/. Biochem. 84, 925-932 (1978)
Resonance Raman Spectra of Riboflavin and Its Derivatives in the Bound State with Egg Riboflavin Binding Proteins Yasuzo NISHINA, Teizo KITAGAWA,* Kiyoshi SHIGA,** Kihachiro HORUKE,"* Yuichi MATSUMURA, Hiroshi WATARI,** and Toshio YAMANO*** Department of Biochemistry, Wakayama Medical College, Wakayama, Wakayama 640, ^Institute for Protein Research, Osaka University, Suita, Osaka 565, "National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444, and * "Department of Biochemistry, Medical School, Osaka University, Kita-ku, Osaka, Osaka 530 Received for publication, May 11, 1978
The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH, RF), 3-carboxymethyl (3-CH,COOH RF), and 7,8-dichloro riboflavins (7,8-Cl RF), in H,O and D,O were observed in the 700-1700 cm"1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm"1 line of RF was shifted to ca. 1300 cm"1 for 3-D RF, 3-CHs RF, and 3-CH.COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm"1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C=O stretching mode was observed in the present resonance Raman spectra.
Resonance Raman spectroscopy has provided a practical method for observing selectively the vibrational spectra of chromophoric groups of biomolecules (7). For hemoproteins, for example,
the technique has revealed the vibrational spectra of iron-porphyrin interacting in situ with the apoprotein, allowing detailed discussion of the structural features of the heme moieties (2, 3). Flavoproteins, i.e., proteins containing flavin Abbreviations: RF, riboflavin; RBP,riboflavinbind- derivatives as a chromophore, play important roles ing protein; 3-D RF, N(3)-deuterated riboflavin; j n various redox processes. The physicochemical
1X!£
N ( 3 > t h
^
r i b f e 3
^
H R F
navoproteins have been studied by
N(3)carboxymethyl nboflavin; 7,8-Cl RF, 7,8dichloro riboflavin; FAD, flavin adenine dinucleot.de; CARS, spectroscopic methods including fluorometry {4coherent anti-Stolces Raman scattering. substitution and 7,8-dichJorination occurred to different lines but the frequency shifts were not dispersed widely to all modes. This suggests that some of the resonance Raman-active modes are moderately localized in each of three rings of isoalloxazine. Accordingly we can discuss the assignments of a few selected Raman lines to each ring. The notation for rings is shown in Fig. 6, and the vibrations of the Q4a)-C(10a) and C(5a)C(9a) bonds are included tentatively as Ring II modes, although those vibrations are spread over two rings and some vibrations of one ring are
930
Y. NISHINA et al.
considerably mixed with those of other rings. 1-methyl uracil upon N(3)-deuteration (24). Thus Ring III Modes—The Raman lines at 1253 we deduce that the unperturbed frequency of Ring and 1407 cm"1 of RF (top in Fig. 2) are Ring m III mode with a major contribution from C(2)modes. The former moves to ca. 1300 cm"1 upon N(3) and N(3)-C(4) stretching modes is located replacement of N(3)-hydrogen with deuterium or near 1295 cm"1 and its frequency is lowered to carbon but the latter shows little frequency shift. 1253 cm"1 for RF by vibrational coupling with the Similar frequency changes are also observed for N(3)-H bending mode. uracil derivatives upon N(3)-substitution (21). The Upon N(3)-deuteration of 1-methyl uracil, a Raman lines of 1-methyl uracil at 1242 cm"1 show new Raman line appears at 1120 cm"1 (21). The 1 a high frequency shift to 1271 cm"1 upon N(3> 1139 cm- line of 3-D RF (Figs. 1 and 2) corre1 1 deuteration, but the 1390 cm" line remains at the sponds to the 1120 cm" line of 1-methyl uracil and thus is assigned to a Ring III mode. The same frequency. 1 According to the interpretation of Spiro et al. Raman line of RF at 789 cm" is shifted to 772 1 1 1 cm" upon N(3)-deuteration, although it is unrec(13), the 1359 cm" line of free FAD (1355 cm" ognizable for 3-CH, RF and 3-CH.COOH RF. for bound RF in HiO) is accidentally degenerate, but the degeneracy is released upon N(3)-deutera- This line of RF is presumably associated with a tion. One component of the degenerate pair of breathing-like mode of Ring 111 since the breathing 1 free FAD involves the N(3)-H in-plane bending mode of 1-methyl uracil is observed at 770 cm" 1 1 vibration and therefore shifts to 1297 cm" in D,O in H,O and at 755 cm" in D,O (21). (1296 cm"1 for bound RF in D,O) while the other Ring II Modes—The Raman lines of RF which component is unaffected by the substitution. How- are little affected by the replacement of peripheral ever, if the 1355 cm"1 line of bound RF in H,O substituents at positions 7, 8, and 3 are presumed were composed of two modes and one were shifted to arise mainly from Ring n . Two intense Raman to 1296 cm"1 in D,O, the intensity of the 1352 cm"1 lines at 1584 and 1355cm - 1 in particular were noted line in D t O would be further reduced, because the as Ring II modes. The 1584 cm"1 mode might intensity of the 1355 cm"1 line in H,O is divided involve vibronic coupling at the electronic excited into the 1352 and 1296 cm"1 lines in D,O, and state for the 370 nm band, since the 1584 cm"1 line moreover the 1296 cm"1 line would show a further is markedly intensified upon excitation at 363 nm, low frequency shift upon replacement of N(3)- but the 1355cm"1 line is not (25). The 1584cm"1 deuterium with carbon. This is not compatible line is considered to correspond to the 1584 cm-1 with the observed results. line of pyrazine (y&i) (26); C(9a), C(4a), C(5a), We assigned the 1296cm"1 line of 3-D RF to and C(10a) are displaced in-phase along the C-N and N(10)]. the vibration corresponding to the 1253 cm"1 mode bonds for resting nitrogen atoms [N(5) 1 of RF and thus to a Ring m mode involving On the other hand, the 1355 cm" line is markedly C(2)-N(3) and C(4)-N(3) stretching vibrations. intensified upon excitation at 488.0 nm. Thus elecSince N(3)-C and N(3)-D bending modes are tronic excitation at the 450 nm band may involve located at much lower frequency, it is mixed little a slight expansion of the pyrazine ring. The ring-1 at 1015 cm" with the C-N stretching vibrations. On the other breathing mode of pyrazine is observed 1 hand, when hydrogen is bound to the N(3) atom, (26) and accordingly the 994 cm" line of RF may the N(3)-H bending frequency is presumably possibly correspond to the breathing-like mode of located around 1420 cm"1, as inferred from the facts Ring H. that Amide II of 1-methyl uracil has been identified Ring I Modes—The Raman lines which are at 1417 cm"1 (22) and also Amide II of a mono- slightly affected by N(3) substitutions, but show substituted amide compound with cis conformation appreciable frequency shift upon 7,8-dichlorination, has been theoretically calculated to be at 1445 cm"1 are presumably associated with Ring I modes. In (23). Therefore a vibrational coupling present be- the Raman spectra of o-xylene and o-dichlorotween the N-H bending and C-N stretching mode benzene (not shown), two Raman lines of o-xylene is expected to lower the frequency of the C-N at 1222 and 1053 cm"1 are apparently replaced by stretching mode. This view is also applicable to one line in o-dichlorobenzene at 1128 cm"1. The the high frequency shift of the 1242 cm"1 line of two lines of o-xylene at 1222 and 1053 cm"1 are J. Biochem.
RESONANCE RAMAN SPECTRA OF RIBOFLAVINS considered to be due to the strongly coupled modes of C-CHj stretching and benzene ring vibrations. Upon replacement of the two CH, groups of oxylene with Cl atoms, the vibrational coupling is removed and thus the benzene ring mode appears at 1128 cm"1. A similar change of frequencies is observed upon dichlorination of RF. The Raman line of RF at 1230 cm"1 (Fig. 2) disappears upon dichlorination and instead an intense line appears at 1191 cm"1. Therefore the 1253 cm"1 line of RF can be assigned to a Ring I mode. In the Raman spectra of both o-xylene and o-dichlorobenzene, a weak line is present at 1158 cm"1. The corresponding line of RF is at 1160 cm"1 and accordingly can be assigned to a Ring I mode. The highest fundamental frequency of benzene ring modes has been reported at 1604 cm"1 for toluene (i/ga, Aj) (27) and at 1583 cm"1 for chlorobenzene (28). The corresponding lines of o-xylene and o-dichlorobenzene were observed at 1607 and 1575 cm"1. This suggests a slight low frequency shift of the benzene ring mode upon replacement of CH, groups with Cl atoms. Comparing the Raman spectrum of RF (Fig. 2) with that of 7,8-Cl RF (Fig. 4), the low frequency shift of the 1632 cm"1 line of RF to 1611 cm"1 upon 7,8-dichlorination is in accord with this tendency. Accordingly, the 1632 cm"1 line of RF can be assigned to a Ring I mode. Previously Spiro et al. (13) suggested that the 1635 cm"1 line of free FAD was possibly due to a C = O stretching mode at C(2) or Q4). Since the C=O stretching mode of amide (Amide I) is usually coupled with N-H bending mode even in the cis conformation (23), the C=O stretching frequency is expected to show a low frequency shift upon N(3)-deuteration. In fact, we confirmed a low frequency shift of the C=O stretching Raman line of uridine from 1681 to 1656 cm"1 upon N(3>deuteration (not shown). The 1632 cm"1 line of RF showed no frequency shift upon N(3)-deuteration. Therefore we deduce that no Raman line mainly due to C=O stretching vibration is present in the spectra. Ring-breathing modes of o-xylene and o-dichlorobenzene are observed at 735 and 1038 cm"1, respectively. However, no Raman line of RF shows such a drastic change of frequency upon 7,8-dichlorination, possibly due to the presence of Ring IL We tentatively assign the Raman line of RF at Vol. 84, No. 4, 1978
931
740 cm"1 to a ring breathing-like mode of Ring I. Some Raman lines assignable to Ring II and Ring m modes also show appreciable frequency shifts upon 7,8-dichlorination. The Raman lines of RF at 1253 and 1407 cm-1 move to 1258 and 1398 cm"1 for 7,8-Cl RF. These slight changes of frequency are presumably caused by a change of electronic state, because n- electrons are so delocalized that a change of electronic state in Ring I due to 7,8-dichlorination can be easily transmitted to Ring m , where it induces a slight change of potential function and thus of stretching force constants. It should be noted that the 1258 cm"1 line of 7,8-Cl RF is shifted to 1292 cm"1 upon N(3>deuteration, but the 1289 cm"1 line of 3-CH3 RF is left unshifted in D,O. These observations also support the present assignments. Flavin-Protein Interaction—In the Raman study of FAD (75),flavin-proteininteractions when FAD is incorporated into glucose oxidase result in the appearance of an extra Raman line at 1371cm"1. The appearance of this line is interpreted in terms of the splitting of the accidentally degenerate line at 1359 cm"1, and the splitting is attributed to hydrogen bonding of the N(3)-H proton to the apoprotein on the basis of assignment of this line to a mode involving N(3)-H in-plane bending mode. In the Raman spectrum of bound RF, on the other hand, the 1355 cm"1 line appears as a single line. This difference suggests some slight difference in flavin-protein interactions between glucose oxidase and RF bound to RBP (29). However, other frequencies of RF bound to egg-yolk RBP (1631,1583, 1407, and 1355 cm"1) are nearly coincident with those of glucose oxidase (1630,1583,1409, and 1352 cm"1). If the flavin-protein interaction is significant, larger frequency differences would be expected between glucose oxidase and RF bound to RBP. Therefore further study is required to interpret the appearance of the 1371 cm"1 line of FAD for glucose oxidase. A change of the visible absorption spectrum and CD spectrum of RF upon binding to RBP (4) clearly indicates an appreciable change of the JT electronic state of isoalloxazine. Since recording of the Raman spectrum of free RF is interfered with strongfluorescence,comparison of vibrational frequencies of isoalloxazine in free RF and bound RF is impossible by conventional methods. Frequencies of Raman lines observed in the
Y. NISHINA et al.
932
present experiment are sufficiently close to those of FAD bound to glucose oxidase, and therefore the assignments of Raman lines for RF should be applicable to other flavin derivatives without major modification. Once the assignments of Raman lines are established, resonance Raman spectroscopy will be powerful tool for elucidation of specific interactions berweeen a flavin and apoprotein. The authors wish to express their gratitude to Dr. Y. Miyake, Osaka University, for his encouragement and to Dr. G. Tollin, Arizona University, for the advice upon synthesizing 7,8-dichloro nboflavin. REFERENCES 1. Spiro, T.G. & Gaber, B.P. (1977) Ann. Rev. Biochem. 46, 553-557 2. Kitagawa, T., Kyogoku, Y., Iizuka, T., & IkedaSaito, M. (1976) / . Am. Chem. Soc. 98, 5169-5173 3. Kitagawa, T., Ozaki, Y., & Kyogoku, Y. (1978) Adv. Biophys. 11, 153-198 4. Nishikinu, M. & Kyogoku, Y. (1973) / . Biochem. 73, 1233-1242 5. Nishina, Y., Horiike, K., Shiga, K., & Yamano, T. (1977) / . Biochem. 82, 1715-1721 6. Nishina, Y. (1977) Osaka Daigaku Igaku Zasshi (in Japanese) 29, 261-269 7. Yagi, K., Ohishi, N., Takai, A., Kawano, K., & Kyogoku, Y. (1976) Biochemistry 15, 2877-2880 8. Yagi, K. & Ozawa, T. (1962) Biochim. Biophys. Ada 56, 420-426 9. Edmondson, D.E. & Tollin, G. (1971) Biochemistry 10, 113-123 10. Shiga, K., Horiike, K., Isomoto, A., & Yamano, T. (1976) / . Biochem. 80, 1101-1108 11. Shiga, K., Honike, K., Nishina, Y., Isomoto, A., & Yamano, T. (1977) / . Biochem. 81, 1465-1472
12. Massey, V. & Ganther, H. (1965) Biochemistry 4, 1161-1173 13. Dutta, P.K., Nestor, J.R., & Spiro, T.G. (1977) Proc. Natl. Acad. Sci. U.S. 74, 4146-4149 14. Hemmerich, P. (1964) Heh. Chim. Ada 47, 464-475 15. Shiga, K. & Tollin, G. (1976) Flavins and Flavoproteins (Singer, T.P., ed.) pp. 422^33. Elsevier, Amsterdam 16. Whitby, L.G. (1953) Biochem. J. 54, 437-442 17. Harbury, H.A. & Foley, K.A. (1958) Proc. Natl. Acad. Sci. U.S. 44, 662-667 18. Steczku, J. & Ostowski, W. (1975) Biochim. Biophys. Ada 393, 253-266 19. Hendra, P.J. & Loader, EJ. (1968) Chem. Ind. 718-719 20. Sun, M., Moore, T.A., & Song, P-S. (1972) /. Am. Chem. Soc. 94, 1730-1740 21. Lord, R.C. & Thomas, GJ., Jr. (1967) Spectrochim. Ada 23A, 2551-2591 22. Miles, H.T., Lewis, T.P., Becker, E.D., & Frazier, J. (1973) J. Biol. Chem. 248, 1115-1117 23. Miyazawa, T. (1960) / . Mol. Spedrosc. 4, 155-167 24. Nishimura, Y., Hirakawa, A., & Tsuboi, M. (1978) Advances in Infrared and Raman Spedroscopy (Clark, R.J.H. & Hester, R.E., eds.) Vol. 5, pp. 217-275, Heyden, London 25. Tsuboi, M. (1976) in Proceedings of the Fifth International Conference on Raman Spedroscopy (Schmid, E.D., Brandmuller, J., Kiefer, W., Schrader, B., & SchrStter, H.W., eds.) pp. 135-143, Hands Ferdinand Schulz Verlag, Freiburg im Breisgau, W. Germany 26. Lord, R.C, Marston, A.L., & Miller, F.A. (1957) Spectrochim. Ada 9, 113-125 27. Fuson, N., Gamgou-Lagrange, C , & Josien, M.L. (1960) Spectrochim. Ada 16, 106-127 28. Whiffen, D.H. (1956) / . Chem. Soc. 1350-1356 29. Blankenhorn, G. (1978) Eur. J. Biochem. 82, 155-160
/. Biochem.
Resonance Raman Spectra of Riboflavin and Its Derivatives in the Bound State with Egg Riboflavin Binding Proteins Yasuzo NISHINA, Teizo KITAGAWA,* Kiyoshi SHIGA,** Kihachiro HORUKE,"* Yuichi MATSUMURA, Hiroshi WATARI,** and Toshio YAMANO*** Department of Biochemistry, Wakayama Medical College, Wakayama, Wakayama 640, ^Institute for Protein Research, Osaka University, Suita, Osaka 565, "National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444, and * "Department of Biochemistry, Medical School, Osaka University, Kita-ku, Osaka, Osaka 530 Received for publication, May 11, 1978
The resonance Raman spectra of riboflavin (RF) and its derivatives, including 3-deuterated (3-D RF), 3-methyl (3-CH, RF), 3-carboxymethyl (3-CH,COOH RF), and 7,8-dichloro riboflavins (7,8-Cl RF), in H,O and D,O were observed in the 700-1700 cm"1 region. The fluorescence problem of riboflavin was overcome by complex formation of riboflavin with riboflavin binding proteins. The observed frequencies of Raman lines of RF are in good agreement with those of glucose oxidase obtained by Spiro et al. by the resonance CARS method, although the present spectral range is extended to much lower frequency with a higher signal-to-noise ratio than that for glucose oxidase. The observed Raman lines were assigned to the individual ring modes of isoalloxazine on the basis of the Raman spectra of appropriate model compounds such as uracil, pyrazine, and o-xylene. The 1253 cm"1 line of RF was shifted to ca. 1300 cm"1 for 3-D RF, 3-CHs RF, and 3-CH.COOH RF, and accordingly can be assigned to the CN stretching mode of Ring III. The 1632 cm"1 line of RF was shifted for 7,8-Cl RF and was assigned to a Ring I mode. No Raman line mainly due to C=O stretching mode was observed in the present resonance Raman spectra.
Resonance Raman spectroscopy has provided a practical method for observing selectively the vibrational spectra of chromophoric groups of biomolecules (7). For hemoproteins, for example,
the technique has revealed the vibrational spectra of iron-porphyrin interacting in situ with the apoprotein, allowing detailed discussion of the structural features of the heme moieties (2, 3). Flavoproteins, i.e., proteins containing flavin Abbreviations: RF, riboflavin; RBP,riboflavinbind- derivatives as a chromophore, play important roles ing protein; 3-D RF, N(3)-deuterated riboflavin; j n various redox processes. The physicochemical
1X!£
N ( 3 > t h
^
r i b f e 3
^
H R F
navoproteins have been studied by
N(3)carboxymethyl nboflavin; 7,8-Cl RF, 7,8dichloro riboflavin; FAD, flavin adenine dinucleot.de; CARS, spectroscopic methods including fluorometry {4coherent anti-Stolces Raman scattering. substitution and 7,8-dichJorination occurred to different lines but the frequency shifts were not dispersed widely to all modes. This suggests that some of the resonance Raman-active modes are moderately localized in each of three rings of isoalloxazine. Accordingly we can discuss the assignments of a few selected Raman lines to each ring. The notation for rings is shown in Fig. 6, and the vibrations of the Q4a)-C(10a) and C(5a)C(9a) bonds are included tentatively as Ring II modes, although those vibrations are spread over two rings and some vibrations of one ring are
930
Y. NISHINA et al.
considerably mixed with those of other rings. 1-methyl uracil upon N(3)-deuteration (24). Thus Ring III Modes—The Raman lines at 1253 we deduce that the unperturbed frequency of Ring and 1407 cm"1 of RF (top in Fig. 2) are Ring m III mode with a major contribution from C(2)modes. The former moves to ca. 1300 cm"1 upon N(3) and N(3)-C(4) stretching modes is located replacement of N(3)-hydrogen with deuterium or near 1295 cm"1 and its frequency is lowered to carbon but the latter shows little frequency shift. 1253 cm"1 for RF by vibrational coupling with the Similar frequency changes are also observed for N(3)-H bending mode. uracil derivatives upon N(3)-substitution (21). The Upon N(3)-deuteration of 1-methyl uracil, a Raman lines of 1-methyl uracil at 1242 cm"1 show new Raman line appears at 1120 cm"1 (21). The 1 a high frequency shift to 1271 cm"1 upon N(3> 1139 cm- line of 3-D RF (Figs. 1 and 2) corre1 1 deuteration, but the 1390 cm" line remains at the sponds to the 1120 cm" line of 1-methyl uracil and thus is assigned to a Ring III mode. The same frequency. 1 According to the interpretation of Spiro et al. Raman line of RF at 789 cm" is shifted to 772 1 1 1 cm" upon N(3)-deuteration, although it is unrec(13), the 1359 cm" line of free FAD (1355 cm" ognizable for 3-CH, RF and 3-CH.COOH RF. for bound RF in HiO) is accidentally degenerate, but the degeneracy is released upon N(3)-deutera- This line of RF is presumably associated with a tion. One component of the degenerate pair of breathing-like mode of Ring 111 since the breathing 1 free FAD involves the N(3)-H in-plane bending mode of 1-methyl uracil is observed at 770 cm" 1 1 vibration and therefore shifts to 1297 cm" in D,O in H,O and at 755 cm" in D,O (21). (1296 cm"1 for bound RF in D,O) while the other Ring II Modes—The Raman lines of RF which component is unaffected by the substitution. How- are little affected by the replacement of peripheral ever, if the 1355 cm"1 line of bound RF in H,O substituents at positions 7, 8, and 3 are presumed were composed of two modes and one were shifted to arise mainly from Ring n . Two intense Raman to 1296 cm"1 in D,O, the intensity of the 1352 cm"1 lines at 1584 and 1355cm - 1 in particular were noted line in D t O would be further reduced, because the as Ring II modes. The 1584 cm"1 mode might intensity of the 1355 cm"1 line in H,O is divided involve vibronic coupling at the electronic excited into the 1352 and 1296 cm"1 lines in D,O, and state for the 370 nm band, since the 1584 cm"1 line moreover the 1296 cm"1 line would show a further is markedly intensified upon excitation at 363 nm, low frequency shift upon replacement of N(3)- but the 1355cm"1 line is not (25). The 1584cm"1 deuterium with carbon. This is not compatible line is considered to correspond to the 1584 cm-1 with the observed results. line of pyrazine (y&i) (26); C(9a), C(4a), C(5a), We assigned the 1296cm"1 line of 3-D RF to and C(10a) are displaced in-phase along the C-N and N(10)]. the vibration corresponding to the 1253 cm"1 mode bonds for resting nitrogen atoms [N(5) 1 of RF and thus to a Ring m mode involving On the other hand, the 1355 cm" line is markedly C(2)-N(3) and C(4)-N(3) stretching vibrations. intensified upon excitation at 488.0 nm. Thus elecSince N(3)-C and N(3)-D bending modes are tronic excitation at the 450 nm band may involve located at much lower frequency, it is mixed little a slight expansion of the pyrazine ring. The ring-1 at 1015 cm" with the C-N stretching vibrations. On the other breathing mode of pyrazine is observed 1 hand, when hydrogen is bound to the N(3) atom, (26) and accordingly the 994 cm" line of RF may the N(3)-H bending frequency is presumably possibly correspond to the breathing-like mode of located around 1420 cm"1, as inferred from the facts Ring H. that Amide II of 1-methyl uracil has been identified Ring I Modes—The Raman lines which are at 1417 cm"1 (22) and also Amide II of a mono- slightly affected by N(3) substitutions, but show substituted amide compound with cis conformation appreciable frequency shift upon 7,8-dichlorination, has been theoretically calculated to be at 1445 cm"1 are presumably associated with Ring I modes. In (23). Therefore a vibrational coupling present be- the Raman spectra of o-xylene and o-dichlorotween the N-H bending and C-N stretching mode benzene (not shown), two Raman lines of o-xylene is expected to lower the frequency of the C-N at 1222 and 1053 cm"1 are apparently replaced by stretching mode. This view is also applicable to one line in o-dichlorobenzene at 1128 cm"1. The the high frequency shift of the 1242 cm"1 line of two lines of o-xylene at 1222 and 1053 cm"1 are J. Biochem.
RESONANCE RAMAN SPECTRA OF RIBOFLAVINS considered to be due to the strongly coupled modes of C-CHj stretching and benzene ring vibrations. Upon replacement of the two CH, groups of oxylene with Cl atoms, the vibrational coupling is removed and thus the benzene ring mode appears at 1128 cm"1. A similar change of frequencies is observed upon dichlorination of RF. The Raman line of RF at 1230 cm"1 (Fig. 2) disappears upon dichlorination and instead an intense line appears at 1191 cm"1. Therefore the 1253 cm"1 line of RF can be assigned to a Ring I mode. In the Raman spectra of both o-xylene and o-dichlorobenzene, a weak line is present at 1158 cm"1. The corresponding line of RF is at 1160 cm"1 and accordingly can be assigned to a Ring I mode. The highest fundamental frequency of benzene ring modes has been reported at 1604 cm"1 for toluene (i/ga, Aj) (27) and at 1583 cm"1 for chlorobenzene (28). The corresponding lines of o-xylene and o-dichlorobenzene were observed at 1607 and 1575 cm"1. This suggests a slight low frequency shift of the benzene ring mode upon replacement of CH, groups with Cl atoms. Comparing the Raman spectrum of RF (Fig. 2) with that of 7,8-Cl RF (Fig. 4), the low frequency shift of the 1632 cm"1 line of RF to 1611 cm"1 upon 7,8-dichlorination is in accord with this tendency. Accordingly, the 1632 cm"1 line of RF can be assigned to a Ring I mode. Previously Spiro et al. (13) suggested that the 1635 cm"1 line of free FAD was possibly due to a C = O stretching mode at C(2) or Q4). Since the C=O stretching mode of amide (Amide I) is usually coupled with N-H bending mode even in the cis conformation (23), the C=O stretching frequency is expected to show a low frequency shift upon N(3)-deuteration. In fact, we confirmed a low frequency shift of the C=O stretching Raman line of uridine from 1681 to 1656 cm"1 upon N(3>deuteration (not shown). The 1632 cm"1 line of RF showed no frequency shift upon N(3)-deuteration. Therefore we deduce that no Raman line mainly due to C=O stretching vibration is present in the spectra. Ring-breathing modes of o-xylene and o-dichlorobenzene are observed at 735 and 1038 cm"1, respectively. However, no Raman line of RF shows such a drastic change of frequency upon 7,8-dichlorination, possibly due to the presence of Ring IL We tentatively assign the Raman line of RF at Vol. 84, No. 4, 1978
931
740 cm"1 to a ring breathing-like mode of Ring I. Some Raman lines assignable to Ring II and Ring m modes also show appreciable frequency shifts upon 7,8-dichlorination. The Raman lines of RF at 1253 and 1407 cm-1 move to 1258 and 1398 cm"1 for 7,8-Cl RF. These slight changes of frequency are presumably caused by a change of electronic state, because n- electrons are so delocalized that a change of electronic state in Ring I due to 7,8-dichlorination can be easily transmitted to Ring m , where it induces a slight change of potential function and thus of stretching force constants. It should be noted that the 1258 cm"1 line of 7,8-Cl RF is shifted to 1292 cm"1 upon N(3>deuteration, but the 1289 cm"1 line of 3-CH3 RF is left unshifted in D,O. These observations also support the present assignments. Flavin-Protein Interaction—In the Raman study of FAD (75),flavin-proteininteractions when FAD is incorporated into glucose oxidase result in the appearance of an extra Raman line at 1371cm"1. The appearance of this line is interpreted in terms of the splitting of the accidentally degenerate line at 1359 cm"1, and the splitting is attributed to hydrogen bonding of the N(3)-H proton to the apoprotein on the basis of assignment of this line to a mode involving N(3)-H in-plane bending mode. In the Raman spectrum of bound RF, on the other hand, the 1355 cm"1 line appears as a single line. This difference suggests some slight difference in flavin-protein interactions between glucose oxidase and RF bound to RBP (29). However, other frequencies of RF bound to egg-yolk RBP (1631,1583, 1407, and 1355 cm"1) are nearly coincident with those of glucose oxidase (1630,1583,1409, and 1352 cm"1). If the flavin-protein interaction is significant, larger frequency differences would be expected between glucose oxidase and RF bound to RBP. Therefore further study is required to interpret the appearance of the 1371 cm"1 line of FAD for glucose oxidase. A change of the visible absorption spectrum and CD spectrum of RF upon binding to RBP (4) clearly indicates an appreciable change of the JT electronic state of isoalloxazine. Since recording of the Raman spectrum of free RF is interfered with strongfluorescence,comparison of vibrational frequencies of isoalloxazine in free RF and bound RF is impossible by conventional methods. Frequencies of Raman lines observed in the
Y. NISHINA et al.
932
present experiment are sufficiently close to those of FAD bound to glucose oxidase, and therefore the assignments of Raman lines for RF should be applicable to other flavin derivatives without major modification. Once the assignments of Raman lines are established, resonance Raman spectroscopy will be powerful tool for elucidation of specific interactions berweeen a flavin and apoprotein. The authors wish to express their gratitude to Dr. Y. Miyake, Osaka University, for his encouragement and to Dr. G. Tollin, Arizona University, for the advice upon synthesizing 7,8-dichloro nboflavin. REFERENCES 1. Spiro, T.G. & Gaber, B.P. (1977) Ann. Rev. Biochem. 46, 553-557 2. Kitagawa, T., Kyogoku, Y., Iizuka, T., & IkedaSaito, M. (1976) / . Am. Chem. Soc. 98, 5169-5173 3. Kitagawa, T., Ozaki, Y., & Kyogoku, Y. (1978) Adv. Biophys. 11, 153-198 4. Nishikinu, M. & Kyogoku, Y. (1973) / . Biochem. 73, 1233-1242 5. Nishina, Y., Horiike, K., Shiga, K., & Yamano, T. (1977) / . Biochem. 82, 1715-1721 6. Nishina, Y. (1977) Osaka Daigaku Igaku Zasshi (in Japanese) 29, 261-269 7. Yagi, K., Ohishi, N., Takai, A., Kawano, K., & Kyogoku, Y. (1976) Biochemistry 15, 2877-2880 8. Yagi, K. & Ozawa, T. (1962) Biochim. Biophys. Ada 56, 420-426 9. Edmondson, D.E. & Tollin, G. (1971) Biochemistry 10, 113-123 10. Shiga, K., Horiike, K., Isomoto, A., & Yamano, T. (1976) / . Biochem. 80, 1101-1108 11. Shiga, K., Honike, K., Nishina, Y., Isomoto, A., & Yamano, T. (1977) / . Biochem. 81, 1465-1472
12. Massey, V. & Ganther, H. (1965) Biochemistry 4, 1161-1173 13. Dutta, P.K., Nestor, J.R., & Spiro, T.G. (1977) Proc. Natl. Acad. Sci. U.S. 74, 4146-4149 14. Hemmerich, P. (1964) Heh. Chim. Ada 47, 464-475 15. Shiga, K. & Tollin, G. (1976) Flavins and Flavoproteins (Singer, T.P., ed.) pp. 422^33. Elsevier, Amsterdam 16. Whitby, L.G. (1953) Biochem. J. 54, 437-442 17. Harbury, H.A. & Foley, K.A. (1958) Proc. Natl. Acad. Sci. U.S. 44, 662-667 18. Steczku, J. & Ostowski, W. (1975) Biochim. Biophys. Ada 393, 253-266 19. Hendra, P.J. & Loader, EJ. (1968) Chem. Ind. 718-719 20. Sun, M., Moore, T.A., & Song, P-S. (1972) /. Am. Chem. Soc. 94, 1730-1740 21. Lord, R.C. & Thomas, GJ., Jr. (1967) Spectrochim. Ada 23A, 2551-2591 22. Miles, H.T., Lewis, T.P., Becker, E.D., & Frazier, J. (1973) J. Biol. Chem. 248, 1115-1117 23. Miyazawa, T. (1960) / . Mol. Spedrosc. 4, 155-167 24. Nishimura, Y., Hirakawa, A., & Tsuboi, M. (1978) Advances in Infrared and Raman Spedroscopy (Clark, R.J.H. & Hester, R.E., eds.) Vol. 5, pp. 217-275, Heyden, London 25. Tsuboi, M. (1976) in Proceedings of the Fifth International Conference on Raman Spedroscopy (Schmid, E.D., Brandmuller, J., Kiefer, W., Schrader, B., & SchrStter, H.W., eds.) pp. 135-143, Hands Ferdinand Schulz Verlag, Freiburg im Breisgau, W. Germany 26. Lord, R.C, Marston, A.L., & Miller, F.A. (1957) Spectrochim. Ada 9, 113-125 27. Fuson, N., Gamgou-Lagrange, C , & Josien, M.L. (1960) Spectrochim. Ada 16, 106-127 28. Whiffen, D.H. (1956) / . Chem. Soc. 1350-1356 29. Blankenhorn, G. (1978) Eur. J. Biochem. 82, 155-160
/. Biochem.
