220
J. Phys. Chem. 1983, 87,220-225
Linear Dichroism Studies of Flavins in Stretched Poiy(viny1 alcohol) Films. Molecular Orientation and Electronic Transition Moment Directions Yuklo Matsuoka and Bengt NordOn’ Institute of Physical C b m k t v , Chalmers University of Technology, S-4 12 96 Gothenburg, Sweden (Received; April 13, 1982; In Final Form: September 9, 1982)
Linear dichroism of 3-methyllumiflavin (MLF) and riboflavin (RF) partially oriented in stretched poly(viny1 alcohol) (PVA) film was measured in the 220-530-nm region to determine the directions of the electronic transition moments. IR dichroism was measured in the same stretched films in the region 1900-1500 cm-* to obtain information about the molecular orientation tensor. The four important T-T* transitions of 3-methyllumiflavin, with absorptions at 448, 360, 270, and 225 nm in poly(viny1 alcohol) medium, were found to be polarized at the angles (counterclockwisedirection) 50°, 74O, 90°, and 40’ with respect to the N5-N10 line. The corresponding transitions of riboflavin, occurring at 448,365, 270, and 225 nm, were found at the angles 45O, 6 8 O , 76”, and 39O, respectively. Besides these four major transitions, evidence for two hidden transitions at about 310 and 250 nm is presented. The transition moment directions were found to be in good agreement with results from single-crystal measurements.
Introduction The electronic and photochemical properties of flavins are matters of basic importance for the understanding of the role of these chromophores in different biological mechanisms. For example, detailed knowledge about the electronic transition moment directions of flavins is necessary for interpretation of circular dichroism and polarization spectroscopy in studies of conformation and of orientation (in membranes) of flavoproteins. Despite a widespread interest in the flavin (isoalloxazine) chromophore, however, many experimental and theoretical results still tend to be in conflict with each other. With the aim of determining transition moment directions, flavins have been studied by means of fluorescence polarization in glassy matrix,’* polarized absorption and reflection spectra in single and with linear dichroism in partially oriented Molecular orbital (MO)calculat i o n ~ ’ ~ -have ’ ~ also been carried out in parallel with the experimental studies. Single-crystal measurements can in principle give absolute directions of transition moments in crystals of (1)Chen, R. F.; Bowman, R. L. Science 1965,147,729. (2)Kurtin, W. E.; Song, P.-S. Photochem. Photobiol. 1968, 7, 263. (3)Gordon-Waker, A.; Penter, G. R.; Radda, G. K. Eur. J. Biochem. 1970,13,313. (4)Sun, M.;Moore, T. A.; Song, P.3. J . Am. Chem. SOC.1972,94, 1730. (5) Song, P.-S.; Moore, T. A.; Kurtin, W. E. 2. Naturforsch. B 1972, 27, 1011. (6) Schmidt, W.Photochem. Photobiol. 1981,34, 7. (7)Eaton, W. A.; Hofrichter, J.; Makinen, M. W.; Andersen, R. D.; Ludwig, M. L. Biochemistry 1975,14, 2146. (8)Yu, M. W.; Fritchie, C. J.; Fucaloro, A. F.; Anex, B. G. J . Am. Chem. SOC.1976,98,6496. (9)Lhoste, J. M.In “Procedings of the First European Biophysics Congress”; Broda, H., Locker, A., Springer-Lederer, H., Eds., Wiener Medizinischen Akademie: Vienna, 1971;p 221. (10)S i h i a k , J.;Frackowiak, D. Photochem. Photobiol. 1972,16,173. (11) Drabent. R.Acta Phvs. Pol. A 1979. 55. 371. (12)Johansson, L. B. A.; Davidsson, A.; Lindblom, G.; Naqvi, K. R. Biochemistry 1979,19,4249. (13)Fox, J. L.;Laberge, S. P.; Nishimoto, K.; Forster, L. S. Biochim. Biophys. Acta 1976,136,544. (14) Song, P.4. Int. J. Quantum Chem. 1968,2,463. (15)Song, P.-S. Int. J. Quantum Chem. 1969,3,303. (16)Grabe, B. Acta Chem. Scand. 1972,26,4084.
known structure; however, electronic interactions between identical chromophores may complicate the interpretation of the observed spectra. This problem of interaction can be eliminated by dilution of the guest molecules in a host crystal or a glassy matrix. The molecules have no preferential orientation in the glass but can be photoselected by excitation with polarized radiation, and the resulting fluorescence polarization can give information about the directions of the transition moments relative to each other. Dichroism methods using anisotropic solvents, such as liquid crystals or stretched polymer films, can provide directions of transition moments when the molecular orientation is known.” Although this dichroism technique is a very direct one, there have been only few attempts to use it for quantitative study of transition moment directions of flavins. This is probably due to the general difficulty of analyzing the orientation of molecules with a low symmetry. In the first film dichroism study of flavins, done by Lhosteg in poly(viny1 alcohol), PVA, the orientation was estimated simply from the shape of the solute molecule, and only a qualitative discussion of the directions of the first two T-K* transitions was possible. For highly symmetric and elongated molecules, it is usually possible to estimate an “orientation axis” which has the highest tendency of becoming aligned parallel to the stretching direction. However, for small, unsymmetrical molecules such as riboflavin, it may no longer be physically meaningful to speak about an orientation axis.ls By studying instead electric dichroism, where the orientation is produced by the interaction of a molecular dipole moment with an applied electric field, one can in principle eliminate the orientation ambiguity and determine the transition moment directions relative to the permanent dipole moment.lg In our laboratory, some transition moments in lumiflavin were recently determined by means of this technique in combination with linear dichroism in lamellar liquid crystals (model membranes20).’* Unfortunately, lack of precise information about the direction of the dipole moment and about the orientation in the anisotropic (1‘7)Norddn, B.Appl. Spectrosc. Reo. 1978,14, 157. (18)Matsuoka, Y.;Norddn, B. J . Phys. Chem. 1982,86,1378. (19)Davidsson, A.; Norddn, B. Spectrosc. Lett. 1977,10,447. (20)Norden B.; Lindblom, G.; Jon%, I. J . Phys. Chem. 1977,81,2086.
0022-3654/83/2087-0220$01.50/00 1983 American Chemical Society
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983 221
Flavins in Stretched Poly(viny1 alcohol) Films
solvent made it necessary to rely on previous crystal results and on a semiempirical MO calculation, and the absolute accuracy of that determination is therefore difficult to estimate. The moment directions of riboflavin have also been studied by Si6dmiak et al.1° and by Drabent’l in stretched PVA film. However, the reported linear dichroism values are conflicting with each other, especially in the 290-350-nm region, and, since very little seems to be known about how the molecules are oriented in the film, the assignments become uncertain. The primary object of this paper is to study the orientation of flavins (3-methyllumiflavinand riboflavin) in the PVA system and to try to determine the electronic transition moment directions. For this purpose the linear dichroism is measured in both the IR (1900-1500 cm-l) and the UV-visible (220-530 nm) regions. The analysis will be based on a general expression for the linear dichroism which does not presume any preferred molecular orientation axis and which has been developed for the study of low-symmetrical planar m o l e ~ u l e s . ~By ~ J the ~ aid of IR dichroism, a molecular coordinate system is first determined providing a diagonal orientation tensor. The directions of the electronic transition moments will finally be discussed in relation to results from single-crystal studies and MO calculations.
Method Section Experimental Section. The 3-methyllumiflavin(MLF) was a gift from Professor J. Koziol, Academy of Economics, Poznan, Poland, and riboflavin (RF) was obtained from Serva Chemical Co. As the UV-visible absorption spectra were in excellent agreement with literature spectra, the samples were used without further purification. Commercially available poly(viny1 alcohol), PVA, was used for the film matrix. Flavin-PVA films were prepared as follows: Aqueous PVA (ca. 10% w/w) and flavin (ca. M) solutions were mixed and stirred in the dark at about 50 “C until a homogeneous solution was obtained. This was spread onto a horizontal glass plate and kept for several days in a dark room to give an isotropic flavin-PVA film with a water content of more than 7% w/w. A reference film without flavin was prepared under the same conditions. The film thickness was varied between and cm to allow study of infrared and UV-visible linear dichroism at varied solute concentrations. Dichroic absorption spectra were measured on a Cary 219 spectrophotometer supplemented with a rotatable Glan air-space polarizer preceding the sample in the light path. The reduced dichroism of flavin in a uniaxially stretched PVA film is given by LD’ = 3(All- AI)/(AII + 2A,), where All and A, are the dichroic absorbances (corrected for background) for linearly polarized light with the electric vector polarized parallel (11 ) and perpendicular (I) to the stretching direction. IR dichroic spectra were recorded on a Nicolet MX-1 Fourier-transform spectrometer. The transmittances TI,,T , of a sample film, and Trll, T,, of a reference film, were measured, providing the absorbances A,, = log T,ll/Tlland A , = log T,,/T,. The stretch ratio R, of the film is defined as before.18 Analysis of Linear Dichroism. For small unsymmetrical molecules, a transition moment direction can be determined by a more general model, starting with a nondiagonal orientation tensor.”J8 If two axes, y’ and z’ ( x ’ is out-of-plane direction), are chosen arbitrarily in the molecular plane and only in-plane polarized transitions are observed, the reduced dichroism can be expressed by LD’ = 3(S,,,, sin2 0’
+ S,,,,
cos2 8’
+ S,,,,
sin 8’ cos 6”) (1)
where 8’ is the angle between the transition moment and the z’axis, and Sylyt,S,, and S,, are order parameters defined as previously.18 If we rotate the system x’y‘z’around the x’ axis an arbitrary angle a into a new system xyz ( x = x’), eq 1 can be rewritten as follows:
+ S,,
+
cos2 (e’ - a) S,, sin (6’ - a) cos (e’ - a)) = 3(S,, sin2 6 + S,, cos2 6 + S,, sin e cos e) (2)
LD’ = 3(S,, sin2 (19’ - a)
where 6 = 6’ - a is the angle between the transition moment and the z axis (e’ and a are taken to be measured counterclockwise relative to the z’axis). We can find such an angle ao,at which S,, in eq 2 disappears and the orientation tensor becomes diagonal, corresponding to extrema in S, and SZz.l8 The angle a. and the diagonalized order parameters S and S,, are directly given by the following equations: tan 2ao = S,~,~/(S,~,~ - S,,,,) S,, = Sytylcos2 cyo
S,, = S , , sin2 a.
+ Sztztsin2 a. - f/2S,,ztsin 2ao + S,,,, cos2 a. + f/2Sytz,sin 2a0 (3)
The axes of the diagonal system xyz are labeled so that the parameters S,,, S,, and S,, (S,, + S,, + S,, = 0) fulfill S,, IS,, IS,,. In the diagonal system, eq 2 thus reduces to LD’ = 3(S,, sin2 e
+ S,, cos2 0)
(4)
Interpretation of LD’ never requires more complicated formalisms than the present one. Introduction of the molecular distribution function in terms of a truncated series generally implies and extra, undesired ambiguity.21
Results Determination of Order Parameters. To determine the directions of the electronic transition moments of 3methyllumiflavin (MLF) and riboflavin (RF), we first need to know the values of S, and S,, in eq 4 and the diagonal coordinate system xyz from some independent measurement. This piece of information can in favorable cases be obtained from the study of IR dichroism.18 As shown in Figure 1,both MLF and RF exhibit in the oriented PVA film a significant linear dichroism in the 1800-1500-~m-~ region, where the respective bands can be identified by correlation with the corresponding isotropic spectra in KBr matrix. The bands at 1700 and 1645 cm-I of MLF (1710 and 1660 cm-l for RF) can be assigned to arise from the C4=0 and C2=0 stretching vibration^^^-^^ (the carbonyl groups of thymine and uracil have also been exploited in dichroism work in PVA18). The distinct peaks at 1590 and 1555 cm-l of MLF (1580 and 1550 cm-’ for RF) are due to the amide I1 vibrational transition^.^^ The LD’ values of the C4=0 and C2=0 bands of MLF were 0.07 f 0.03 and 0.11 f 0.02, respectively, and 0.18 f 0.02 and 0.15 f 0.02 for RF. The LIY of the amide I1 band was 0.58 f 0.02 (at 1555 cm-’) for MLF, and 0.40 f 0.02 (at 1550 cm-’) for RF. Substitution of these data into eq 1 for the three (21)Andersson, L.;NordBn, B. Chem. Phys. Lett. 1980,75, 398. (22)Hemmerich, P.;Lauterwein, J. In ”Inorganic Biochemistry”; Eichhorn, G. L., Ed.; Elsevier: Amsterdam, 1973;Vol. 11, p 1169. (23)Hemmerich, P.; Prijs, B.; Erlenmeyer, H. Helu. Chim. Acta 1960, 43,312. (24)Spence, J. T.;Peterson, E. R. J.Inorg. Nucl. Chem. 1962,24,601.
222
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983
Matsuoka and Norden
R1
60
I
c
\
\
Figure 2. (a) Definition of the z'axis and the rotation angle CY. (b) Diagonal (Sn,S,) values of MLF and RF in the orientation triangle PQR," and the corresponding diagonal system xyz at S,, = 0.
I
I
TABLE I: Order Parameters Calculated for Various Transition Moment Angles p of Amide I1 Band" PI
deg
Sztzt Syiyi Syizi
@,I
deg
,S,
S ,,
S,,
-0.049 -0.085 -0.098 -0.206
-0.216 -0.290 -0.316 -0.532
MLF 1900
1700
c m-'
1500
Flgure 1. Infrared dichroic spectra ( T , , / T r(-) l , and T , / T , , (---)) of 3-methyllumiflavin (a) and riboflavin (b) in PVA film at R , = 4.3. (c) Infrared spectrum of riboflavin in KBr.
in-plane vibrations (C4=0, C2=0, and amide 11) gives in the case of MLF 0.07 = 3(SYry.sin2 0'
+ S,,,,
cos2 0"
+ S,,,,, sin 0" cos 0') (5)
0.11 = 3(S,,,, sin2 120" + S , , cos2 120' + S , , sin 120' cos 120") (6) 0.58 = 3(SY,,,sin2 p
+ SzlZlcos2 p + S,,,,, sin p cos p )
(7)
with the (arbitrary) z'axis defined to be parallel to the N1-C4 line, as shown in Figure 2a (corresponding to CY = 0"). The transition moments of the C4=0 and C2=0 bands are assumed to be directed parallel to the respective bonds, i.e., at the angles 0" and 120" relative to the z 'axis. With the three equations 5-7 we should in principle be able to determine the three independent order parameters Sfy, S,,, and Sf,,.Unfortunately there is a considerable ambiguity concerning the moment direction of the amide I1 band ( p in eq 7), which arises mainly from C-N bondstretching and N-H bond-bending motions. From this assignment and the fact that the amide I1 transition is polarized almost exactly perpendicular to the CY helix of poly-y-benzyl-~-glutamate,~~ we preliminarily estimate p (25) Tsuboi, M. J . Polym. Sci. 1962,59, 139.
90 100 102 110
0.023 0.023 0.023 0.023
0.193 0.267 0.293 0.510
0.263 0.390 0.435 0.811
90 99 100 110
0.060 0.060 0.060 0.060
0.133 0.169 0.174 0.306
0.149 0.211 0.221 0.449
61.4 61.0 60.9 60.5
0.265 0.375 0.414 0.738
RF 58.0 58.6 58.6 59.3
0.180 0.013 0.233 -0.004 0.241 -0.007 0.439 -0.073
-0.193 -0.229 -0.234 -0.366
" Angle CY,, for which S y t = 0. to be approximately 100" (f20"). Table I shows the solutions of eq 5-7 for selected p values in the interval 90-110° (correspondingdiagonal solutions obtained by use of eq 3). The determination of the order parameters can in fact be refined by considering the physical limits of the respective Siiparameters. First, the out-of-plane parameter must satisfy the condition -0.50 IS,, I 0. Second, we have that S,, 5 1/3(LDrmin) < 1/3(LDrm,) IS,,, where LDrminand LD', are the minimum and maximum reduced dichroisms observed among all in-plane UV-visible and IR transitions. This gives S,, I1/3(l.19)= 0.397 for MLF, and S,, 1 1/3(0.68) = 0.227 for RF, and we obtain @ very close to 100" for both molecules. This conclusion is supported by the corresponding S,, values (-0.30 for MLF and -0.23 for RF) which are in very good agreement with those obtained for acridine orange (-0.31) and acridine (-0.21) in PVA at R, = 4.3.26 The obtained diagonal system xyz and the corresponding order parameters (S,,, s,,) are shown in Figure 2b. (26) Matauoka, Y.; Yamaoka, K. Bull. Chem. SOC.Jpn. 1979,52,3163.
Flavins in Stretched Poly(viny1 alcohol) Films
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983 223
41.0
-08
-06
-34
x
nm Flguw 3. (a) Isotropic absorption spectrum (-) and reduced dichroism (0) of MLF in PVA film at R , = 4.3. The two posslble moment directions (double-headed arrows) of the transition I are shown relative to the diagonal z axis. (b) Isotropic absorption spectrum (-) and of RF in PVA film at R , = 4.3. The sum of reduced dichroism (0) contributions from the six Gaussian bands e/ (---), and the corresponding reduced dichroism LD' = C,,,ee,(LDr),/C,,,ot, are represented by dotted curves.
From the position in the orientation triangle (Figure 2b) it can be seen that RF shows a rather disklike17orientation compared to MLF, which can be understood as an effect of the ribityl side chain at the 10-position.n The direction of the diagonalizing z axis, nearly parallel to the N3-NI0 line, is in agreement with the "orientation axis" assumed in earlier work on 3-methylcarboxylumiflavin.g Determination of Transition Moment Directions. The isotropic absorption spectrum Ai, = (All+ 2A,)/3 and the reduced dichroism LD' = (A,,- A,)/Ai, of MLF and RF are shown in Figure 3. The Ai, spectra were practically identical with those observed in aqueous solution at room temperature. Each indicated LIY value is the average from three sample films with different flavin concentrations, of which the one with highest concentration was used for the IR measurement. (The corresponding three Ai, spectra had practically identical shapes, and only one of them is therefore shown in the figure.) MLF and RF show positive LD' in the studied wavelength region which is expected as the absorption is dominated by in-plane polarized transitions. Theoretically, the transition moments in low-symmetry planar molecules can have any direction parallel to the molecular plane or exactly perpendicular to it.17 Any existing out-of-plane (n-r*) transition in the region 220-530 nm of MLF and RF should be very weaka and will therefore be ignored in the following analysis (27) Thulstrup, E. W. 'Aspects of the Linear and Magnetic Circular Dichroism of Planar Organic Molecules";Springer-Verlag: Heiderberg, 1980.
which means that we can directly apply eq 4 for the determination of the transition moment directions. As seen from eq 4, LD' as a function of wavelength will be flat over an isolated absorption band only if 8 is a constant (SYyand S,, are, of course, independent of wavelength). The LD' curve can therefore provide a test of whether there is an admixture of different electronic transitions in the wavelength region. Unsymmetric vibrations can also change the polarization of an electronic transition: superposition of different vibronic transitions then generally leads to LD' changing gradually over the absorption band. Comparison of the LD' curves with the isotropic spectra indicates the presence of six transitions (designated I-VI) in the 220-530-nm region of both MLF and RF. The apparent wavelength positions of these transitions are in reasonable agreement with the results of circular dichroism28 and molecular orbital calculations.4y5J3J5Transitions I11 and V are hidden near 310 and 250 nm, but their presence is revealed by abrupt changes in the LD' curve at these positions. Magnetic circular dichroism (MCD) spectra also support the presence of these additional UV bands,29corresponding to the transitions I11 and V in Figure 3a (these transitions will be further discussed below). Note the difference in magnitude of LD' between MLF and RF, an effect of their different orientation in the PVA film. The estimated set of diagonal order parameters Syyand S,, of MLF and RF (Figure 2b) now allows the electronic transition moment angle 8 to be determined. For example, for transition I of MLF, where LD' is nearly flat over the corresponding absorption region, eq 4 can be directly applied 1.19 = 3(-0.10 sin2 0
+ 0.41 cos2 8)
which gives 181 = 9.3" relative to the diagonal z axis (see the inset in Figure 3a). For the region below 400 nm, however, the continuously varying LD' indicates that bands of differently polarized transitions overlap each other. Using a computer-aided trial-and-error method, described we have first resolved the observed isotropic spectrum into component bands (Gaussian on a wavenumber scale), and then the observed LD' curve was reconstructed by using the Gaussian bands and the transition moment angles. In this way intrinsic LIY values due to the respective transitions could be evaluated. In Figure 3b the component bands and the calculated LD' curve are shown together with the experimental spectra for the case of RF. The shoulders observed at about 480 and 420 nm are parts of a vibrational structure of the first absorption band of RF;4 we have therefore approximated the band with a single Gaussian envelope, which was sufficient to reproduce the observed LD' in the corresponding wavelength region. The abrupt decrease of LD' near 300 nm can be satisfactorily explained by the in-plane polarized transition 111; however, a small difference between observed and calculated LD' curves still remains near 350 nm (this difference was also observed for MLF). The gradually decreasing LD' over the absorption band of transition I1 (320-360 nm) can indicate that the difference is an effect of vibronic coupling. The results in terms of LD' and 0 values for MLF and RF are presented in Table 11. As is seen from this table, there are two possible moment directions for each transition. However, as will be shown below, the unphysical (28) Miles, D. W.; Urry, D. W . Biochemistry 1968, 7, 2791. (29) Tollin, G. Biochemistry 1968, 7, 1720. (30) Matauoka, Y.; Yamaoka, K. Boll. Chem. SOC.Jpn. 1980,53,2146. (31) Matauoka, Y.; Norden, B. Biopolymers, in press.
The Journal of Physical Chemistry, Vol. 87, No. 2, 1983
224
Matsuoka and NordBn
TABLE 11: Reduced Dichroism of Resolved Absorption Bands and Transition Moment Directions in 3-Methyllumiflavin (MLF) and Riboflavin ( R F )
c ~ l a t i o n s , and ~ ~ ~we J ~therefore take this solution as the most probable one. In the corresponding discussion for RF, we can by the same arguments eliminate the solutions 72 f 4' (for I) and 50 f 4' (for 111,in favor of 45 f 4' (for I) and 68 f 4O (for 11). Transition III. From magnetic circular dichroism (MCD) measurements on RF and its analogues TollinZ9 suggested that the second absorption band (370 nm) of RF is due to two transitions. The presence of more than one transition is in fact manifested through the significant variations of LD' at the short-wavelengthside of the second band (Figure 3). The LD' curve shows a minimum near 300 nm, in accordance with the linear dichroism results of Drabent." In order to explain and reproduce the LD' behavior in the 300-370-nm region, it was necessary to introduce in the curve analysis a third, weak in-plane polarized transition (denoted 111) with LD' = 0.35 at the blue edge of the second absorption band of RF (see Figure 3b and Table 11). Its wavelength position and moment direction (nearly parallel to the Nl-C7 line) are both in reasonable agreement with the results of MO calculation~.~-'~ Transition IV. From fluorescence polarization data of flavins,1~2,6,'oJ2 the angle between the transition moments of the first and third absorption bands (at 450 and 270 nm) can be estimated to about 35 f loo, provided that the 270-nm absorption arises purely from a single transition. As seen from Figure 3b, the effect of overlap from neighboring absorption bands is almost negligible at 270 nm and we shall accept the value 35 f 10' as the angle between the transitions I and IV. This result is in agreement with the solution 76 f 3' for the transition IV of RF (which makes 31' between the transitions) and we can discard the solution 42 f 3' as unphysical. For the same reason the solution 32 f 3' for MLF is eliminated. Transitions V and VI. The assignment of polarizations in the far-UV region is less clear, both because of superposition of differently polarized electronic transitions (as seen from the varying LD' below 270 nm) and also because of lack of reliable fluorescence polarization data in this region. A decrease of LD' between the third (270 nm) and fourth (225 nm) absorption bands suggests the presence of a fifth transition at about 250 nm. The existence of such a transition (V) is in fact supported by MCD measurementsm We have no fluorescence data for the transitions V and VI; however, on the basis of polarized reflectance measurements on bis(l0-methylisoalloxazine)copper(II) perchlorate tetrahydrate crystal by Yu et ala,*we shall eliminate the following solutions: 27 f 3' for the transition V, and 82 f 3' for the transition VI of MLF. From the same arguments, we can discard the solutions 37 f 3' and 78 f 4' for the transitions V and VI of RF. The eliminated solutions are all very different from the physical ones and
moment directions leia/
transitions
A/ nm
(LDr)i
I I1
448 360
1.19 1.16
deg
6 /deg
MLF 11 i 4
50 i 4 o r ( 7 1 i 4 ) (48f 4)or74i 4 (19i 4)orl03i 4
I11
313
0.55
13+4 421 4
IV
270
0.88
29i 3
(32
V VI
249 225
0.76 1.04
34 21
(27 i 3 ) o r 9 5 i 3 4 0 + 3 o r ( 8 2 i 3)
I
448
0.66
I1
365
I11 IV
310 270
V VI
249 225
0.68 0.35 0.64 0.60 0.62
RF 14 i 4 9i 4
i f
3 3
44i 4 17f 3 22i 3 19 f 4
i
3)or90*3
45i 4 o r ( 7 2 * 4 ) (50 i 4 ) o r 6 8 = 4 ( 1 4 i 4 ) o r 103 = 4 (42 f 3 ) o r 7 6 i 3 (37 i 3 ) o r 8 1 2 3 39 i d o r ( 7 8 i 4 )
a Angles relative t o the diagonal z axis. Angles with respect t o the N,-N,, line toward the C, atom (counterclockwise direction), the solutions in parentheses have been eliminated as unphysical, from arguments given in the text; values in italics represent the concluded angles.
solution can usually be discarded after considering the result in relation to fluorescence polarization data and single-crystal measurements.
Discussion Transitions I and II. A constant L D in the 400-500-nm region, for both MLF and RF, is evidence that the first absorption band is due to a single transition (with vibrational structure). This conclusion is in agreement with the fluorescence polarization data of riboflavin and lumiflavin reported by Song et aL4 In their study, the polarized fluorescence excitation was nearly constant (about 0.4) across the first absorption band but decreased to 0.25-0.30 in the second band region. This is also clear evidence that the second absorption band arises from a different electronic transition. Different fluorescence polarization studies have obtained the two transitions a t roughly 20 f 10' to each other.14J2 We therefore get only two possible solutions for the transitions I and I1 of MLF: solution 1 is 50 f 4' (for transition I) and 74 f 4O (for 11),and solution 2 is 71 f 4O (for I) and 48 f 4' (for 11). Solution 1 is in good agreement with the results of single-crystal measurements7s8and does not conflict with the MO cal-
TABLE 111: Observed and Calculated Transition Moments in Lumiflavin and Related Compounds experiments
a
MLF in PVAa
transitions
~f
I I1 I11 IV V VI
448 360 313 270 249 225
This work.
hf
6g
50 i 74 i 103 c 90i 95i 40i
R F in PVAa
4 4 4 3 3 3
6g
448 45 i 4 365 68 i 4 310 103 i 4 270 76+3 249 81 i 3 225 39 i 4 Reference 8. Reference 12.
bis( 10methylisoalloxazine)copper crystalb
calculations lumiflavin in CHCl,-CCl, Af 6g
7,8-dimethylisoalloxazined
if
6g
507 395
64 89
450 350
58 97
i
4 3
284 87 257 109 221 48 Reference 1 5 .
260
119
i
2
e
Reference 4.
f
lumiflavine
hf
hg
Af
444 357 287 274
81 115 163 127
442 345 298 280 265
In nanometers.
g
In degrees.
6
80 100 118 139
The Journal of Physical Chemistfy, Vol. 87, No. 2, 1983 225
Flavins in Stretched Poly(viny1 alcohol) Films
MLF
RF
-
VI
-VI
Figure 4. Concluded transltlon moment directions of MLF and RF.
from the crystal moments, so the risk of mistakes should be negligible even with a possible uncertainty in the crystal results due to exciton interactions. We shall finally comment on the possibility of n-a* transitions, which can arise from the lone-pair orbitals of the aza nitrogens or of the carbonyl oxygens. Although n-r* transitions have been discussed for a long time-they may play an important role in determining the luminescence properties of the flavin systemseit is still unknown if they contribute to any significant extent to the apparent absorption spectrum. The shoulder at about 480 nm has been suspected, as it disappears in polar solvents; 32 however, low-temperaturestudies4indicate that it is rather due (32) Kotaki, A.; Naoi, M.; Okuda, J.; Yagi, K. J. Biochem. (Tokyo) 1967, 61, 404.
to a resolved vibrational structure of the a-a* transition. Our constant LD' is also supporting this conclusion. The n-a* transitions of aza nitrogens are polarized perpendicular to the molecular plane (LD' = 3S,) and even weak out-of-plane polarized intensities are generally effectively revealed by their strong negative LD contributions." The peak in negative direction at about 300 nm in the LD' curve might in principle arise from an out-of-plane polarized (n-a*) transition; however, curve analyses of LD' and isotropic absorption of MLF and RF showed that it was not possible to account for more than a small fraction of the oscillator strength, above ascribed to transition 111, in terms of an out-of-plane transition. Therefore the conclusion about an extra a-a* transition (111)between 280 and 330 nm still remains and the LD' behavior can be explained without introducing any out-of-plane transition. The concluded polarizations of the six electronic transitions, resolved in the 220-530-nm region, are in Table I11 compared with the results from previous studies. Our polarization directions are in good agreement with the assignments from single crystals; however, they are in qualitative agreement with the MO calculation. This is not directly surprising since transition moment directions, calculated within even the most extensive semiempirical methods, tend to become fairly uncertain for small, unsymmetrical heterocyclics (cf. the DNA basesla).
Conclusion The transition moment directions of MLF and RF, as assigned from the results, are shown in Figure 4. They are in good agreement with the results of single-crystal studies. The orientation of MLF and RF observed in the PVA film demonstrates that a more general treatment starting with a nondiagonal orientation tensor is necessary for the quantitative analysis of the dichroic spectra of low symmetrical planar molecules. Acknowledgment. We are grateful to Professor J. Koziol for the gift of MLF and to Dr. T. Kurucsev (Visiting Professor of Physical Chemistry) for valuable discussions. This project is supported by the Swedish Institute (grant to Y.M.) and the Swedish Natural Science Research Council. Registry No. MLF, 18636-32-3; RF, 83-88-5; PVA, 9002-89-5.
