- Email: [email protected]
Interpretation of the resonance Raman spectra of linear tetrapyrroles based on DFT calculations
8 October 1999
Chemical Physics Letters 311 Ž1999. 479–484 www.elsevier.nlrlocatercplett
Interpretation of the resonance Raman spectra of linear tetrapyrroles based on DFT calculations Christa Kneip, Peter Hildebrandt ) , Karoly Nemeth, Franz Mark, Kurt Schaffner ´ ´ Max-Planck-Institut fur Germany ¨ Strahlenchemie, Postfach 101365, D-45413 Mulheim, ¨ Received 9 July 1999
Abstract Raman spectra of linear methine-bridged tetrapyrroles in different conformational and protonation states were calculated on the basis of scaled force fields obtained by density functional theory. Results are reported for protonated phycocyanobilin in the extended ZZZasa configuration, as it is found in C-phycocyanin of cyanobacteria. The calculated spectra are in good agreement with experimental spectra of the protein-bound chromophore in the a-subunit of C-phycocyanin and allow a plausible and consistent assignment of most of the observed resonance Raman bands in the region between 1000 and 1700 cmy1. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction The physiological response of the plant photoreceptor phytochrome is associated with the light-induced interconversion of two stable states, Pr and Pfr , that is initiated by a photoreaction of the chromophore phytochromobilin ŽPFB; Fig. 1., a linear tetrapyrrole w1x. To understand the functioning of phytochrome on a molecular level, a detailed knowledge of the chromophore structure in the various states of the photocycle is required. In this respect, resonance Raman ŽRR. spectroscopy is a promising tool since it selectively probes the vibrational spectrum of the chromophore w2–5x. However, the interpretation of the vibrational spectra is hampered by a lack of reliable band assignments. Normal mode
) Corresponding author. Fax: q49-208-306-3951; e-mail: [email protected]
analyses have been performed so far only at the semi-empirical level, and for unprotonated tetrapyrroles or protonated tetrapyrroles with truncated substituents w2,5,6x. Furthermore, since the chromophore conformation in phytochrome is still unknown, extrapolations from the free to the proteinbound chromophore are speculative. There are strong indications, albeit no proofs, that the chromophore in the Pr state is protonated and adopts an extended conformation w7,8x similar to the phycocyanobilin chromophore ŽPCB; Fig. 1. in the C-phycocyanin ŽCPC. of cyanobacteria w9x. The crystal structure of this pigment reveals a protonated Žcationic. chromophore ŽPCBHq. in a ZZZasa configuration. PCB differs from PFB only by the ethyl substituent at ring D Žinstead of a vinyl. and it can readily be assembled with recombinant apophytochrome A w10x. The resulting holoprotein undergoes a photoreaction similar to the native phytochrome, and in the parent states the structures of the protein-bound PCB are
0009-2614r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 9 . 0 0 8 6 8 - 4
480
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
Fig. 1. Structural formulas of PCB ŽR s ethyl. and PFB ŽR s vinyl. chromophores in the protein-bound protonated form Ž ZZZasa, left. and in the esterified form Ž ZZZsss, right..
the same as for PFB as revealed by the RR spectra w11x. Thus, normal mode analyses of PCB in different configurations and protonation states should substantially support the interpretation of the RR spectra of phytochrome. In this work, Raman spectra were calculated for PCB in the cationic ZZZasa and in the neutral ZZZsss configuration and compared with the experimental RR spectra of the a-subunit of CPC Ž a-CPC. and phycocyanobilin dimethyl ester ŽPCBE.. The goal of the study is to assess the potential of this approach for determining the tetrapyrrole structure in the various state of phytochrome. We have employed density functional theory ŽDFT. to calculate the force fields which were subsequently corrected for the intrinsic systematic errors of the method using a global set of scaling factors, previously determined for a series of small training molecules w12x. Furthermore, Raman intensities were calculated as additional criteria for the vibrational assignment.
2. Methods DFT calculations were performed with the 6-31G ) basis set and the B3LYP exchange-correlation functional using the Gaussian94 program package w12x. Force fields were calculated by numerical differentiation of energy gradients at displaced geometries ˚ .. The Ždisplacement in Cartesian coordinates 0.002 A
scaling procedure and the calculation of the Raman intensities by means of the finite electric field method were described in detail previously w12x. Raman spectra were measured with 1064 nm excitation using the Fourier-transform technique. All spectra were obtained at y1408C. Details of the experiments have been published previously w13x.
3. Results and discussion For the geometry optimization of PCBHq Ž ZZZasa., the starting geometry was taken from the crystal structure of a-CPC, where the tetrapyrrole is protonated by Asp87 located near rings B and C w9x. In the present calculations w14x, these interactions have been mimicked by replacing Asp87 by chloride acting as a counterion. In the optimized geometry, the positive charge is delocalized on the rings B and C and the chloride is located equidistant to both N–H groups. For the propionate side chains we have chosen the neutral Žprotonated. form to avoid hydrogen-bonding interactions with the pyrrole nitrogens of rings B and C since these interactions stabilize the ZZZaaa configuration w14x. Molecules of the size of tetrapyrroles exhibit a close spacing of the calculated normal modes in the region of interest Ži.e., between 300 and 1700 cmy1 . which for PCBHq was ; 8 cmy1 at average. Thus, even assuming an error of the wavenumber calcula-
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
tions comparable to that achieved for small molecules Ži.e., "11 cmy1 . w12x, an assignment of the modes to the observed bands is not possible solely based on the comparison of calculated and experimental wavenumbers. Therefore, intensities were calculated to provide further assignment criteria. Fig. 2A displays the calculated Raman spectrum of PCBHq. Although the experimental spectrum of a-CPC ŽFig. 2B. is obtained under preresonance conditions w13x, non-resonance Raman intensities evidently represent a good approximation as indicated by the satisfactory overall agreement between the calculated and the experimental spectra for both the non-deuterated and
Fig. 2. Calculated Raman spectra of ŽA. PCBHq and ŽD. PCBDq in the ZZZasa configuration compared with the experimental RR spectra of ŽB. a-CPCŽH. and ŽC. a-CPCŽD. in the spectral range from 1000 to 1700 cmy1 .
481
deuterated species Žvide infra.. A selection of calculated and observed bands is listed in Table 1. The spectral region between 1450 and 1750 cmy1 which is displayed in an expanded view in Fig. 3 is of particular interest as it includes modes which are regarded to be sensitive markers for the configuration, conformation, and protonation state of the tetrapyrrole w2–5x. The experimental spectrum of a-CPC in H 2 O w a-CPCŽH.x is dominated by a strong and asymmetric peak with two components at 1635 and 1645 cmy1 . These bands are readily assigned to the modes n47 Ž1635 cmy1 . and n46 Ž1640 cmy1 . for which high Raman intensities are predicted in contrast to the nearby mode n48 . These assignments are in line with the predicted and measured HrD effects ŽFig. 3C and D. and indicate that the strongest RR band originate from modes localized in the methine bridges A–B, C–D, and ring D ŽTable 1.. The modes n 50 and n 51 which both predominantly include the N–H in-plane bending Žip. of the rings B and C are assigned to the RR bands at 1590 and 1521 cmy1 . These bands vanish in the RR spectrum of a-CPC in D 2 O w a-CPCŽD.x confirming these assignments. Also the weak 1567 cmy1 band of a-CPCŽH. disappears upon HrD exchange. Since no fundamental is calculated in this region, this band as well may originate from a N–H ip Ž n 50 ., suggesting that there two subpopulations of the tetrapyrrole chromophore which only differ by hydrogen-bonding interactions of rings B and C with the protein environment. The N–H ip coordinates of rings A and D are distributed among several modes between 1350 and 1300 cmy1 with the largest contributions to n 91 Ž1337 cmy1 . and n 95 Ž1313 cmy1 ., respectively. Taking into account the calculated Raman intensities, these modes as well as two adjacent modes are ascribed to the RR bands of a-CPCŽH. at 1337 and 1318 cmy1 . The only modes of PCBDq that contain substantial contributions of the N–D ip coordinates are n 131 Žring D; 1009 cmy1 . and n 121 Žrings B and C; 1073 cmy1 . but only the latter is predicted to be Ramanactive and can in fact be assigned to the 1071 cmy1 band of a-CPCŽD.. For the calculations of the unprotonated Žneutral. PCBE in the ZZZsss configuration, the starting geometry was adopted from the crystal structure of the closely related biliverdin dimethyl ester w15x. This
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
482
Table 1 Assignments of the RR bands of a-CPC measured in H 2 O and D 2 O PCBHq, ZZZasa, calc. y1 .
a-CPCŽD., exp.Žcmy1 .n ŽI RR . a
a-CPCŽH., exp. PCBDq, ZZZasa, calc. b
mode
Žcm n Ž IRa . a
PED
46 47 48 49 50
1640 Ž98. 1635 Ž100. 1628 Ž9. 1610 Ž2. 1595 Ž54.
C5C str; A–B C5C str; C–D C5C str; D C–C str; B–C N–H ip; C, B
51 52 53 59 68 83 84 91 92 93 94 95 101 106 110 111 112 118
1534 Ž15. 1530 Ž2. 1513 Ž3. 1467 Ž4. 1452 Ž14. 1374 Ž18. 1371 Ž19. 1337 Ž3. 1334 Ž10. 1322 Ž7. 1319 Ž16. 1313 Ž16. 1278 Ž7. 1241 Ž35. 1210 Ž1. 1193 Ž8. 1178 Ž5. 1121 Ž7.
N–H ip; B, C C–C str; B C5C str; C CH 3 def; B, C CH 3 def; B CH 3 ŽEt. def; D CH 3 ŽEt. def; D N–H ip; A CH 2 ŽEt. wag; D NH ip; B, A CH 2 ŽEt. wag; D N–H ip; D CH 2 ŽEt. twi; D C–H ip; C–D O–H Žprop. ip; B C–N str; A C–N str; C C–N str; C
y1 .
y1
b
Žcm n Ž IRR . a
mode
Žcm n Ž IRa . a
PED
1645 Žvs. 1635 Žvs.
46 47 48 49 50
1631 Ž100. 1629 Ž24. 1627 Ž83. 1598 Ž1. 1529 Ž2.
C5C str; A–B C5C str; C–D; D C5C str; D; C–D C–C str; B–C C–C str; B
51 52 53 54 62 70 71 73 74 76 79 86 94 95 97 103 118 121
1512 Ž1. 1490 Ž31. 1478 Ž2. 1477 Ž8. 1462 Ž15. 1447 Ž14. 1442 Ž17. 1425 Ž3. 1421 Ž3. 1404 Ž35. 1385 Ž14. 1353 Ž5. 1305 Ž5. 1299 Ž5. 1278 Ž4. 1250 Ž10. 1111 Ž2. 1073 Ž26.
C5C str; C CH 3 def; C CH 3 def; D CH 3 def; B, C CH 3 def; B CH 2 ŽEt. sci; D CH 2 ŽEt. sci; D C–H ip; B–C C–H ip; C–D C–H ip; A–B CH 3 def; B C–N str; C CH 2 Žprop. twi; B CH 2 Žprop. twi; B CH 2 ŽEt. twi; D C–N str; A C–C ŽEt. str; A N–D ip; B, C
1590 Žm. 1567 Žw. 1521 Žw. 1512 Žw. 1470 Žm. 1452 Žw. 1370 Žs. 1337 Žm.
1318 Žw. 1270 Žm. 1257 Žm. 1217 Žm. 1187 Žw. 1158 Žvw. 1108 Žm.
1637 Žs. 1629 Žvs. 1595 Žvw. 1537 Žvw. 1518 Žw. 1497 Žm. 1477 Žw. 1469 Žm. 1448 Žs. 1427 Žm. 1402 Žw. 1376 Žw. 1360 Žw. 1309 Žw. 1279 Žm. 1264 Žw. 1118 Žw. 1072 Žm.
a
Wavenumbers are expressed in cmy1 ; intensities are given in parentheses. Calculated Raman intensities are related to the strongest mode Ž IRa s 100. whereas the experimental RR bands are characterized by very strong Žvs., strong Žs., medium Žm., weak Žw., and very weak Žvw.. b Only the largest contribution in terms of potential energy distribution ŽPED. is listed for each mode. A, B, C, and D denote the individual tetrapyrrole rings and A–B, B–C, and C–D the corresponding methine bridges ŽFig. 1.. Stretching, in-plane bending, deformation, twisting, scissoring, and wagging coordinates are abbreviated by str, ip, twi, sci, and wag, respectively. The ethyl and propionic substituents are abbreviated by Et and prop, respectively.
tetrapyrrole forms hydrogen-bonded dimers via the N–H and C5O groups of the rings A and D w16x. PCBE most likely forms dimers as well. Since centrosymmetric dimers can be constructed from the calculated monomer structure at ring A but not at ring D without steric interference between the monomeric entities, an A–A dimer was assumed. The effect of hydrogen-bonding interactions on the structure and force constants were taken into account by modifying a posteriori the F and G matrices calculated for monomeric PCBE. These corrections were approximated by the differences in the bond lengths ŽG matrix. and force constants ŽF matrix. of monomeric and dimeric maleimide which served as models for the non-hydrogen-bonded and hydrogen-
bonded terminal pyrrole rings of PCBE, respectively w14x. Also for PCBE, there is a good agreement between the calculated and the experimental spectra, and no further improvement is achieved by calculating RR intensities using the ‘bond order’ approximation w6,12,14x. Both the calculated spectra of the ZZZsss configuration and the experimental RR spectra of the non-deuterated PCBE wPCBEŽH.x differ substantially from those of the cationic ZZZasa PCBHq ŽFig. 4.. Compared to a-CPC, the strongest RR band of PCBEŽH. is found at substantially lower wavenumbers Ž1597 cmy1 .. It is assigned to a mode at 1588 cmy1 including the stretching of the B–C methine bridge, whereas for the C–D stretching
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
483
Based on the comparison with the calculated wavenumbers, Raman intensities, and HrD isotopic effects, it is possible to achieve a plausible and consistent assignment for most of the observed bands of PCBEŽH. and a-CPC. For a-CPC there is a worse agreement in the spectral regions between 1200 and 1300 cmy1 and 800 and 900 cmy1 which include modes involving vibrations of the propionate side chains and the C–H out-of-plane coordinates of the methine bridges, respectively. These modes may be particularly sensitive towards molecular interactions with the protein environment which cannot be considered by the calculations. A detailed vibrational
Fig. 3. Expanded view of the calculated Raman spectra of ŽA. PCBHq and ŽD. PCBDq in the ZZZasa configuration and the experimental RR spectra of ŽB. a-CPCŽH. and ŽC. a-CPCŽD. in the spectral range from 1450 to 1750 cmy1 .
mode, calculated at 1629 cmy1 and assigned to the 1641 cmy1 band, a much lower Raman intensity is predicted which is in agreement with the experiment. In a-CPC, the corresponding mode, albeit at nearly the same wavenumber, gives rise to one of the strongest RR bands in the spectrum. The second characteristic feature of the spectrum of PCBEŽH. is the lack of a prominent N–H band at ; 1590 cmy1 which, hence, can be taken as a characteristic marker for the protonation state of the tetrapyrrole. The only band in this region which disappears upon HrD exchange Žspectrum not shown. is the weak band at ; 1536 cmy1 which is assigned to the mode at 1531 cmy1 involving the N–H ip coordinate of ring C.
Fig. 4. Calculated Raman spectrum of ŽB. PCBEŽH. in the ZZZsss configuration compared with the experimental RR spectra of ŽA. PCBEŽH. and ŽC. a-CPCŽH..
484
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
analysis of these tetrapyrroles will be published elsewhere w14x.
4. Conclusions It is shown that DFT calculations can reproduce the experimental spectra of linear tetrapyrroles in different conformations and protonation states. There are no obvious restrictions to extend this approach to further configurational and conformational isomers. Thus, it is expected that based on the comparison of such spectra with the experimental RR spectra, it will be possible to determine the tetrapyrrole structures in different Žintermediate. states of phytochrome. The present results provide a key for extracting structural information from the experimental spectra. Since many normal modes are largely localized in the individual rings and adjacent methine bridges, the identification of these modes in the RR spectra of phytochrome may contribute to the elucidation of structural changes in specific parts of the chromophore during the phototransformation.
Acknowledgements P.H. acknowledges a Heisenberg fellowship by the Deutsche Forschungsgemeinschaft.
References w1x G.C. Whitelam, N.T. Harberd, Plant Cell Environ. 17 Ž1994. 615. w2x S.P.A. Fodor, J.C. Lagarias, R.A. Mathies, Biochemistry 29 Ž1990. 11141. w3x Y. Mizutani, S. Tokutomi, T. Kitagawa, Biochemistry 33 Ž1994. 153. w4x J. Matysik, P. Hildebrandt, W. Schlamann, S.E. Braslavsky, K. Schaffner, Biochemistry 34 Ž1995. 10497. w5x F. Andel III, J.C. Lagarias, R.A. Mathies, Biochemistry 35 Ž1996. 15997. w6x K. Smit, J. Matysik, P. Hildebrandt, F. Mark, J. Phys. Chem. 97 Ž1993. 11887. w7x H. Foerstendorf, E. Mummert, E. Schafer, H. Scheer, F. ¨ Siebert, Biochemistry 35 Ž1996. 10793. w8x J. Burke, D.C. Pratt, A. Moscowitz, Biochemistry 22 Ž1972. 4025. w9x M. Duerring, R. Huber, W. Bode, R. Ruembeli, H. Zuber, J. Mol. Biol. 211 Ž1990. 633. w10x J.T. Murphy, J.C. Lagarias, Photochem. Photobiol. 65 Ž1997. 750. w11x C. Kneip, D. Mozley, P. Hildebrandt, W. Gartner, S.E. ¨ Braslavsky, K. Schaffner, FEBS Lett. 414 Ž1997. 23. w12x I. Magdo, ´ K.F. Mark, P. Hildebrandt, K. Schaffner, J. Phys. Chem. A 103 Ž1999. 289. w13x C. Kneip, A. Parbel, H. Foerstendorf, H. Scheer, F. Siebert, P. Hildebrandt, J. Raman Spectrosc. 29 Ž1998. 939. w14x K. Nemeth, C. Kneip, P. Hildebrandt, F. Mark Žto be pub´ lished.. w15x W.S. Sheldrick, J. Chem. Soc., Perkin Trans. II Ž1976. 1457. w16x H. Takahashi, K. Kuroyanagi, O. Yamada, N. Kaneko, Chem. Phys. Lett. 94 Ž1983. 38.
Chemical Physics Letters 311 Ž1999. 479–484 www.elsevier.nlrlocatercplett
Interpretation of the resonance Raman spectra of linear tetrapyrroles based on DFT calculations Christa Kneip, Peter Hildebrandt ) , Karoly Nemeth, Franz Mark, Kurt Schaffner ´ ´ Max-Planck-Institut fur Germany ¨ Strahlenchemie, Postfach 101365, D-45413 Mulheim, ¨ Received 9 July 1999
Abstract Raman spectra of linear methine-bridged tetrapyrroles in different conformational and protonation states were calculated on the basis of scaled force fields obtained by density functional theory. Results are reported for protonated phycocyanobilin in the extended ZZZasa configuration, as it is found in C-phycocyanin of cyanobacteria. The calculated spectra are in good agreement with experimental spectra of the protein-bound chromophore in the a-subunit of C-phycocyanin and allow a plausible and consistent assignment of most of the observed resonance Raman bands in the region between 1000 and 1700 cmy1. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction The physiological response of the plant photoreceptor phytochrome is associated with the light-induced interconversion of two stable states, Pr and Pfr , that is initiated by a photoreaction of the chromophore phytochromobilin ŽPFB; Fig. 1., a linear tetrapyrrole w1x. To understand the functioning of phytochrome on a molecular level, a detailed knowledge of the chromophore structure in the various states of the photocycle is required. In this respect, resonance Raman ŽRR. spectroscopy is a promising tool since it selectively probes the vibrational spectrum of the chromophore w2–5x. However, the interpretation of the vibrational spectra is hampered by a lack of reliable band assignments. Normal mode
) Corresponding author. Fax: q49-208-306-3951; e-mail: [email protected]
analyses have been performed so far only at the semi-empirical level, and for unprotonated tetrapyrroles or protonated tetrapyrroles with truncated substituents w2,5,6x. Furthermore, since the chromophore conformation in phytochrome is still unknown, extrapolations from the free to the proteinbound chromophore are speculative. There are strong indications, albeit no proofs, that the chromophore in the Pr state is protonated and adopts an extended conformation w7,8x similar to the phycocyanobilin chromophore ŽPCB; Fig. 1. in the C-phycocyanin ŽCPC. of cyanobacteria w9x. The crystal structure of this pigment reveals a protonated Žcationic. chromophore ŽPCBHq. in a ZZZasa configuration. PCB differs from PFB only by the ethyl substituent at ring D Žinstead of a vinyl. and it can readily be assembled with recombinant apophytochrome A w10x. The resulting holoprotein undergoes a photoreaction similar to the native phytochrome, and in the parent states the structures of the protein-bound PCB are
0009-2614r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 Ž 9 9 . 0 0 8 6 8 - 4
480
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
Fig. 1. Structural formulas of PCB ŽR s ethyl. and PFB ŽR s vinyl. chromophores in the protein-bound protonated form Ž ZZZasa, left. and in the esterified form Ž ZZZsss, right..
the same as for PFB as revealed by the RR spectra w11x. Thus, normal mode analyses of PCB in different configurations and protonation states should substantially support the interpretation of the RR spectra of phytochrome. In this work, Raman spectra were calculated for PCB in the cationic ZZZasa and in the neutral ZZZsss configuration and compared with the experimental RR spectra of the a-subunit of CPC Ž a-CPC. and phycocyanobilin dimethyl ester ŽPCBE.. The goal of the study is to assess the potential of this approach for determining the tetrapyrrole structure in the various state of phytochrome. We have employed density functional theory ŽDFT. to calculate the force fields which were subsequently corrected for the intrinsic systematic errors of the method using a global set of scaling factors, previously determined for a series of small training molecules w12x. Furthermore, Raman intensities were calculated as additional criteria for the vibrational assignment.
2. Methods DFT calculations were performed with the 6-31G ) basis set and the B3LYP exchange-correlation functional using the Gaussian94 program package w12x. Force fields were calculated by numerical differentiation of energy gradients at displaced geometries ˚ .. The Ždisplacement in Cartesian coordinates 0.002 A
scaling procedure and the calculation of the Raman intensities by means of the finite electric field method were described in detail previously w12x. Raman spectra were measured with 1064 nm excitation using the Fourier-transform technique. All spectra were obtained at y1408C. Details of the experiments have been published previously w13x.
3. Results and discussion For the geometry optimization of PCBHq Ž ZZZasa., the starting geometry was taken from the crystal structure of a-CPC, where the tetrapyrrole is protonated by Asp87 located near rings B and C w9x. In the present calculations w14x, these interactions have been mimicked by replacing Asp87 by chloride acting as a counterion. In the optimized geometry, the positive charge is delocalized on the rings B and C and the chloride is located equidistant to both N–H groups. For the propionate side chains we have chosen the neutral Žprotonated. form to avoid hydrogen-bonding interactions with the pyrrole nitrogens of rings B and C since these interactions stabilize the ZZZaaa configuration w14x. Molecules of the size of tetrapyrroles exhibit a close spacing of the calculated normal modes in the region of interest Ži.e., between 300 and 1700 cmy1 . which for PCBHq was ; 8 cmy1 at average. Thus, even assuming an error of the wavenumber calcula-
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
tions comparable to that achieved for small molecules Ži.e., "11 cmy1 . w12x, an assignment of the modes to the observed bands is not possible solely based on the comparison of calculated and experimental wavenumbers. Therefore, intensities were calculated to provide further assignment criteria. Fig. 2A displays the calculated Raman spectrum of PCBHq. Although the experimental spectrum of a-CPC ŽFig. 2B. is obtained under preresonance conditions w13x, non-resonance Raman intensities evidently represent a good approximation as indicated by the satisfactory overall agreement between the calculated and the experimental spectra for both the non-deuterated and
Fig. 2. Calculated Raman spectra of ŽA. PCBHq and ŽD. PCBDq in the ZZZasa configuration compared with the experimental RR spectra of ŽB. a-CPCŽH. and ŽC. a-CPCŽD. in the spectral range from 1000 to 1700 cmy1 .
481
deuterated species Žvide infra.. A selection of calculated and observed bands is listed in Table 1. The spectral region between 1450 and 1750 cmy1 which is displayed in an expanded view in Fig. 3 is of particular interest as it includes modes which are regarded to be sensitive markers for the configuration, conformation, and protonation state of the tetrapyrrole w2–5x. The experimental spectrum of a-CPC in H 2 O w a-CPCŽH.x is dominated by a strong and asymmetric peak with two components at 1635 and 1645 cmy1 . These bands are readily assigned to the modes n47 Ž1635 cmy1 . and n46 Ž1640 cmy1 . for which high Raman intensities are predicted in contrast to the nearby mode n48 . These assignments are in line with the predicted and measured HrD effects ŽFig. 3C and D. and indicate that the strongest RR band originate from modes localized in the methine bridges A–B, C–D, and ring D ŽTable 1.. The modes n 50 and n 51 which both predominantly include the N–H in-plane bending Žip. of the rings B and C are assigned to the RR bands at 1590 and 1521 cmy1 . These bands vanish in the RR spectrum of a-CPC in D 2 O w a-CPCŽD.x confirming these assignments. Also the weak 1567 cmy1 band of a-CPCŽH. disappears upon HrD exchange. Since no fundamental is calculated in this region, this band as well may originate from a N–H ip Ž n 50 ., suggesting that there two subpopulations of the tetrapyrrole chromophore which only differ by hydrogen-bonding interactions of rings B and C with the protein environment. The N–H ip coordinates of rings A and D are distributed among several modes between 1350 and 1300 cmy1 with the largest contributions to n 91 Ž1337 cmy1 . and n 95 Ž1313 cmy1 ., respectively. Taking into account the calculated Raman intensities, these modes as well as two adjacent modes are ascribed to the RR bands of a-CPCŽH. at 1337 and 1318 cmy1 . The only modes of PCBDq that contain substantial contributions of the N–D ip coordinates are n 131 Žring D; 1009 cmy1 . and n 121 Žrings B and C; 1073 cmy1 . but only the latter is predicted to be Ramanactive and can in fact be assigned to the 1071 cmy1 band of a-CPCŽD.. For the calculations of the unprotonated Žneutral. PCBE in the ZZZsss configuration, the starting geometry was adopted from the crystal structure of the closely related biliverdin dimethyl ester w15x. This
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
482
Table 1 Assignments of the RR bands of a-CPC measured in H 2 O and D 2 O PCBHq, ZZZasa, calc. y1 .
a-CPCŽD., exp.Žcmy1 .n ŽI RR . a
a-CPCŽH., exp. PCBDq, ZZZasa, calc. b
mode
Žcm n Ž IRa . a
PED
46 47 48 49 50
1640 Ž98. 1635 Ž100. 1628 Ž9. 1610 Ž2. 1595 Ž54.
C5C str; A–B C5C str; C–D C5C str; D C–C str; B–C N–H ip; C, B
51 52 53 59 68 83 84 91 92 93 94 95 101 106 110 111 112 118
1534 Ž15. 1530 Ž2. 1513 Ž3. 1467 Ž4. 1452 Ž14. 1374 Ž18. 1371 Ž19. 1337 Ž3. 1334 Ž10. 1322 Ž7. 1319 Ž16. 1313 Ž16. 1278 Ž7. 1241 Ž35. 1210 Ž1. 1193 Ž8. 1178 Ž5. 1121 Ž7.
N–H ip; B, C C–C str; B C5C str; C CH 3 def; B, C CH 3 def; B CH 3 ŽEt. def; D CH 3 ŽEt. def; D N–H ip; A CH 2 ŽEt. wag; D NH ip; B, A CH 2 ŽEt. wag; D N–H ip; D CH 2 ŽEt. twi; D C–H ip; C–D O–H Žprop. ip; B C–N str; A C–N str; C C–N str; C
y1 .
y1
b
Žcm n Ž IRR . a
mode
Žcm n Ž IRa . a
PED
1645 Žvs. 1635 Žvs.
46 47 48 49 50
1631 Ž100. 1629 Ž24. 1627 Ž83. 1598 Ž1. 1529 Ž2.
C5C str; A–B C5C str; C–D; D C5C str; D; C–D C–C str; B–C C–C str; B
51 52 53 54 62 70 71 73 74 76 79 86 94 95 97 103 118 121
1512 Ž1. 1490 Ž31. 1478 Ž2. 1477 Ž8. 1462 Ž15. 1447 Ž14. 1442 Ž17. 1425 Ž3. 1421 Ž3. 1404 Ž35. 1385 Ž14. 1353 Ž5. 1305 Ž5. 1299 Ž5. 1278 Ž4. 1250 Ž10. 1111 Ž2. 1073 Ž26.
C5C str; C CH 3 def; C CH 3 def; D CH 3 def; B, C CH 3 def; B CH 2 ŽEt. sci; D CH 2 ŽEt. sci; D C–H ip; B–C C–H ip; C–D C–H ip; A–B CH 3 def; B C–N str; C CH 2 Žprop. twi; B CH 2 Žprop. twi; B CH 2 ŽEt. twi; D C–N str; A C–C ŽEt. str; A N–D ip; B, C
1590 Žm. 1567 Žw. 1521 Žw. 1512 Žw. 1470 Žm. 1452 Žw. 1370 Žs. 1337 Žm.
1318 Žw. 1270 Žm. 1257 Žm. 1217 Žm. 1187 Žw. 1158 Žvw. 1108 Žm.
1637 Žs. 1629 Žvs. 1595 Žvw. 1537 Žvw. 1518 Žw. 1497 Žm. 1477 Žw. 1469 Žm. 1448 Žs. 1427 Žm. 1402 Žw. 1376 Žw. 1360 Žw. 1309 Žw. 1279 Žm. 1264 Žw. 1118 Žw. 1072 Žm.
a
Wavenumbers are expressed in cmy1 ; intensities are given in parentheses. Calculated Raman intensities are related to the strongest mode Ž IRa s 100. whereas the experimental RR bands are characterized by very strong Žvs., strong Žs., medium Žm., weak Žw., and very weak Žvw.. b Only the largest contribution in terms of potential energy distribution ŽPED. is listed for each mode. A, B, C, and D denote the individual tetrapyrrole rings and A–B, B–C, and C–D the corresponding methine bridges ŽFig. 1.. Stretching, in-plane bending, deformation, twisting, scissoring, and wagging coordinates are abbreviated by str, ip, twi, sci, and wag, respectively. The ethyl and propionic substituents are abbreviated by Et and prop, respectively.
tetrapyrrole forms hydrogen-bonded dimers via the N–H and C5O groups of the rings A and D w16x. PCBE most likely forms dimers as well. Since centrosymmetric dimers can be constructed from the calculated monomer structure at ring A but not at ring D without steric interference between the monomeric entities, an A–A dimer was assumed. The effect of hydrogen-bonding interactions on the structure and force constants were taken into account by modifying a posteriori the F and G matrices calculated for monomeric PCBE. These corrections were approximated by the differences in the bond lengths ŽG matrix. and force constants ŽF matrix. of monomeric and dimeric maleimide which served as models for the non-hydrogen-bonded and hydrogen-
bonded terminal pyrrole rings of PCBE, respectively w14x. Also for PCBE, there is a good agreement between the calculated and the experimental spectra, and no further improvement is achieved by calculating RR intensities using the ‘bond order’ approximation w6,12,14x. Both the calculated spectra of the ZZZsss configuration and the experimental RR spectra of the non-deuterated PCBE wPCBEŽH.x differ substantially from those of the cationic ZZZasa PCBHq ŽFig. 4.. Compared to a-CPC, the strongest RR band of PCBEŽH. is found at substantially lower wavenumbers Ž1597 cmy1 .. It is assigned to a mode at 1588 cmy1 including the stretching of the B–C methine bridge, whereas for the C–D stretching
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
483
Based on the comparison with the calculated wavenumbers, Raman intensities, and HrD isotopic effects, it is possible to achieve a plausible and consistent assignment for most of the observed bands of PCBEŽH. and a-CPC. For a-CPC there is a worse agreement in the spectral regions between 1200 and 1300 cmy1 and 800 and 900 cmy1 which include modes involving vibrations of the propionate side chains and the C–H out-of-plane coordinates of the methine bridges, respectively. These modes may be particularly sensitive towards molecular interactions with the protein environment which cannot be considered by the calculations. A detailed vibrational
Fig. 3. Expanded view of the calculated Raman spectra of ŽA. PCBHq and ŽD. PCBDq in the ZZZasa configuration and the experimental RR spectra of ŽB. a-CPCŽH. and ŽC. a-CPCŽD. in the spectral range from 1450 to 1750 cmy1 .
mode, calculated at 1629 cmy1 and assigned to the 1641 cmy1 band, a much lower Raman intensity is predicted which is in agreement with the experiment. In a-CPC, the corresponding mode, albeit at nearly the same wavenumber, gives rise to one of the strongest RR bands in the spectrum. The second characteristic feature of the spectrum of PCBEŽH. is the lack of a prominent N–H band at ; 1590 cmy1 which, hence, can be taken as a characteristic marker for the protonation state of the tetrapyrrole. The only band in this region which disappears upon HrD exchange Žspectrum not shown. is the weak band at ; 1536 cmy1 which is assigned to the mode at 1531 cmy1 involving the N–H ip coordinate of ring C.
Fig. 4. Calculated Raman spectrum of ŽB. PCBEŽH. in the ZZZsss configuration compared with the experimental RR spectra of ŽA. PCBEŽH. and ŽC. a-CPCŽH..
484
C. Kneip et al.r Chemical Physics Letters 311 (1999) 479–484
analysis of these tetrapyrroles will be published elsewhere w14x.
4. Conclusions It is shown that DFT calculations can reproduce the experimental spectra of linear tetrapyrroles in different conformations and protonation states. There are no obvious restrictions to extend this approach to further configurational and conformational isomers. Thus, it is expected that based on the comparison of such spectra with the experimental RR spectra, it will be possible to determine the tetrapyrrole structures in different Žintermediate. states of phytochrome. The present results provide a key for extracting structural information from the experimental spectra. Since many normal modes are largely localized in the individual rings and adjacent methine bridges, the identification of these modes in the RR spectra of phytochrome may contribute to the elucidation of structural changes in specific parts of the chromophore during the phototransformation.
Acknowledgements P.H. acknowledges a Heisenberg fellowship by the Deutsche Forschungsgemeinschaft.
References w1x G.C. Whitelam, N.T. Harberd, Plant Cell Environ. 17 Ž1994. 615. w2x S.P.A. Fodor, J.C. Lagarias, R.A. Mathies, Biochemistry 29 Ž1990. 11141. w3x Y. Mizutani, S. Tokutomi, T. Kitagawa, Biochemistry 33 Ž1994. 153. w4x J. Matysik, P. Hildebrandt, W. Schlamann, S.E. Braslavsky, K. Schaffner, Biochemistry 34 Ž1995. 10497. w5x F. Andel III, J.C. Lagarias, R.A. Mathies, Biochemistry 35 Ž1996. 15997. w6x K. Smit, J. Matysik, P. Hildebrandt, F. Mark, J. Phys. Chem. 97 Ž1993. 11887. w7x H. Foerstendorf, E. Mummert, E. Schafer, H. Scheer, F. ¨ Siebert, Biochemistry 35 Ž1996. 10793. w8x J. Burke, D.C. Pratt, A. Moscowitz, Biochemistry 22 Ž1972. 4025. w9x M. Duerring, R. Huber, W. Bode, R. Ruembeli, H. Zuber, J. Mol. Biol. 211 Ž1990. 633. w10x J.T. Murphy, J.C. Lagarias, Photochem. Photobiol. 65 Ž1997. 750. w11x C. Kneip, D. Mozley, P. Hildebrandt, W. Gartner, S.E. ¨ Braslavsky, K. Schaffner, FEBS Lett. 414 Ž1997. 23. w12x I. Magdo, ´ K.F. Mark, P. Hildebrandt, K. Schaffner, J. Phys. Chem. A 103 Ž1999. 289. w13x C. Kneip, A. Parbel, H. Foerstendorf, H. Scheer, F. Siebert, P. Hildebrandt, J. Raman Spectrosc. 29 Ž1998. 939. w14x K. Nemeth, C. Kneip, P. Hildebrandt, F. Mark Žto be pub´ lished.. w15x W.S. Sheldrick, J. Chem. Soc., Perkin Trans. II Ž1976. 1457. w16x H. Takahashi, K. Kuroyanagi, O. Yamada, N. Kaneko, Chem. Phys. Lett. 94 Ž1983. 38.