants changed from the force field as a result of CI3=Cl4 and C=N isomerization. Symbols used: K,diagonal stretch; H, diagonal bend; F, Urey-Bradley quadratic constant (linear term set equal to -0.lF); k, stretch-stretch interaction; h, bend-bend interaction (two common atoms); m,stretch-bend interaction (one common atom); t, chain torsion; (t,w),, wag-torsion interaction across a double bond; (t,w), wag-torsion interaction across a single bond; I(t,CCH), chain torsion-methyl bend interaction; I(w,CCH), chain wag-methyl bend interaction. Stretching force constants in mdyn/A, stretch-bend cross terms in mdyn/rad, bending constants in mdyn A/rad2. bApplies to all CCC or CCCH, bends that are cis to another such bend. cF(H12-.H15)is a central force quadratic constant representing the through space repulsion between HI, and His. The corresponding linear term was set to zero. "Applies to pairs of chain stretches separated by three or five bonds. Interactions involving the c5=c6 bond have not been included. 'This term applies to all interactions between chain CC stretches and CCC or CCH bends which have a nonvertex atom of the bending angle in common. Its sign is positive if the substituents are trans and negative if they are cis. 'Applies to bend-bend interactions across trans double bonds. gApplies to bend-bend interactions across trans single bonds. derivatives, 4 cm-' in 8-[13C] BR548, 7 cm-' in 7-[I3C] BR548, and 5 cm-l in 10-['3C] BR5,*. T h u s the major contribution to the 1536-cm-l mode comes from the C l l = C 1 2 and C,=C8 stretching coordinates. T h e r e is some contribution from C,=Clo a s well.
In the 11- and 12-[I3C] derivatives the 1533-cm-' line is calculated to shift 1 3 a n d 14 cm-I, respectively, while shifts of 5 and 6 cm-' a r e calculated in 7- a n d 8-[I3C] BR5,,. T h e close agreement between the calculated and observed I3C shifts indicates that the
Smith et al.
808 The Journal of Physical Chemistry, Vol. 91, No. 4, 1987 TABLE II: Freauencies ( c d ) and Assignments for BRqra calcd obsd 1634 1599 1570 1536 1515 1461 1461 1447 1447 1412 1375 1375 1344 1328 1312 1301 1279 1234 1202 1183 1167 1050 1012
1633 1598 1593 1558 1533 1516 1439 1435 1433 1433 1410 1401 1392 1392 1379 1345 1321 1313 1303 1277 1253 1234 1201 1184 1165 1122 1041 1026 1004 997 980
description"
+ +
0.34(C=N) - 0.14(14-15) - O.lO(N-C) - 0.56(NH) 0.52(15H) 0.19(5=6) - 0.10(11=12) - 0.14(6-7) 0.62(7H) - 0.55(8H) 0.27(7=8) 0.09(13=14) 0.11(8-9) 0.48(8H) 0.32(5=6) - 0.15(7=8) - 0.12(9=10) 0.45(14H) 0.26(13=14) - 0.18(9=10) - 0.14(14-15) - 0.12(12-13) 0.25(11=12) 0.15(9=10) 0.10(5=6) 0.09(7=8) 0.08(13=14) 0.17(10-11) - 0.17(12-13) 0.62(11H) - 0.60(12H) 0.24(9-10) 0.15(13=14) - 0.13(11=12) - 0.15(14-15) - 0.12(12--13) - 0.57(148) ( 19-CH,) in-plane deformation (20-CH,) in-plane deformation (19-CH3) out-of-planedeformation (20-CH3) out-of-planedeformation 0.80(15H) 0.23(NH) + 0.25(14H) (19-CH3) symmetric deformation (20-CH,) symmetric deformation 0,37(14H) 0,55(15H) - 0.04(12-13) 0.15(13-CH,) 0.48(10H) - 0.30(14H) - 0.09(9-CH,) 1.07(NH) - 0.11(14-15) - 0.16(15H) 0.45(7H) 0.50(8H) - 0.57(12H) - O.16(9-CH,) 0.35(8H) - 0.20(7H) 0.64(12H) 0.45(11H) 0.06(8-9) 0.71(7H) - 0.46(8H) 0.38(12H) 0.77(11H) -0,19(12H) - O.ll(10-11) 0.89(lysine rock) 0.03(14-15) - 0.02(12-13) 0.09(12-13) - 0.09(14-15) 0.71(14H) 0.23(lysine rock) - 0.14(13-CH,) 0.21(8-9) O.lO(14-15) - 0.58(10H) - 0.23(12H) - O.lO(9-CH3) 0.16(14-15) - 0.12(8-9) + 0.36(11H) 0.21(10-11) 0.12(12-13) + 0.19(NH) 0.16(14-15) 0.3 1(6-7) 0.78( 19-CH30r) 0.80(20-CH30r) 0.57(20-CH,r) -- 0.22(N-C) 0.55( 19-CH3r) 0.22(N-C) 0.56(20-CH3r)
+
+
+ +
+
+ +
+ + +
+
+
+
+
+ +
+
+
+
+
+
+
+
+ +
+
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"Coefficients (dS/dQ) of internal coordinates S in the normal modes Q. Symbols used: H, in-plane hydrogen rock; or, out-of-plane methyl rock; 1 / d + 6 of CCH rocking coefficients having comparable potential energy contributions due to their greater reduced mass. r, in-plane methyl rock; def, deformation. Coefficients of CC stretches are
TABLE III: C==CStretches in Native and 13CC-Substituted BRw native 1 5-13C 14-13C
14,15-I3C
1 3-I3C
1634a (7)b 1599 (1) 1570 (1) 1550 (5) 1536 (74) 1515 (12) 10-13c
1622 (4) 1599 (3) 1570 (3) 1552 (4) 1533 (78) 1514 (8) 10,i 1-13c
1632 (7) 1597 (1) 1563 (2) 1545 (4) 1535 (82) 1499 (4) 9-13C
1621 (4) 1597 (1) 1563 (5) 1540 (3) 1531 (82) 1500 (5) 8-I3C
1634 (6)
1632 (8) 1590 (3) 1570 (2)
1633 (6) 1589 (9) 1564 (1) 1548 (2) 1521 (77) I509 (5)
1634 1595 1575 1551 1536 1503
1637 (7) 1595 (2) 1571 (3) 1555 (11) 1532 (77)
1634 (7)
1531 (72) 1512 (IS)
(6) (2) (I) (3) (88) (1 1)
1563 (5) 1550 (5) 1535 (79) 1495 (5) 7-"C
1568 (3) 1550 (12) 1529 (78)
12-13c 1632 (8) 1594 (2) 1570 (4) 1551 (12) 1524 (74)
11-13c
1636 (7) 1594 (7) 1553 (6) 1524 (80)
6-I'C
5-'3C
1636 (8) 1591 (1) 1570 ( I )
1634 (9) 1589 (1) 1564 (3)
1536 (83)
1537 (79) IS13 (8)
-1515 (7)
"All freauencies are in wavenumbers. *Raman intensity obtained by deconvolution of the 1500-1600-cm-' region of the Raman spectrum. Total Raman intknsity in this region was normalized to 100.
calculated contribution of the C , ,=C,, and C7=C8 stretches to the 1536-cm mode is approximately correct. The intensities of the C=C vibrations are of interest because they provide information about the sign of the C=C internal coordinates in the normal modes. In an in-phase combination of C=C stretches the intrinsic resonance Raman intensities of the individual stretches add to give an intense band, while in an out-of-phase combination the component C=C stretching intensities tend to cancel. Thus, the high intensity of the 1536-cm-' line is consistent with our description of this mode as an in-phase combination of the five C=C stretches. The 1515-cm-' shoulder on the intense 1536-cm-I band is calculated to be an in-phase combination of the C9=Clo a,nd C13- /
511
Y
-d
PBends
438
+
Figure 10. Calculated vibrational frequencies for planar all-trans-, 13cis-, and 13,15-di-cis-PSBfragments showing the effect of isomerization on the Cl4-ClSstretching frequency. Cloand Clyswere represented by atoms of mass 15. Force constants for the trans calculation were taken from normal-mode calculations on the all-trans-PSB." Changes in the force field were made to account for isomerization about the C,3=Cl, and C=N bonds based on model compound studies (see text). The shift of the C,4-CIs stretch is due to a drop in frequency of the symmetric combination of the skeletal bends flanking the isomerized bond with which it is kinetically coupled. 13-trans 13-cis isomerization lowers the symmetric bend combination p + a,while 15-trans 15-cis isomerization lowers the symmetric combination y + 4. (Only the bending modes having large frequency changes have been included, and they are labeled by the largest bend components in the mode.)
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isomerization is larger than that caused by C I 3 = C l 4isomerization because there are two effects. The CI4-Cl5stretch experiences reduced coupling with the C C C bends, and increased coupling with the N H rock and C=N stretch.I2 The magnitude of the observed downshift (9 cm-I) is less than the 20 cm-' predicted because there is an opposing upshift of the C-C stretches in response to increased x-electron delocalization upon proteinbinding. Comparison of the C-C stretching frequencies between BRSa and the oll-trans-PSB (both of which have the same C=N configuration) shows an IO-cm-' increase in each C-C freshift of the Cl4-C15 stretch in BR548would q ~ e n c y . l ~A* 'similar ~ quantitatively account for the observed 9-cm-' drop in the C14-C15 frequency. The 17-cm-I increase of the Cl0-Cll stretch probably results from increased x-electron delocalization as well as a change in coupling with the CI4-CI5stretch. Since the CI4-Cl5stretch is below the ClO-Cl1mode in BR548it will push the Clo-CIIup. Increased r-electron delocalization also accounts for the 8-cm-' increase in the Cl2-Cl3 stretching mode. Only the c&9 stretching mode remains unchanged in frequency in going from the 13-cis-PSB to BRS48. This presumably arises because mode mixing effects counteract the increase due to enhanced delocalization.
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J. Phys. Chem. 1987, 91, 819-822 of coupling and intensity is characteristic of CCH rocks associated with cis bonds.15 What are the implications of these results for the structure of the retinal chromophore in BR548? The observation that both the CI3=Cl4 and C=N bonds in BR548 are in the cis configuration implies that the thermal BR568 BR548 transition involves a concerted “bicycle pedal” isomerization as initially proposed by Orlandi and S ~ h u l t e n . ~The ~ formation of the 13,15-di-cis structure in the dark also shows that the protein preferentially binds the di-cis chromophore since the all-trans geometry is the lowest energy form of the chromophore in solution. However, once the effects of CI3=CI4 and C=N isomerization are accounted for, the vibrational structure of BR548 is very similar to that of BR568indicating that differences in protein-chromophore interactions between BR568and BR548 are not pronounced. Recent solid-state N M R studies on BRS6*and BR548 have established the presence of a negative protein charge (or the minus end of a dipole) near Cs and a positive charge (or the plus end of a dipole) near C7.29aThere is also evidence for weak hydrogen bonding between the Schiff base and its protein c o u n t e r i ~ n ,and ~ ~ -it~appears ~ that this weak hydrogen bond is the dominant mechanism in the “opsin shift” of BR.36-38 The similar I3C-chemical shifts for the two pigments support the idea that electrostatic protein-chromophore interactions in BR568and BR548 are not markedly different and suggest that steric differences in the retinal binding site may be responsible for stabilizing the BR548 chromophore. One of the most pronounced differences in the Raman spectra of and BRs48 is the large intensity of the CI4HHOOP mode in BR548. The intensity of this mode is probably induced by a local twist in the ground-state structure of the chromophore near
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(33) Orlandi, G.; Schulten, K. Chem. Phys. Left.1979; 64, 370. (34) Hildebrandt, P.; Stockburger, M. Biochemistry 1984, 23, 5539. (35) Harbison, G. S.;Herzfeld, J.; Griffin, R. G. Biochemistry 1983, 22, 1.
(36) Nakanishi, K.; Balogh-Nair, V.;Arnaboldi, M.; Tsujimoto, K.; Honig, B. J . Am. Chem. SOC.1980, 102, 7945. (37) Lugtenburg, J.; Muradin-Szweykowska, M.; Harbison, G. S.;Smith, S. 0.;Heeremans, C.; Pardoen, J. A,; Herzfeld, J.; Griffin, R. G.; Mathies, R. A. J . Am. Chem. SOC.1986, 108, 3104. (38)Spudich. J. L.; McCain, D. A,; Nakanishi, K.; Okabe, M.; Shimizu, N.; Rodman, H.; Honig, B.; Bogomolni, R. A. Biophys. J . 1986, 49, 479.
819
C14, forcing the C I 4 Hhydrogen out of the plane of the conjugated T-system. This suggests that steric constraints in BRs48 do not allow the chromophore to fully relax following the BR568 BR548 conversion. This is consistent with the large upfield shift of the l3CI4N M R resonance which is attributed to steric interaction of the C14Hproton with the C-CH2 lysine protons across the cis C=N bond.20 Steric constraints near CI4 may also explain the reduced coupling between the C=N stretch and C15Hrock. In BR548 the C=N stretching mode shifts 5 cm-I upon 15-deuteriation, significantly less than the 11-cm-’ shift seen in BR56818 and the 12-15-cm-l shifts seen in the all-trans- and 1 3 - ~ i s - P S B . ~ ~ , ” In summary, the major differences between the resonance Raman spectra of BR568and BR548 can be directly explained by isomerization about the C I 3 = C l 4and C=N bonds. Characteristic vibrational frequencies and isotopic shifts previously described in all-trans- and 13-cis-retinal are observed in the spectra of the protein-bound PSB chromophores, demonstrating that the methods developed for determining chromophore structure in retinal model compounds are applicable for the retinal pigments. Thus, the results presented here should be transferable to the interpretation of the vibrational spectra of the other BR photocycle intermediates, as well as halorhodopsin and sensory rhodopsin.
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Acknowledgment. We thank Anne Myers and Mark Braiman for insightful discussions and critical comments. Patrick Mulder, Chris Winkel, Jacques Courtin, Marcel van der Brugge, Albert Broek, Gerard Lam, and Ellen van den Berg synthesized the retinal isotopic derivatives employed in this work. This research was supported by the National Science Foundation (CHE-8 116042), the National Institutes of Health (EY-02051 and GM 27057), the Netherlands Foundation for Chemical Research (SON), and the Netherlands Organization for the Advancement of Pure Research (ZWO). R.M. is an N I H Research Career Development Awardee (EY-00219). Registry No. 13-cis-Retina1, 472-86-6.
Supplementary Material Available: The Cartesian and internal coordinates used in the calculations and a complete description of the force field will be found in Tables XIV and XV (7 pages). Ordering information is given on any current masthead page.
Polarized Absorption and Phosphorescence Spectra of Xanthone in Stretched Polyethylene Films Robert E. Connors,* Robert J. Sweeney, and Frank Cerio Department of Chemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609 (Received: July 21, 1986: I n Final Form: October 6, 1986)
Polarized absorption and phosphorescence spectra have been measured at several temperatures for xanthone embedded in stretched polyethylene films. On the basis of experimental results and molecular orbital calculations, a portion of the absorption spectrum has been reassigned. At 4.2 K the phosphorescence data indicate that T,(TT*),of 3A1symmetry, obtains the major source of its radiative strength through a vibronic spin-orbit coupling mechanism involving the mixing of S,(nr*), of ‘A2 symmetry, and ‘ B 2 ( m * ) states through bl vibrations. Thermally activated phosphorescence is observed from T, which is separated from T! by 145 cm-’. Polarized phosphorescence spectra at 165 K support the view that T,(na*), of 3A2symmetry, derives its radiative activity through direct spin-orbit coupling to S 2 ( r r * ) ,of IAl symmetry.
Introduction Recent studies of the phosphorescenceof xanthone in n-hexane1V2 have shown that the lowest triplet state, Tl(?r?r*),of 3A1symmetry and the second triplet state, T2(nT*),of 3A2symmetry are very (1) Connors, R. E.; Christian, W. R. J . Phys. Chem. 1982, 86, 1524. (2) Griesser, H . J.; Bramely, R. Chem. Phys. Left. 1981, 83, 287.
closely spaced (A& = 29 cm-I). Griesser and Bramley3 have recorded and analyzed high-resolution phosphorescence spectra for the two emitting states in n-hexane. At 4.2 K they found that the T I emission could be resolved into two components, one arising from the TI, sublevel and the other from one or both sublevels (3) Griesser, H. J.; Bramely, R. Chem. Phys. 1982, 67, 373.
0022-3654/87/209 1-0819$01.50/0 0 1987 American Chemical Society
