2 Physical Methods and Techniques Part (iii) Ultraviolet and Visible Spectroscopy of Bio-organic Molecules By P.-S. SONG Department of Chemistry Texas Tech University Lubbock Texas 79409 U.S.A. 1 Introduction Although recent developments in various spectroscopic properties of organic molecules have been reviewed in these Reports there has not been a specific review of the literature on the U.V. and visible spectroscopy of bio-organic mole- cules in recent years. The present Report attempts to review selected literature in this field. In reviewing the literature luminescence spectroscopy of biomolecules will be largely excluded unless it is directly relevant to the discussion of the u .v.-visible spectroscopy of molecules under consideration. Furthermore inter- molecular interactions (e.g.chlorophyll aggregates excitons complexes etc.) and effects of proteins on u.v.-visible spectra of chromophores (e.g. retinal in rhodop- sin) will not be discussed. 2 Cyclic Polyenes Porphyrins.-Porphyrins including metalloporphyrins can be treated as cyclic conjugated polyenes. Chlorophylls (chl) and bacteriochlorophylls (Bchl) are struc- turally and spectroscopically derived from the chlorin and bacteriochlorin chromophores respectively. The absorption spectra of these compounds are characterized by visible bands in the 500-800 nm region and near-u.v. bands (Soret) in the region of 350450nm. The latter are considerably more intense (E =1-3 x lo5,f=1-2) than the former (E = 1-8 x lo4,f = 0.01-0.3) par-ticularly in symmetrical porphyrins.This spectral shape contrasts with the well known spectral feature (i.e.intense visible band with weak or no near-u.v. bands) of linear polyenes such as carotenoids. The genealogy of theoretical models for explaining the visible (and u.v.) spectral characteristics of porphyrins is as follows Free-electron perimeter model of Simpson (1949) and Kuhn (1949) 1 Huckel molecular orbital (MO) model of Longuet-Higgins Rector and Platt (1950) 1 Four-orbital configuration interaction (CI) model of Gouterman (1959) 18 Ph ysica 1 Methods-Part (iii) Spectroscopy of Bio-organ ic Molecules 19 1 SCF MO CI Pariser-Parr-Pople model of Weiss Kobayashi and Gouterman (1965) 1 All-valence-electron MO model of Schaffer and Gouterman (1972) 1 Ab initio SCF CI model of Christoffersen and his co-workers (1977)' In the free-electron perimeter model porphyrin of D4hsymmetry can be viewed as an 18~-electron cyclic polyene with an approximately circular field around the perimeter.The highest occupied (orbital angular momentum quantum number q = f4) and lowest empty orbital (q = f5) levels are doubly degenerate. Between these degenerate levels four excited electron configurations are possible account- ing for T -+ T* transitions to doubly degenerate B,, (from Aq = f1 configura-tions) and QX. (from Aq = *9 configurations) states. These transitions are assigned to the Soret and visible bands respectively. The degenerate Q and B bands split into Q, Q and B, By,respectively as the symmetry of the metalloporphyrin-like system descends from D4h to DZh(e.g.free-base porphyrin) and C2,(e.g.chlorin). The splits between Q and Q and B and By are about 3000cm-' and 250cm-' respectively as predicted from semi- empirical SCF MO CI calculations including electron correlation.2 Previous MO calculations and ab initio CI' tended to overestimate the B,-By split. In addition to the four-state (Q,, and BX.,)absorption spectrum one prominent vibronic satellite appears for each Q 0-0 and Q 0-0 origin thus producing the well known visible bands I(C?, 0-0) II(Qx 1-0) III(Q,,,0-0) and IV(Q, 1-0) for free-base porphyrins. Four similar bands (I-IV) appear in the spectra of chlorins and bacteriochlorins except that Bands I and I11 are designated as Qy(O-0) and Q (0-0) respectively in accordance with the polarization axes.Qualitatively speaking Q,. and B,, bands of porphyrins may be compared with La.b(from Aq = f5 configurations) and Ba,6(from Aq = f1 configurations) bands of naph- thalene and other polyacenes respectively. Although the above model is qualitatively satisfactory in describing the major spectral features of Soret and visible bands in porphyrins the weak- and near-u.v. (Nx,,,L,,, M,,, erc.) bands n -+ v* transitions and effects of metal ions are not at all represented. The assignment of bands I1 and IV as vibronic satellites of the Q,. transitions is by no means Alternatively band IV may be assigned to a third 7r + T* transition (e.g. a band at 485 nm with E = 3.4 X lo4 for meso- tetraphenylporphin and at 495 nm for octaethylporphin in the vapour phase).6 The SCF MO CI theory also predicts such a transition,' but ab initio CI theory (see ' (a)J.D. Petke G. M. Maggiora L. L. Shipman and R. E. Christoffersen J. Mof. Spectroscopy. in press; (b)D. Spangler G. M. Maggiora L. L. Shipman and R. E. Christoffersen J. Amer. Chem. SOC. 1977,99,7470,7478. K. Tomono and K. Nishimoto Bull. Chem. SOC.Japan. 1976,49 1179. R. Plus and M. Lutz Spectroscopy Letters 1974 7,73. J. Bohandy B. F. Kim and C. K. Jen J. Mol. Spectroscopy 1973 43 199. A. H. Corwin A. B. Chivois R. W. Poor D. G. Whitten and E. W. Baker J. Amer. Chem. SOC. 1968 90 6577. J. B. Kim J. J. Leonard and F. R. Longo J. Amer. Chem. SOC., 1974,94 3986. ' A. J. McHugh M.Gouterman and C. Weiss jun. Theor. Chim.Acta 1972 24 346. 20 P.-S.Song below) does not support this. Furthermore this assignment is not likely in view of the low-temperature Shpol'skii spectroscopic analysis of the vibronic origin for this band.' The ab initio CI calculations' predict that one electronic transition (to the doubly degenerate 2lE state) in Mg2'-porphyrin accounts for the major Soret intensity while the higher-lying states and several forbidden 7r -+T* transitions make rela- tively minor contributions to the Soret band at 387 nm. The N band at ca. 327 nm is identified as arising from T -+ T* transition to another doubly degenerzte 4lE state. In free-base porphyrin five 7r -+ T* transitions (to 2'B2, 21B3U, llBlu 31Bzu and are predicted to contribute to the Soret band intensity with a maximum at 385 nm.The N band at 315 nm may consist of two allowed transitions 41B3 and 41B2,. Absorption bands of porphyrins in the vacuum-u.v. region are apparently diffuse.' No evidence for Rydberg states was revealed in the vacuum-u.v. spectra of tetraphenylporphin and its metalloporphyrin derivatives." The vapour-phase absorption bands of porphins from 640-210 nm have been assignedxo Q, Qy,B N L,and M(Q B N and L for Mg2'-porphin). The n -+T* transitions in porphyrins including chlorins can originate from the pyridine-like nitrogen n -orbitals and from formyl or aza substituents at the cyclo- pentanone ring and methine bridge positions. A different definition of n -+ T* assignment for porphyrins and chlorins has also been described." A PPP MO variant taking into account n-bonding orbital electrons predicts an n + T*tran-sition of free-base porphin at 316 nm.I2 Similarly the extended HMO method predicts an n -+ T* transition in the near u.v.' in contrast to the CNDO/S predic-tion at 269 nm for porphins and chlorin~.~~ There is indirect evidence that the Soret band broadening in tetra-azaporphin and phthalocyanine is attributable to the underlying n T* state which is localized at the methine bridge.14 Linear dichroism of free-base and Mn2'-porphyrins shows all transitions in the range 350-900nm to be in-plane polarized," in contrast to an earlier report which showed a negative dichroism at ca.400 nm in tetraphenylporphin. It now appears however that dichroism at 400nm is definitely negativeI6 and that it represents either n -+ T* or a vibronic component of T -+ T* origin.Various studies do not generally support the n +T* assignment for the visible bands I and 111 which has been made by some workers. It is clear that information concerning the location and intensity of n +T*bands in porphyrins (and chlorophylls see below) is either lacking or only tentative. However it is quite likely that 'n T* state(s) lie in the vicinity of the Soret and near U.V.region resulting in band broadening of the T +7r* transitions." Ab initio CI calculation also predicts an n -+ T* (1'B1,) transition under the Soret band.' A. T. Grudyushko K. N. Solov'ev and A. S. Starukhin Optika i Spectroskopiya 1976,40,267.B. H. Schechtman and W. E. Spicer J Mol. Spectroscopy 1970 33.28. lo L. Edwards D. H. Dolphin M. Gouterman and A. E. Adler J. Mol. Spectroscopy 1970,35,90; ibid. 1971 38 16. S. G. Boxer G. L. Closs and J. J. Katz J. Amer. Chem. SOC.,1974,96 7058. l2 M. Sundbom Acta Chem. Scand. 1968,22 1317. l3 G. M. Maggiora and L. J. Weimann Chem. Phys. Letters 1973 22 297. 14 M. Gouterman in 'Excited States of Matter' ed. C. W. Shoppee Texas Tech. University Press Lubbock Texas 1973 p. 63. R. Gale R. D. Peacock and B. Samori Chem. Phys. Letters 1976 37,430. l6 B. Norden and A. Davidson Chem. Phys. Letters 1976 37,433. K. N. Solov'ev A. T. Gradushko and M. P. Tsvirko J. Luminescence 1976,14 365. Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules 21 The visible spectrum of porphin with its characteristic four-band (I-IV) system changes to the typical D4h-porphyrin spectrum of two-band (aand /3 bands) upon metal substitution,I8 as indicated earlier.The effects of different metal substitu- tions on the porphyrin absorption spectrum are partly attributed to perturbations on orbital energy levels of bl (or a2 in symmetry notation) cl(e,) and c2(eg) molecular orbitals which are substantially localized on the central N atoms. Effects of different metal ions on the D4h porphyrin spectrum are generally not drastic with some exceptions such as Mn2'- Fe2'- and Fe3'-porphyrins. Band I (Qx,0-0) usually shifts to the red with increasing atomic number of the central metal ion but spectral shifts of Q tA and B tA bands in both direction and magnitude are determined by ligand (Lkporphyrin interactions in L-Me-porphyrin-type complexes (for example see ref.5). Also the Bx-By band split by fifth and sixth ligands (e.g. cytochome c) could be larger than the Qx-Qysplit in contrast to the case of free-base porphyrin. In general high-spin metalloporphyrins show stronger charge-transfer (CT) bands than low-spin complexes as has been found for Fe2'-tetraphenylpor- phyrin." CT bands arising from the occupied porphyrin MO (azuin particular) to the empty metal d-orbitals (e,) may occur throughout the near-u.v.-near-i.r. region depending on the strength of ligand field and the nature of ligands. For example porphyrin + Fe3' CT bands have been assigned for Fe3'-myoglobin using polarized absorption spectroscopy and the weak band at 695 nm in Fe3+-cyto- chrome c (low spin) was found to be out-of-plane polarized consistent with the ligand (azu)-+metal (d,z; al,) CT transition,*' whereas ligand (a2,or ulu)-+ metal (d ; e,) CT transitions are predicted to occur in the near-u.v.region (320400 nm) in Cr3+-tetraphenylporphyrinin chloroform.21 The d -+ d transitions are generally so weak that their resolution is usually masked by the strong porphyrin bands. Metalloporphyrins of d3,d4,d6 and ds configurations show d -+ d transitions of low frequency in the near-i.r. or i.r. region (e.g. Cr3"-tetraphenylporphyrin21),as can be predicted from porphyrin being a weak-field ligand. The d + d transitions for various metalloporphyrin camplexes have been assigned but the assignment of these transitions are often not unam- biguous owing to CT and porphyrin bands which are more intense.It is well known that the visible band Q +A of porphyrins with D4, symmetry can be described as a planar oscillator as a consequence of the two-fold degeneracy for Qx,ystates. Thus the degree of fluorescence polarization with respect to the visible absorption in porphyrins is independent of excitation wavelengths within the visible band and is in the neighbourhood of f a value characteristic of a planar oscillator.22 The fluorescence polarization degree increases substantially as the porphyrin symmetry is lowered from D4,,to D2,,or Cz0 as in free-base porphins and chlorins respectively.The SztSo transition (Q in free-base porphyrins and 0 in hydrogenated porphyrins) is then polarized perpendicular to the S1tSo absorption or fluorescence polarization axis yielding a negative degree of M. Gouterrnan L. K. McCaffery and M. D. Rowe J.C.S. Dalron 1972 596. l9 H. Kobayashi and Y. Yanagawa Bull. Chem. SOC.,Japan 1972,45,450. 2o W. A. Eaton and R. M. Hochstrasser J. Chem. Phys. 1967,46 2533; ibid. 1968,49,985. M. Gouterman L. K. Hanson G. E. Khalil and W. R. Leenstra J. Chem. Phys. 1975,62 2343. 22 G. D. Yegorova V. A. Mashenkov K. N. Solov'ev and N. A. Yushkevich Biofizika 1973,18,40. 22 P.-S. Song fluorescence polarization. However fluorescence polarization does not yield absolute orientations of these transition moment dipoles.The lowest transition 0 tA in free-base porphins is theoretically predicted to be polarized along the NH-NH axis (x) and the Q +A transition is perpen- dicular to the former. This is consistent with the effects of substitution along the x-and y-axes on the hyperchromicity of the Q (band I) and 0,(band 111) bands respectively and with the polarized reflectance spectroscopy of free-base tetra- phenylporphin single and the polarized quasi-line absorption spectrum of Zn2’-porphin in triphenylene.24”5 Vibronic coupling between electronic states (e.g. Q state with higher electronic states) can be invoked to explain mixed polarization characteristics of the porphyrin absorption bands. According to the vibronic theory the Q 1-0 band gains its intensity from the y-component of the Soret transitions through bl vibration-induced mixing whereas the al* vibiation mixes the x-component of the Soret band with the Q 1-0 band.26 High-resolution vibrational analysis of the absorp- tion spe~tra,~’-~’ site-selection fluorescence spectroscopy,28 and resonance Raman ~pectroscopy~~ provide powerful tools to carry out vibronic analysis of the visible and Soret bands of metalloporphyrins.Fluorescence polarization degree varies over the Soret band of porphins (e.g. octaethylporphin) suggesting that there are two more or less perpendicularly polarized transitions to B and Bystates,” but other electronic transitions which are not readily resolved by the fluorescence polarization technique are also likely to contribute to the Soret band as mentioned earlier.Ch1orophylls.-Chls and Bchls show absorption spectra similar to those of chlorin and bacteriochlorin respectively. Chl-a shows the characteristic four visible bands system [band I at 660 nm= 0 (0-0); I1 at 615 nm = Q (1-0); 111 at 575 nm= Q (0-0); IV at ca. 530 nm = Q (1-0)] and the Soret band (429- and 410 nm) with absorbance peak ratios of 1.0:0.16 0.1 :0.06 1.31:0.87 in ether. The oscillator strength f,of the Q (0-0) averaged from absorption spectra in different solvents is 0.151.30 Attempts have been made to assign the u..v. bands of chls in the 200410 nm regi~n,~’,~~ but they remain to be established. The Q +A band of Bchl-a occurs at 769 nm and the Soret bands at 392 and 357 nm with absorbance peak ratios of 1:0.55 :0.8 in ether.Protochl-a chl-a and Bchl-a assume approximately circular triangular and rectangular v-electron fields respectively. Thus the Q tA band shows a pro- gressive red shift whereas the Q +A band shows a relatively small blue shift. The Qy-Q split is also expected to increase in going from protochl-a to Bchl-a. 23 B. G. Anex and R. S. Umans J. Arner. Chem. SOC.,1964,86 5026. 24 B. F. Kim J. Bohandy and C. K. Jen Specrrochirn. Acta 1974,30A 2031. 2s B. F. Kim and J. Bohandy J. Mol. Spectroscopy 1977,65,90. 26 Y.J. Aronowitz and M. Gouterman J. Mol. Spectroscopy 1977,64 267. 27 J. A. Shelnutt D. C. O’Shea N. Y. Yu. L. D. Cheng and R. H. Felton J. Chern. Phys. 1977,66,3387. 28 J. Funfschilling and D. F. Williams Photochem. and Photobiol.1975 22 151; ibid. 1977 26 109. 29 T. G.Spiro Accounts Chem. Res. 1974,7 339. 30 L. L. Shipman Photochem. and Photobiol. 1977 26 287. 31 C. Weiss jun. J. Mol. Spectroscopy 1972. 44 37. 32 P.-S. Song T. A. Moore and M. Sun in ‘The Chemistry of Plant Pigments’ ed. C. 0.Chichester Academic Press New York,1972 pp. 33-74. Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules 23 Configuration analysis of wavefunctions for chl-a and Bchl-a suggests that the Q states of these molecules are very similar as the peripheral C=C (3b-4b) bond in chl-a is not locally excited to any appreciable extent. The isocyclic ring C=O group is also unimportant in substantially perturbing the Q +A transition of chlorin and bacteriochlorin macrocycles.Furthermore the vinyl substituent does not significantly contribute to the Q state functions of chls. These analyses are therefore consistent with the use of the four-otbital model of porphyrins for chls in that the low-energy electronic states Q,, and BX7,can be described by two degenerate MOs of the macrocylic ring.33 It is possible that a shallow hump at 325 nm in the absorption spectrum of chl-a is an n + T* band. Since cyclopentanone shows the n + n* band in this general region this possibility is energetically feasible. However the intensity at 325 nm is too high to be attributed. to an n + T* band of chi-a although perturbations generated by nearby states can enhance its intensity considerably. It should also be noted that in deuterioporphyrins N eA (n+ r*)transitions with E S lo4occur in the same region (320-350 nm).15N N.m.r. studies” suggest that the lowest n + T* transition is localized in ring IV of chl-a and pheophytin-a but the suggestion that the n + n* transition might occur near the Q band with relatively strong intensity has not been directly confirmed. The absorption band maximum (I,Q, 0-0) of chl-a is polarized (Po> 0.4) parallel to the fluorescence emission oscillator and its polarization approximately corresponds to the y-axis according to the PPP MO calc~lation.~~ Band I1 (Q, 1-0) at 615 nm is made up of two components one at ca. 632 nm with Po-0 and another at 613 nm with Po-0.28. These bands may be assigned to Q (1-0) and Q (2-0) respectively. If this assignment is correct the 575 nm band with Po -0.03 is most likely assigned to Q (0-0) (band 111).M.c.d. results are consistent with the assignment here.35 Pheophytin-a chlorin and porphins at 77 K also show negative polarization for this band. The Q tA transition is thus polarized (approximately along the x-axis) nearly perpendicular to the 0 tA tran~ition.~~ However Shipman et assign the 635 nm component (or ca. 632 nm34) of chl-a in ethanol at 77 K to the Q,(O-0) on the basis of spectral analogies with Bchl-a and an apparent shift of the room-temperature absorption band 111(Q, 0-0) to ca. 635 nm at 77 K. Although this is possibly due to the effect of an anisotropic polarizability of the medium with increased refractive index at 77 K preferentially red-shifting the Q (0-0) band the Q (1-0) assignment for this band with a negative polariza- tion degree is more likely on the basis of the following observation.High-resolution fluorescence excitation and polarization spectra of chl-a in ether at 15 K revealed a well-resolved peak at 632 nm.37 If this peak were Q,(O-0) one would not see its mirror image in the fluorescence spectrum. However the 723 cm-’ vibrational mode of the 632 nm excitation band shows its mirror image (729 cm-’) in the fluorescence emission spectrum at 15 K. 33 P.-S.Song C. A. Chin I. Yamazaki and H. Baba Znternat. J. Quantum Chem. Quantum Biol. Symp. 1977,4 305; ibid. 1976,3,89. 34 P. Koka and P.-S. Song Biochim. Biophys. Acta 1977 495 220. ” C. Houssier and K. Sauer J. Amer. Chem. SOC.,1970,92 779.36 L. L. Shipman T. M. Cotton J. R. Norris and J. J. Katz J. Amer. Chem. SOC. 1976,98 8222. ” W. W. Mantulin and P.-S.Song unpublished results. 24 P.-S.Song The minimum polarization at 435 nm in chl-a is attributable to the B +A transition in agreement with theoretical predictions and m.c.d. Absorp-tion and polarization characteristics of protochl-a chl-6 and pheophytins are qualitively similar to those of chl-a. In particular chl-b shows a fluorescence excitation polarization spectrum closely resembling that of chl-a in ether at 77 K except that the Q (0-0) band shows a lower Po value (0.35) than that of ~h1-a.~~ The large red shift of Q relative to that of chl-a is accompanied by increase in the Q,-Q gap which contributes to improved resolution of fluorescence polarization in Bchl-a.Thus Po for Q (0-0) of Bchl-a is 0.4 in ethanol at 77 K whereas Po for Q (0-0) at 625 nm is 0.28.39 The above discussion is based on the simplifying assumption that each of the resolved vibronic bands Q (0-0) and (1-0) Q (0-0) and (1-0) contains no strong contribution due to other overlapping vibronic transitions. However low-temperature site-selection spectroscopy of chl-a and -b in which the fluorescence spectra are resolved into a number of vibronic components28 clearly indicates complexity in interpreting the polarization spectra of chlorophyls and porphyrins. Corrins and Tetrapyrro1es.-Corrins show two visible bands designated as cy -and p-bands along with the intense near-u.v. y-band (==Soret) and moderately intense U.V.bands. The a-and @-bands are the 0-0 and 1-0 components of the first allowed electronic transition. Kuhn (1959) was the first to describe the corrin spectrum in terms of the free electron model. The four-orbital model equivalent to the Gouterman model for porphyrins was proposed by Day4’ within the framework of the HMO and PPP MO approximations. The latter was also applied to corrins by other^.^',^^ The HMO method was found to be inadequate for describing the spectral intensities of corrins. On the basis of these results it is now well established that the visible and near-u.v. band intensities in corrins are mainly of the T +T*type. This conclusion is generally valid for corrinoids such as vitamin BI2 since ‘free-base’ corrins (e.g.descobalt-B12) show the characteristic visible and near-u.v. spectral intensities. The T +T* assignment for the entire u.v.-visible spectrum of free-base corrins is clearly confirmed by the polarized phos-phorescence excitation spectrum of descobalt-B12 which shows Po< 0.1 for all visible and U.V. peaks with respect to the out-of-plane polarized phosphorescence of 372 T* The low-temperature absorption spectrum of descobalt-BI2 in ethanol shows well resolved peaks at 525 493 465(sh) 435(sh) 395 375 358(sh) 326 314 300(sh) 285 280(sh) 268 260(sh) and 238nm with peak ratios of 1 0.76 0.36 0.12 0.16 0.17 0.12 2.09 1.04 0.64 0.48 0.54 1.39 0.76 0.43. Although calculated oscillator strengths are invariably greater than experimental f values the f ratios produced by the PPP SCF MO CI calculation are 1(Q,):0.07(Qx):6.8 (near U.V.&) which agrees with the obser ed ratios of 1:0.07:6.5 in descobalt B12. In analogy to the porphyrin notation we lave assigned 38 C. A. Chin Ph.D. Thesis Texas Tech University Lubbock Texas 1975. 39 T. G. Ebrey and R. K. Clayton Photochem. and Photobiol. 1969,10 109. ‘O P. Day Theor. Chim. Acta 1967,7 328. 41 H. Johansen and L.L. Ingraham J. Theor. Biol. 1969.23 191. 42 P. 0.Offenhartz B. H. Offenhartz and M. M. Fung J. Amer. Chem. SOC.,1970,92 2966. 43 R. D. Fugate C. A. Chin and P.-S.Song Biochim. Biophys. Actu 1976,421 1. Physical Methods-Part (iii)Spectroscopyof Bio-organicMolecules 25 the 525 nm peak and its vibrational satellites at 493,465 and 435 nm to Q 0-0,l-0 2-0 and 3-0 respectively while the 395,375 and 358 nm peaks are designated as Q 0-0 1-0 and 2-0 respectively.The subscripts x and y correspond to the polarization axes.43 The 362 nm maximum may then be designated as a R equivalent. These designations are consistent with the relative polarization data discussed below. The lowest-energy transition Q, arises mainly from the 7(HOMO) -+ 8(LEMO) configuration while Q and B transitions are contributed to mainly by 6 -D 8 and 7 +9 configuration The fourth (S4tSo)and fifth (SseSo)transitions are mainly due to 7 +10 and 6 +9 configurations respectively. The latter is polarized nearly parallel to the Q,-axis in descobalt BI2,whereas the S4transition is polarized between the x-and y-axes.43 The predicted polarization axes for Q and Q bands are the y-and x-axis respectively.The fluorescence polarization spec- trum of desc0ba1t-B~~ shows that Q and B transitions are polarized perpendicular to the Q reminiscent of the chl-a Although the linear dichroism of vitamin BI2in stretched poly(viny1 alcohol) film is too small to deduce the absolute polarization axes of the visible and near-u.v. bands,4s the negative dichroism at the near-u.v. band is indicative of the perpendicular polarization of the B transition with respect to the Q polarization direction. Both ~.d.~~*~~~~~ and m.~.d.~' spectra also suggest this polarization picture. The absolute polarizations of the absorption spectrum of a single-crystal Ni2'-corrin (nirrin) indicate that the visible band is polarized along the y-axis and the near-u.v.band is oppositely polari~ed.~' Thus the polarized reflection spectroscopy result is consistent with the PPP prediction^.^^ Biliverdin an open tetrapyrrole bile pigment derived from haems shows an absorption spectrum similar to those of porphyrins with the visible band maximum at 670 nm (E -1.1x lo4) and a near U.V. band at 376 nm (E -3.8 x lo4) in water (pH 11.8) at room temperat~re.~~ The absorption bands are not resolved. The low-temperature absorption spectrum of biliverdin in ethanol at 77 K resolves shoulders at 660 nm (f = 0.14) and 435 nm (f-0.09) along with maxima at 707 nm (f = 0.19) and 380 nm (f = 1.06)?' These spectral characteristics are reminiscent of the spectra of porphyrins.For descriptive purposes we will use the porphyrin notations Qx., for the visible and near-u.v. ('Soret') bands of bili~erdin.~' Thus the long-wavelength band at 707 nm is designated as Q which is predominantly due to the 16(HOMO)+ 17 (LEMO) configuration. The second band at 660 nm resolved in the low-temperature ethanol glass is largely contributed by the 14 -+ 17 configuration while the strong near-u.v. transition arises from the 16+18 configuration is predicted to occur between the Q and near-u.v. regions. It is generally agreed that the conformation of biliverdin is neither fully linear nor fully c~clic.~~*~' The most likely conformation is probably 'semi-circular' since 44 A. J. Thompson J. Amer. Chem. SOC.,1969 91 2780. 45 R. Eckert and H.Kuhn 2.Elektrochem. 1960,64 356. ''R. Bonnett D. M.Godfrey V. B. Math P. M. Scopes and R. N. Thomas J.C.S. Perkin I 1973 252 47 B. Briat and C. Djerassi Nature 1968 217 918. 48 B. G. Anex and G. J. Eckhardt Abstracts Symposium on Molecular Structure and Spectroscopy Columbus Ohio 1966. 49 G. Blauer and G. Wagniere J. Amer. Chem. SOC. 1975 97 1949; ibid. 1976,98 7806. Q. Chae and P.-S. Song J. Amer. Chem. SOC.,1975,97,4176. P.-S.Song the oscillator strength ratio fvisible/fnear u.v. is inconsistent with either extreme conformation. It should be noted that this ratio is a sensitive measure of the chromophore conformation with the maximum ratio that for linear polyenes and the minimum ratio that for cyclic polyenes (see above). In addition the fluorescence excitation polarization spectrum of biliverdin yields a value of ca.SO"for the angle between the polarization axes of the visible and near-u.v. bands consistent with a semi-circular c~nformation.~' The spectroscopic assignments and conformational analysis of other phycobilin chromophores are now in progress using the methods described for biliverdin (J. Jung and P.-S. Song to be published). The absolute polarization directions of the visible and near-u.v. absorption bands are not known. However approximate polarization directions for these bands have been deduced from the linear dichroic data which show negative and positive dichroism for the 675 and 370nm bands respectively of biliverdin in stretched PVA film." By assigning the PVA stretching axis to the long axis of the semi- circular conformation of biliverdin the polarization of the visible band at 675 nm has been determined to be along the A-C ring axis while the 380nm band is polarized perpendicular to the former.It should be noted that a number of simplifying assumptions are involved in these deductions although the relative polarizations of these two bands are consistent with the polarized fluorescence excitation spectrum mentioned earlier. Further refinements in the data analysis of linear dichroic spectra and a polarized single-crystal spectroscopic study are needed for definitive assignments of the polarization axes. 3 Linear Polyenes Simple Po1yenes.-In contrast to cyclic polyenes linear polyenes show strong absorption at the first absorption band with considerably weaker absorption at shorter wavelengths.Until recently the electronic spectra of simple linear poly- enes of conjugated C=C bonds (N =2-6 where N is the number of conjugated C=C bonds) have been 'satisfactorily' described in terms of free-electron and simple MO theories. For a polyene within the 'four-orbital' framework (e.g.butadiene) the electronic transitions arise from electron configurations corresponding to the change in orbital quantum number An = 1 for N(HOM0) +(N+l)(LEMO) and An = 2 for (N-1)+(N+ 1) and N+ (N+2) configurations. The degeneracy of the latter excitations (c.f.Coulson-Rushbrooke theorem) is lifted via configuration inter- actions between them. It is well known that the first transition 'B t'A (or 'Bt'A in Platt notation) is strongly electric-dipole allowed with its polarization direction along the long molecular axis.Both oscillator strength [f -0.74 for all-trans-butadiene -3.4 for all-trans-@-carotene] and A max increase with con- jugation. The latter converges to an asymptotic value of 610 nm for 'infinitely' long polyenes as a result of C-C and C=C bond-order alternations which restrict the electron motion in a one-dimensional periodic potential and/or v-electron cor- relation effects." The 'A tA and 'A +A transitions are symmetry-forbidden but the former can be readily observable even in trans-polyenes and it becomes strongly A. Ovchinnikov I. I. Ukrainskii and G. V. Kventsel Sou. Phys. Uspekhi,1973 15 575. Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules 27 allowed in cis-polyenes ('cis peak') due to the loss of the centre of symmetry.The doubly forbidden (g +g,-+-) 'A +-A transition is mainly the result of a doubly excited configuration. In contrast to the 'B,state which is ionic in charac- ter the 'A excited state is covalent with a high degree of double-bond character for the C-C single bond relative to the 'B or A ground state. The long held traditional assignment of polyene spectra described above has been questioned on both experimental and theoretical grounds in recent years.52 The new assignment puts the 'A; excited state below the strongly allowed 'B state The lowering of the 'A state arises from configuration interactions among singly and multiply excited ~onfigurations.~~*~~ The quantum mechanical prediction for the location of 'A is by no means definitive at the present state of compu-tation since both ab initio and semi-empirical MO-CI (singly plus doubly excited configurations included) used involve several approximations that may affect the accuracy of the predicted location of the 'A; state; some of these approximations include neglect of the 0-core and its redistribution restriction of basis set etc.It is also possible that the order of the 'A state reverses upon inclusion of triplyexcited configurations in the CI calculations. Nonetheless the new order for the 'A state is strongly supported on both theoretical and experimental grounds as mentioned below. The gas-phase study of cis and trans-hexa-l,3,5-triene showed no detectable absorption on the red edge of the main band and that results of PPP-type cal- culations including only singly excited configurations matched more closely the experimental U.V.spectra than the singly +doubly excited configuration interaction calculations (see Gavin and Rice in refs. 52 and 55). Karplus et al? subsequently argued that the failure to detect the 'A absorption band in the gas-phase spectrum of the hexatriene is not inconsistent with the original PPP (single+double CI) prediction for the low-lying 'A state since the transition to 'A occurs too close to the allowed 'B band and it is symmetry-f~rbidden.~~ Apparently such a 'masking' is not a problem for diphenylpolyenes at low temperature where the weak absorption has been seen.52 The low-lying 'A state is consistent with the valence-bond picture of polyenes in contrast to the free-electron and simple LCAO MO methods.The valence-bond picture indicates that the 'A,-state is lowered in energy as the conjugation of polyenes is extended and that the 'A +-A transition may occur below the 'B tA tran~ition.~~ The assignment of 'A to the lowest excited IT T* state has a profound impli- cation in re-interpreting spectroscopic and photochemical properties of linear polyenes. The former includes several anomalies (e.g. almost no overlap between absorption and emission bands) which had been largely explained on the basis of Franck-Condon arguments (e.g. distortion of the polyene molecule by a low- frequency mode).In addition to the near 'non-overlap' of the 'B,+-A absorp-tion and the fluorescence bands the spectroscopic anomalies of polyenes include 52 (a) B. Hudson and B. Kohler Ann. Rev. Phys. Chem. 1974 25 437; see original references therein (1972-1974); (6)R. L. Christensen and B. E. Kohler J. Phys. Chem. 1976,80,2197. '' R. J. Buenker and J. L. Whitten J. Chem. Phys. 1968,49 5381. 54 K. Schulten and M. Karplus Chem. Phys. Letters 1972 14 305. " M. Karplus R. M. Gavin jun. and S. A. Rice J. Chem. Phys.. 1975.63 5507. 28 P.-S.Song (a) anomalously long T~ and (b) apparent differential solvent shifts of the main 'B absorption and fluorescence bands. Thus the former shifts to the red as the solvent polarizability (n -l)/(n +2) increases but the fluorescence maximum of diphenyloctatetraene shifts to the red only ~lightly.'~ As a result the Stokes shift ranges from 2000 to 6000 cm-'.In general polyenes show a large Stokes shift of 3000-7000~m-'.~~ Along with such a large Stokes shift there is an apparent separation between the 'B t-A 0-0 and the first observed line of the fluores- cence. This separation is understandable in mixed-crystal and Shpol'skii quasi-line spectra of diphenyloctatetraene at 1.84.2 K.52 A series of new quasi-lines can be observed between the red edge of the absorption band and the blue edge of fluorescence under high-resolution conditions. Such weak quasi-lines are not attributable to the 'B tA tran~ition.'~ These new absorption lines are rich in low-frequency vibrations (<500 cm-').The weak sharp absorption shows an apparent 0-0 frequency at 452.24 nm for diphenyloctatetraene in a biphenyl host coincident with the first emission line of the fluorescence spectrum. The weak absorption and fluorescence show an approximate mirror image. In contrast to the strong 'B eA absorption band the weak band cf =0.05f0.02) is relatively insensitive to the solvent matrix. On the basis of this and other less direct evidence the weak absorption has been assigned to the 'A tA transition consistent with the single +double CI PPP calculations. It is concluded that this new assignment is generally valid for all polyenes particularly for diphenylpolyenes of 3-6 conjugated C=C bonds decapentaene carboxylic acid 2,10-dimethylundecapentaene parinaric acid," and retinyl polyenes (see Furthermore it is predicted that the 'A state is lowered in energy with increasing conjugation in contrast to the allowed 'B state which converges to the asymptotic level.The 'A tA absorption and its fluorescence spectrum of 2 lo-dimethylun- decapentaene in n-nonane matrices at 4.2 K have been recorded at high resolution revealing several predominant low frequency non-totally symmetric vibrations (see above). These vibrations apparently induce the forbidden 'A tA transition in agreement with the Herzberg-Teller vibronic theory which predicts the mixing of the closely lying 'A and 'B The fluorescence polarization at the weak edge of the absorption in diphenyl- octatetraene remains high (>0.4) indicating that the transition moment is parallel to the emission and 'B transition moment^.'^ Earlier this was used as an argument against the 'A assignment^.'^ However the polarization data can be accommodated in terms of the Herzberg-Teller-type vibronic mixing of the two states 'A and 'B,.The vibronic mixing is expected to decrease with the extent of conjugation as the gap between the two states increases resulting in a quadratic decrease in the oscillator strength of the former transiti~n.~~'~' In an attempt to obtain an independent evidence for the 'A state assignment in polyene electron scattering (ES) spectroscopy has been applied to hexatriene. A shoulder at 282nm in the ES spectrum increases its intensity as the momentum transfer is raised.This is taken as evidence for the 'A assignment of the lowest 56 M. Kovner Acta Physicochim. U.R.S.S. 1944 19 385. " L. A. Sklar B. S. Hudson M. Peterson and J Diamond Biochemistry 1977,16 813. '* R. L. Christensen and B. E. Kohler J. Chem. Phys. 1975,63 1837. 59 T. A. Moore and P.-S. Song Chem. Phys. Letters 1973 19 128. 6o J. B. Birks and D. J. S. Birch Chem. Phys. Letters 1975 31 608. Physical Methods -Part (iii) Spectroscopy ofBio- organ ic Molecules 29 singlet excited state in polyenes.61 Several attempts to resolve the 'A state of polyenes have also been made without conclusive evidence for or against the assignment by using two-photon absorption for which the 'A tA transition is allowed. In 1,4-diphenylbutadiene it is possible that the two-photon absorption occurs at ca.500cm-' below the 'B state at 347.2nm.62 The 'A state in diphenylhexatriene and diphenyloctatetraene has also been located at 386 and 442.5 nm respectively.62 However two-photon absorption with the thermal blooming technique did not reveal the 'A state for hexa-1 3,5-triene and more recent studies of electron-impact and multiphoton ionization spectra of hexatriene did not provide conclusive evidence for the low-lying 'A state in the 270-300 nm region.63 Nonetheless the failure to observe such a low-lying state in hexatriene does not necessarily entail rejection of the new state ordering in polyenes since the two-photon absorption cross-section could be too small to be measured and/or masked by nearby intense two-photon absorption bands (e.g.Rydberg band). Another line of evidence for the low-lying 'A state has been explored using c.d. and m.c.d. A weak absorption at the red edge of the main absorption band of cycloheptatriene shows an m.c.d. sign opposite to that of the latter. This weak band has been assigned to the 'Ai-type band.52 However m.c.d. has not yielded unambiguous conclusions in several polyene systems including those complexed at inducible optical active sites on proteins. The induced c.d. spectra of a-and P-parinaric acid octadeca-9,11,13,15-tetraenoicacidtbovine serum albumin complexes showed a negative c.d. band at the long-wavelength edge and a positive c.d. band at hma,.64In addition no mirror image between the main absorption and fluorescence spectra was observed.These results are consistent with the 'A assignment for the weak absorption. However interpretation of the induced c.d. spectra is complicated by the exciton contribution arising from inter-chromophore interaction^.^^ It appears that the 'A assignment as the lowest 7r +7r* transition in polyenes is an attractive proposal which is able to accommodate characteristic anomalies (see above) of polyene spectra. There still seem to be some spectroscopic features not readily resolved at present however. These include the apparent absence of the 'A; band intensity in unsubstituted hexatriene in the gas phase uncertainties as to accurate values of the 'A +A oscillator strength and the location of the 'A state in longer polyenes with six or more conjugated C=C bonds.Apparently the end aryl group substitution can affect the relative ordering of 'A and 'B It is concluded that 'A lies below '8,for all orientations of the phenyl group in diphenylhexatriene and diphenyloctatetraene.60 Accepting the 'A; nature for the weak red edge absorption of polyenes (e.g. diphenyloctatetraene) and assuming the mirror image between the 'A absorption and fluroescence which overlap just slightly one would then expect the intensity ratio of the absorption at A,, to that 61 D. E. Post W. M. Hetherington and B. Hudson J. Chem. Phys. 1976,64 4020. (a)R. L. Swofford and W. M. McClain J. Chern. Phys. 1973 59,5740; (b)G. R. Holtom and W. M. McClain Chem. Phys. Letters 1976 44 436. (a)A. J. Towarowski and D.S. Kliger Chem. Phys. Lerfers 1977 50 36; (6) W. M. Flicker 0.A. Mosher and A. Kupperman ibid. 1977,45,492;(c)D. H. Parker S. J. Sheng and M. A. El-Sayed J. Chem. Phys. 1976,65 5534. (a)L. A. Sklar B. S. Hudson and R. D. Simoni Proc Nut. Acad. Sci. U.S.A.,1975,72,1649;(b)L. A. Sklar B. S. Hudson and R. D. Simoni. Biochemistry 1977 16 5100. 30 P.-S.Song of 'A 0-0 to be roughly comparable to that of the fluorescence at AF,max and 'A 0-0. The long-wavelength absorption and fluorescence of -apo-8'-carotenal and astacene in the solid state have been assigned to the 'A However these are likely to be due to impurities3* or reaction products. Retinyl Po1yenes.-Retinyl polyenes such as retinol and retinal show broad struc- tureless absorption spectra at room temperature and at 77 K.The main absorption band 'B +A, A,, == 330-335 nm,f= 1.36 of all-trans-retinol is due to the 'B,-type (or 'B in Platt notation) transition. (Hereafter we will retain the CZhor free-electron notation for genealogical correlation with the previous section.) The cis band 'A; tA (or 'C +A) occurs at 240-250 nm. and its intensity is substantially enhanced in cis-isomers (f =0.1-0.2). The 'B transition is most likely polarized along the long molecular axis while the former is polarized nearly perpendicular to the latter (-55" from the fluorescence and 65" from PPP-CI calculation). Because of the lack of symmetry and distortion of the planarity at the C-6-C-7 bond extensive configuration mixing of various excited states is expected.Except for the above two states 'B and two 'A states of retinol that may lie in the 210-290 nm region have not been resolved. One of the 'A states has recently been assigned as the lowest T,T* state (352 nm; i.e. 1600 cm-' lower than 'I?,) in all-trans-retinol on the basis of two-photon absorp- tion.66 This assignment is consistent with the earlier assignment and with spec- troscopic anomalies of retinol which are shared by other polyenes (see aboue) including 2,1O-dimethylundecapentaene,axerophtene anhydro-vitamin A and all-tr~ns-retinal.~~~~~ The 'A level in all-trans-retinol is ca. 1600 cm-' below the 'B state as mentioned above. This gap is less than in 2,lO-dimethylundecapen- taene (3250 cm-' for the latter).67 The absorption bands of retinyl polyenes containing p -ionone are usually diffuse and structureless even at low temperature.The diffuseness is expected from vibronic interactions between close-lying states but the torsional potential of the C-6-C-7 bond in the ground and excited states appears to play the most important Thus it is noted that anhydrovitamin A at 77 K67 and all-trans-retinol bound specifically to P-lactoglobulin (unpublished results) show structured absorp- tion spectra possibly as the result of a fixed torsional angle about the C-6-C-7 bond. Retinals exhibit absorption spectra similar to that of retinol with A,, at ca. 360-380 nm (f= 1) which is due to the 'B tA genealogy. The 'B transition is nearly maximally polarized with respect to the fluorescence oscillator under pho- toselection conditions at 77 K.69 This transition is most likely polarized along the (a) K.Mandal and T. N. Misra Bull. Chem. Soc. Japan 1976,49 198,975; (6)B. Mallik K. M. Jain K. Mandal and T. N. Misra Indian J. Pure Appl. Phys. 1975 13 699. 66 (a)R. R. Birge J. A. Bennett B. M. Pierce and T. M. Thomas J. Amer. Chem. Soc. 1978 in press; (6) R. R. Birge K. Schulten and M. Karplus Chem. Phys. Letters 1975,31,451. 67 (a)R. L. Christensen and B. E. Kohler Photochem. and Phoroobiof.,1973 18 293; (6) ibid. 1974 19 401. B. Honig A. Warshel and M. Karplus Accourps Chem. Res. 1975 8 92 and references therein. 69 (a)P.-S. Song Q. Chae M. Fujita and H. Baba J. Amer. Chem. Soc. 1976,98,819;(b)T. A. Moore Ph.D. Thesis Texas Tech.University Lubbock Texas 1975;(c) T. A. Moore and P. S. Song Nature (NewBiol.) 1973 242 30. Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules long molecular axis and shorter-wavelength transitions are expected to be polarized at some angle to the former on the basis of the declining degrees of fluorescence polarization at A <320 nm. The PPP calculation predicts an angle of 52" between 'B and 'A; (cis peak) polarization axes.69 The lowest excited state is suggested to be of 'A; type in all-trans-retinal and 1l-cis-l2-s-tran~-retinal.~~~~~ Although this suggestion is consistent with the spec- tral anomalies (long & large Stokes shift etc.) shared by other polyenes and retinol its frequency and intensity are not accurately known at the present partly owing to the lack of highly resolved absorption and fluorescence spectra.making it difficult Furthermore the fluorescence of retinal is A,,-dependent,67*69*70 to utilize fluorescence as a probe for the 'A state. Retinal (e.g. all-trans-isomer) does not fluoresce in dry non-polar solvents but the H-bonded fraction of retinal in H-bonding solvents is apparently responsible for the fluorescence. This accounts for the A,,-dependence of fluorescence in the ~olvent.~' The lowest excited singlet state of all-trans-retinal in dry hydrocarbon solvents7' and of 1l-~is-retinal~~ is then assigned to the 'n77r* state. The *A,tA transition in retinal is no longer symmetry-forbidden. For example the PPP calculation of f values ranges from 0.16 for the planar 11-cis-s- trans-retinal ('A; =S1) to 0.22 for the planar 11-cis-s-cis-retinal ('A; = S2).The calculated f values for other retinal isomers range from 0.176 for 13-cis- and 0.288 for 1l-cis-12-s-(150")-trans-retinal.The half-bandwidth of the main absorption band of the trans- and cis-isomers remains approximately the Thus the predicted strong absorption due to the 'A transition is not readily apparent in the low-temperature absorption spectra. Without identifying the symmetry ('A; us. '&) we have argued for the lowest singlet state to be a non-discrete T,T*state admixed with a nearby n,T* state on the basis of the observation that retinal carbonyl-cation binding enhances the fluorescence quantum yield in a manner analogous to the H-bonding effect noted above.69 However '7ry7r*-n,7r* perturbation is apparently not responsible for the band broadness since the torsional potential about the C-6-C-7 bond plays an important role in the spectral diffuseness in retinals.68 Cation binding to the all-trans-retinal carbonyl results in less than a 3 nm red shift.69 However the H-bonded retinal-phenol complex shows a substantial spec- tral shift (A,, =385 nm in 3-methylpentane and 420 nm in 3-methylpentane- 1mM-phenol; A F.max =520 and 600 nm re~pectively).~~ Furthermore a significant overlap between the main absorption and fluorescence uncharacteristic of other polyenes is observed.Thus it is possible that the strong H-bonding has either lowered the '8,state below the 'A and 'n,7r* states or introduced a charge- transfer character into the lowest singlet state.The 77 K absorption spectra of retinals show structured band(s) over the 260-320 nm region which have been assigned to an n +7r* transition.'* The intensity of this band is enhanced for cis-isomers. However other assignments (second B, A, a;T*,etc.) for this region cannot be ruled out. The cis peak (of 'A 'O R. S. Becker K. Inuzuka J. King and D. E. Balke J. Amer. Chem. Sm.,1971,93 43. 71 (a)T. Takemura P. K. Das G. Hug and R. S. Becker J. Amer. Chem. Suc. 98 7099; (6) R. S. Becker G. Hug P. K. Das A. M. Schaffer T. Takemura N. Yamamoto and W. Waddell J. Phys. Chem. 1976,80,2265. 72 R. R. Birge M. J. Sullivan and B. E. Kohler J. Amer. Chem. Suc. 1976.98 358.32 P.-S.Song genealogy) is located at ca. 250 nm and is strongly enhanced in 11-cis-retinal at 77 K.72 The PPP double-CI calculation of 11-cis-retinal predicts two additional 'A states (3'A and 4'A or 'B in 11-cis-12-s-cis) and the 'A; state.66 Thus the structured absorption band at the 260-320 nm region (A,, =280 nm)73 may be assigned to one or both of these 'A transitions. From the foregoing review it is clear that the assignments of various bands other than the main absorption band (B,)are still tentative. The spectroscopy of retinal Schiff's bases is even less characterized than that of retinals although general features of the absorption spectra of Schiff's bases at room termperature and 77 K are similar to those of corresponding retinal~.~~ Recently a unusually large red shift of protonated retinal Schiff's base has been observed at 77K showing absorption maximum at ca.540 nm. It is suggested that the red shift results from a lowering of the transition energy of the protonated Schiff's base as a result of the preferential lowering of the excited singlet state due to its interaction with the hydrogen halide solvent cage. Carotenoids.-Carotenoid aldehydes containing one /3 -ionone end are called P-apo-carotenals of which retinal (five C=C bonds) may be regarded as the shortest carotenaI with A,, -360 nm. The main band maximum shifts to the red with increasing conjugation (e.g. 414 nm for P-apo-l2'-carotenal with seven C=C bonds and 508 nm for torularhodinaldehyde or 3' 4'-dehydro-@,t,b-caroten-l7'-al with thirteen C=C bonds).This shift is usually accompanied by enhanced vibra- tional resolution in the visible absorption band. This trend is also observed with carotenoids with two p-ionone ends (e.g. P-carotene). The vibrational structure is even more prominent at 77 K.74*75 Thus the torsional potential around the C-6-C-7 s-bond is no longer the dominant factor for the spectral diffuseness as the spectral diffuseness decreases with the number of C=C bonds in carotenoid~.~~ The total bandwidth (SF/crn-') is empirically given by SY = A + 64000N X 6/n* where n N and S(=0.244*0.021 cm-') are the number of double bonds the number of p-ionone rings and the reduction in the effective number of double bonds per p-ionone ring contributing to the spectral width respectively.Thus the second term gives the width contribution due to the torsional potential around the C-6-C-7 s-bond while the first term A = 549k 14 cm-' is of all contributions to the bandwidth from factors other than the distribution of torsional angles. The main absorption band 'B (f -3) is probably polarized along the long axis of the carotenoid conjugation in p-carotene and related carotenoids. The polarized absorption and reflectance spectra of all-trans -p -carotene single crystals have been measured. The 'B band is polarized at an angle of between 0 and 40" to the long molecular axis.77 Linear dichroic results of carotenoids in stretched 73 (a)A. M. Schaffer W. H. Waddell and R. S. Becker J. Amer. Chem. SOC. 1974,96,2063; (b)W.H. Waddell A. M. Schaffer and R. S. Becker ibid. 1977 99 8456. 74 (a)P.4. Song and T. A. Moore Photochem. and Photobiol. 1974,19,435; (b)Q. Chae P.3. Song J. E. Johansen and S. Liaaen-Jensen J. Amer. Chem. SOC. 1977,99 5609; (c)P. Koka and P.-S. Song Biochim. Biophys. Acta 1977,495 220. 7s B. Ke F. Imsgard H. Kj~sen,and S. Liaaen-Jensen Biochim. Biophys. Acta 1970 210 139. 76 R. Hemley and B. E. Kohler Biophys. J. 1978 in press. 77 (a)D. Chapman R. J. Cherry and A. Morrison Proc. Roy. SOC.1967 A301,173; (b) L. J. Parkhurst and B. G. Anex J. Chem. Phys. 1966,45.862. Physical Methods-Part (iii) Spectroscopy ofBio-organic Molecules polyethylene films and in liquid crystals are interpretable in terms of the long molecular axis polarization of the main band.74*78 The main absorption polarization in cis-carotenoids is also expected to be along the long molecular axis.According to the new a~signment,~~'~~ the lowest singlet state in carotenoids is also of the A symmetry. While the frequencies of the transitions to the allowed excited singlet state 'B, can be fitted to the equation i;lcm-' = 16500+64 100/n where n is the effective number of double bonds in carotenoids and Y reaches an assymptotic value for long carotenoids it is predicted that the lowest singlet ('A;) state continues to be lowered in energy with n. Unfortunately carotenoids are non-fluorescent thus making it difficult to confirm this prediction directly. At present there are no theoretical (e.g. dduble CI) or experimental data on the exact location of 'A in carotenoids.Not surprisingly the PPP singly excited CI predicts the 'A state to be at an energy above 'A:. The linear dichroism remains constant over the main absorption band and its red edge in cis-carotenal Since the oscillator strength of the 'A band is not expected to be negligible as is the case for the dichroic ratio measurements may have detected unique polarization characteristics. However the failure to resolve different dichroic ratios does not necessarily rule out the presence of a 'A band. Thus the weak absorption at A >A,, observed for cis-carotenals at 77 K may be attributable to either a 'A; or another T +T* transition strongly perturbed by the carbonyl group or by exciton interactions (e.g.see ref. 69) since carotenols do not show such long-wavelength ab~orption.~~ character is also a An n -* r* transition strongly admixed with ~,r* po~sibility.~~ Thus no definite assignment for the weak long-wavelength band in carotenals can be made at present. The nature of the weak absorption bands at wavelengths shorter than A,, in carotenoids is not well established except for the 'cis peak' (e.g. 337nm for 15 15'-cis-6-carotene in hexane at room remperature; 350 nm at 77 K) which becomes strongly allowed in cis-carotenoids. The cis peak can be safely assigned to the 'A; state (lC+ 'A in Platt notation) consistent with the PPP MO prediction.32778 The polarization of the cis-band has been deduced from linear dichroic spec- troscopy of /3 -carotene and several other car~tenals,~~*~~.~~ and its polarization is perpendicular to the long molecular axis.The 375 nm band in 13-cis-rhodopinal at 77 K is highly structured analogous to the structured band at ca. 280nm of retina171 and renierapurpurin-20-a1 (A =376 nm and 320 nm both having some structure and essentially identical dichroic ratios). It is not certain which of these bands is of the 'A genealogy; the former is tentatively identified as the 'C('AZ) transition. The shorter-wavelength band (lDt'A) is then assignable to the second 'B,+-A; transition. Carotenoids with chiral centres usually yield three distinct c.d. bands of alternating signs in the region of 200-300 nm. It is clear that several weakly absorbing transitions occur in U.V.spectra of carotenoids and more detailed studies are needed to delineate the number and identity of these tran- sitions in carotenoids. In contrast to carotenals spectra of the Schiff's bases of carotenals and carotenones lack any vibrational structure. ''T. A. Moore and P.-S. Song J. Mol. Spectroscopy 1974,52 209 216 224 and references therein. 79 (a)V. R. Salares R. Mendelsohn P. R. Carex and H. J. Bernstein J. Phys. Chern.,1976,80 1137; (b) V. R. Salares N. M. Young H. J. Bernstein and P. R. Carey Biochemistry 1977 16,4751. 34 P.-S. Song The wavelength maxima of near-u.v. (including cis-peak) and U.V. bands arising from excited configurations involving higher empty MOs in carotenoids can be predicted by Dales's rule which states that A (x = 2nd 3rd transition erc.) lies very close to the A ,, (the main long-wavelength band) of a polyene with n/x where n is the number of double bonds.This empirical rule is useful but it fails in longer and substituted carotenoids particularly at low temperature. Although several PPP-type calculations have been made the spectroscopic characterization of shorter-wavelength bands of carotenoids remains to be carried out as mentioned above. As it stands now the state ordering in a typical carotenoid (e.g. all-rrans-P-carotene) is 'B,< 'A < 2'B, with the location of the .symmetry-forbidden 'A; state yet to be spectroscopically resolved. However the validity of the low-lying 'A state for organic vapour-absorbed solid samples of P -apo-8'-carotenal and other p01yenes~~ is questionable.The recent theoretical calculations of absorption and fluorescence spectra of P-carotene suggest that the 'B state relaxes prior to emission by changing bond alternation along the relaxation co-ordinate in the excited state. This implies that the relaxed 'B state is the lowest and emitting state.80 However the fluorescence spectrum used is probably in error since pure p-carotene does not fluoresce. 4 Heterocyclic Compounds Indoles and Tryptophan.-The T -+ n* transitions 'Lb and 'La are not well resolved in indoles owing to considerable overlap and vibronic mixing between these two The U.V. absorption in the region of 260-275 nm is assigned to the 'Latransition although the 'Lb intensity (maximum at 287- 290 nm) contributes to the overall extinction and spectral shape in this region.It has been possible to resolve the 'Laand 'Lb bands of indole and tryptophan from fluorescence excitation polarization (P) measurements in propylene glycol at -58 "C by monitoring the long-wavelength fluorescence where the 'La-+ 'A emission predominates (cf. indole shows dual fluorescence due to 'Lb-+ 'A and 'La-+ The long-wavelength edge at 305-310 nm with P -0.4 is assigned to the 'Lae'A transition in agreement with previous studies. The 'Lbt'A 0-0 maximum in indole is located at 289.5 nm where the fluorescence polarization degree is minimal (P= O.l) with its vibrational satellites at 286 (0+ 538.3 cm-') and 282.5 nm (0+717.6 or 735.7 ~m-').*~ The P value then reaches a maximum (P== 0.3) at 260 nm.Both 'L and 'Lb contribute more or less equally to the absorption at A 6250 nm. Tryptophan shows essentially similar polarization characteristics. The 'Lb A (0-0) is resolved at 291 nm whereas the 'LaA,, is at 267 nm. The angle 9 between 'La and 'Lb transition moments can be calculated from the PPP MO method and it ranges from 54" with CI to 78" without CL8' If we assign the long-wavelength edge of the absorption to the 'La(0-0) transition which possesses a maximum P with respect to the 'L + 'A emission an estimate of 9 can T. Kakitani and H. Kakitani J. Phys. Soc. Japan 1977.42 1287. '' (a)P.-S. Song and W. E. Kurtin J. Amer. Chem. Soc. 1969 91 4892; (b)M. Sun and P.-S.Song Photochem. and Photobiol. 1977 25 3. B. Valeur and G.Weber Photochem. and Photobiol. 1977 25 441. E. H. Strickland. J. Horwitz. and C. Billups Biochemistry 1970 9 4914. Physical Methods-Part (iii) Spectroscopy ofBio-organic Molecules 35 be anywhere between 45 and 90° depending on the assumptions made concerning the linearity of the oscillators and the resolution of 'Laand 'Lbintensities in the excitation and emission spectra. The lower value results if the 290 nm absorption with minimum P with respect to the 'La+'A fluorescence is assumed to be exclusively due to 'Lb whereas the higher value results if both 'La and 'Lb contribute equally to this absorption. The actual composition of extinction at this peak is probably somewhere between these assumptions. According to a polarized single-crystal absorption the 'La and 'Lb transition moments are oriented at -38" and 54" to the long molecular axis respectively.On the other hand the PPP CI method" yields the 'Lapolarization in agreement with the above determination (polarization along N-1 -C-4 axis) but the 'Lbtransition is nearly short-axis polarized. Nucleic Acid Bases.-In spite of the obvious simplicity the genealogy of U.V. bands of purine and pyrimidine in terms of the benzene notation ('Lb,'La, and or 'BZu, 'Blu,and has been heuristically useful. Thus the U.V. bands of purine in trimethyl phosphate (TMP) at 265 nm (E =6.9 X lo3),240 nm (E == 3 X lo3),and 200 (E == 18.1x 103)-188(& -21.1 x lo3)nm are correlated to 1Lb(B2u), 'La(Blu) and lBa,b(Elu)bands respectively and the U.V. bands of pyrimidine in methyl- cyclohexane at 242 nm (& =2 x lo3),210 nm (E = 1X lo3),and 190 nm (E =6 x lo3) are also correlated respectively.According to this genealogical correlation for example the U.V. bands of 9-ethyl guanine in TMP at 275 nm (E ~9.4~ lo3) 256 nm (E = 15.4x lo3) and 203(~ == 20 X 103)-190(~ =27.4 X lo3)nm are cor- related to Lb(BZu),La(Blu),and Ba.b (Elu),respectively and similar correlations are made to the absorption bands of cytosine at 277 nm (E ~7.5 x lo3) 237 nm (E == 3.5 x lo3) and 204 (E = 11.9x 103)-185 (E =12.2x lo3)nm. In addition polarization directions of these bands can be correlated with those of benzene. For example the 260 nm band (lLb)of 1-methyl-thymine is polarized along the axis intersecting N-1-C-2 and C-4-C-5 bonds (inclined by 11*2" toward N-1).86 With the above description of the gross features of the U.V.spectra of bases more recent studies will be selectively reviewed in the following sections. Purine Bases Analysis of polarized reflection spectra of the(1UU) face of purine crystals yields the transition moment directions of both T +T* and n 3T* transition^.^' The first band at 294 nm (f=00.0057 in the crystal 0.003-0.005 in solution) is out-of-plane polarized and is due to an n +T* transition which probably is highly localized at the N-3 position of purine.85 The second band at 263 nm ('Lb),is in-plane polarized at i-48" from the C-4-C-5 axis (toward C-6). The weak band resolved at 250 nm in the crystal is out-of-plane polarized and is assigned to the second n -+T* transition which is probably localized at N-1.The strong absorption at 200 nm is probably due to Bb,as it is polarized nearly parallel to the 263 nm band but the identity of the out-of-plane polarized 190 nm band remains to be established. An n +T* assignment for this band is unlikely owing to 84 Y. Yarnarnoto and J. Tanaka Bull. Chem. SOC.,Japan 1972,45 1362. '' (a)L. B. Clark and I. Tinoco jun. J. Amer. Chem. SOC., 1965.87 11; (b)W. Hug and I. Tinoco jun. ibid. 1973 95 2803; (c) W. Hug and I. Tinoco jun. ibid. 1974,96 665. 86 R. F. Stewart and N. Davidson J. Chem. Phys. 1963,39 255. " (a)H. H. Chen and L. B. Clark J. Chem. Phys. 1969,51,1862;(b)L. B. Clark J. Arner. Chem. Soc. 1977,99,3934. 36 P.-S. Song its relatively strong intensity (E == 6 X lo3).The experimental polarization direc- tions reported here are in good agreement with PPP-type results but agreement is less satisfactory in purine bases containing additional functional groups such as C=O or NH2. The n +n* transitions in purine bases have been calculated by the CNDO-CI meth~d.~'.~~ Agreement with available experimental n -+ n-* transition energies is not sufficiently quantitative but it is possible to use theoretical data in aiding the resolution of weak n +n* bands in purines. The lowest n + n* transition is predicted at 256 nm for purine 228 nm for adenine and 239 nm for guanine-N-1 which are theoretically above the lowest n -+ n* (lLb)transition in the respective bases.85 The calculations based on the same CNDO method predict an n +n-* transition to be the lowest in adenine thymine and cytosine but not in guanine.88 At least for purine there is no doubt that the lowest singlet excited state is an n,n* type as the high-resolution spectra of purine in crystal and in inert-gas matrices at 4-20 K exhibit vibrational progression characteristic of an n +n-* transition (318.5nm in low-temperature matrix).89 The assigned wavelengths of the lowest n -+ n* transitions in adenine and guanine are 280 and 264 nm respectively by means of polarized absorption and reflectance spectra of crystals solvent effects and c.d.spectroscopy. Poly(dA) shows a positive c.d. peak at 280 nm which can be assigned to an n +n* transition of adenine.90 Similarly the 270 nm absorption in deoxyadenosine is assigned to an n +T* transition.However the identification of an n +n* transition at 264 nm is not definitive since this region is significantly masked by n -+ n* transitions. The SCrRPA method predicts the second n +n* at 259nm however. It is reasonable to conclude that the lowest singlet transition is of an n -+n-* type in adenine.91 The lowest n +T* transition appears to be strongly localized on N-3.85 The location of the lowest n +n-* transition in guanine remains to be resolved. Recent polarized single-crystal reflectance data suggest an n -+ n-* transition at 300.3 nm for 9-ethylguanine at 77 K.*'However the possibility of an exciton and matrix-shifted 'BZu(0-0) band at 77 K cannot be ruled out. The long-wavelength band (A = 274 nm) of single crystals of 9-methyl-adenine 1-methylthymine (AT dimer) is due to the lowest n-+n-* transition of the former.Its polarization is along the short molecular axis C-5-C-6) while the weaker band with A,, -255 nm is long-axis polarized.86 According to the com- monly used convention the transition moment directions (8) for purines are measured as positive towards C-6 (clockwise) with respect to the C-4-C-5 axis. The first n +n* transition moment of adenine is then at an angle of -12* 3". Thus the two n +n* transitions are polarized at least 45" from each other. This conclusion is further confirmed by the dichroic analysis of the spectra of adenine and 9-met hyladenine .91 88 N. V. Zheltovskii and V. I. Danilov Biofizika 1974 19 784.89 (a)J. J. Smith Photochem. and Photobiol. 1976,23 365;(b)M.J. Robey and I. G. Ross ibid. 1975 21 363. 90 (a)C.A. Bush and H. A. Scheraga Biopolymers 1969,7 395; (6)C.A.Bush J. Amer. Chem. SOC. 1973 95,214. 9' (a)A. F. Fucaloro and L. S. Forster J. Amer. Chem. SOC. 1971,93,6443;(b) Spectrochim. Acta 1974 30A 883. Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules 37 The 260 nm band of adenine in solution is correlated to 'B2u(Lb) but it is apparently composed of two electronic transitions (La+ Lb) as predicted by theoretical treatments and spectroscopic measurements such as m.c.d. The linear dichroism along the short-wavelength tail of the 260 nm band decreases indicating the presence of a transition corresponding to the second T +T* transition (La) which is polarized at some angle to the Lb transition moment direction.The difficulty with the Laassignment is that its f value appears to be weaker than that of the Lb band in contrast to theory and to what is expected from correlation with the benzene and naphthalene spectra. 9-Ethylguanine in TMP shows absorption bands at 275 nm (shoulder E =9.4X lo3 Lb or B2,) 25.6 nm (E = 15.4X lo3,Laor Blu),and 203 (E =2OX 1O3b19O (E =27.4 x lo3,Ba.6 or El,)nm. In aqueous solution pH 6.8 is the 256 nm band of 9-ethylguanine shifts to 249 nm.87 Based on combined information from polarized specular reflectance measurements on single crystals of 9-ethylguanine and from fluorescence polarization data the first band (B2,)is either polarized nearly short-axis or close to the N-1-N-3 direction (i.e.44*5" or -14*5" with the N-3-C-6 direction) while the second band (Blu)is polarized close to the C-2-C-8 axis (i.e. 115f10" or 95 * 10" with the N-3-C-6 dire~tion).~~ Newer results from polarized single-crystal reflection spectra of 9-ethylguanine yield polarization angles of -4" or +35" for B2 and -75" for Blu,87in general agreement with the above results. Polarization directions for guanine HCl 2H20 are also a~ailable.~' Although PPP generates reasonable r +r transition energies (e.g. 277 nm for B2 and 246 nm for Blu),the calculated polarization directions are not satisfactory as their directions are reversed relative to the observed directions. Both experiments based on polarized reflectance c.d.m.c.d. and fluorescence polarization and theories confirm that the first two r -+ T* transitions in guanine and guanosine are polarized nearly perpendicular to each other. Pyrimidine Bases. Uracil in TMP shows 'B2,(Lb)at 258 nm (E ~7.8 X lO',f= 0.175) and 'El (Ba.6)at 203 (E == 8.2 x lo3)-181 (E = 11.8X lo3) nm. The 'n,v* state probably is at higher energy (250 nm)8' than 'B2,. The c.d. of uridine in water (pH 7) resolves a band at 240 nm with negative ellipticity which may be assigned to 1 93 B1,,in agreement with some theoretical treatments (e.g.B1,at 224 nm by PPP but 195 nm by CNDO-CI or 210 nm estimated by analogy with the 237 nm band of cytosine8'). The 258 nm band of uracil may be of composite nature as revealed by (a)the electric field effectg4 and (b) the polarized single-crystal reflectance spectra of its model 6-azauracil which shows resolved bands at 256 and 278nm in addition to the weak band.95 The latter appears to correspond to the weak absorption at ca.230 nm in solution. 1,3-Dimethyluracil and thymines show simiIar absorption spectra. However the nature of the weak band hidden under the strong absorption band is yet to be elucidated. Several attempts using c.d. solvent perturbation and other spectroscopic methods have not been successful in resolving n +T* transitions in uracil. 92 (a)P. R. Callis B. Fanconi and W. T. Simpson J. Amer. Chem. SOC.,1971 93,6679; (b)P. R. Callis and W. T. Simpson ibid. 1970,92,3593. 93 K. K. Cheong Y. C. Fu R. K. Robins and H. Eyring J.Phys. Chem. 1969,73,4219. 94 K. Seibold and H. Labhart Biopolymers 1971 10 2063. 95 J. N. Brown L. M. Trefonas. A. F. Fucaloro and B. G. Anex J. Amer. Chem. SOC.,1974,96 1597. 38 P.-S. Song However the polarized single-crystal absorption spectrum of 1-methyluracil suggests a weak n +T* transition (E = 247) at 264 nm which is polarized perpen- dicular to the molecular plane; the first T -+ T* transition (&) in the crystal is at 275.5 nm96 The longest-wavelength band in 1-methyluracil and 1-methylthymine is polarized almost parallel to the N-1-C-4 direction (0" or 7" and 19" respectively) with respect to the N-1-C-4 direction with clockwise rotation denoted as +). Semi-empirical MO calculation^^^ are in satisfactory agreement with the experi- mental directions.Another polarized absorption study reports a polarization angle of -38* 14" for the main band at 260 nm for thymine itself and the second absorption band at 220 nm is polarized along the short molecular axis (-86°).98 The angle of 48" between these two transition moment directions is substantially lower than the value for uracil and thymine estimated from the linear dichroic data.91 Cytosine in TMP shows A,, at -277 nm ('BZu,E -7.5 x lo3) a shoulder at E 237 nm ('B1,,== 3.5 x lo3) and 204 nm (E = 11.9x 103)-185 nm (E = 12.2 x lo3,'El,,). The first two T -B T* transitions are polarized nearly parallel accord- ing to the polarized reflectance study on cytosine," cytosine monohydrate and l-methylcyt~sine.~~ The 'BZuband is polarized at an angle of 14* lo and 'Bluis polarized at -5 * 3°.96 Although agreement between observed and MO-calculated polarization angles is good (within -10") for thymine and uracil this is not the case with cytosine.However CNDO-CI seems to yield better angles.85 From polarized absorption measurements on oriented films of polycytidylic acid an n -B T* transition was located at 278 nm.99 Such an n +T* transition may be strongly localized on N-3.89 However both CNDO-CI (calc. n -+ T* at 253 nm) and polarized single crystal ab~orption~~ studies fail to resolve an n -+ T* at A longer than the first T* +T* band. Flavins.-Flavins (e.g. riboflavin) show four major absorption bands at 445 nm (E = 12x lo3) 360 nm (E == 10x lo3) 270 nm (E =32 x lo3) and 230 nm.All four bands are attributable to the n+~*-type transition although other types of transition particularly n -+T* may contribute to some of these band intensities. The PPP calculations on the isoalloxazine nucleus support the T +T* nature of the major absorption bands in flavins. loo Calculated transition energies and relative oscillator strengths of the flavin nucleus 7,8-dimethylisoalloxazine are in agreement with observed absorption spectra of flavins."' The PPP theory predicts a weak T +T* transition (S3)at ca. 300 nm where a fiuorescence polarization minimum is also recorded indicating a separate transition in this region."' M.c.d. provides indirect evidence for this transition as the m.c.d. 96 (a)W. Eaton and T. P. Lewis J. Chem. Phys.1970,53 2164; (b)T.P.Lewis and W. Eaton J.Amer. Chem. SOC. 1971,93 2054. 97 J. S. Kwiatkowski and B. Pullman Adv. Heterocyclic Chem. 1975 18 199; for theoretical results see references therein. 98 M. Tanaka and J. Tanaka Bull. Chern. SOC. Japan 1971,44,672,938. 99 A. Rich and M. Kasha J. Amer. Chem. SOC.,1960 82 6197. loo (a)J. L. Fox S. P. Laberge K. Nishimoto and L. S. Forster Biochim. Biophys. Acta 1967 136 544; (b)K."ishimoto Bull. Chem. SOC. Japan 1967,40 2493. lo' (b)P.4. Song Internat. J. Quantum Chem. (a)P.-S. Song Ann. New York Acad. Sci. 1969,158,410; 1969 3,,303; (c)P.-S. Song T. A. Moore and W. E. Kurtin 2.Nuturforsch. 1972,B27,1011 Physical Methods-Part (iii) Spectroscopy of Bio-organic Molecules maximum is at A <A,, =370 nm for riboflavin.lo2 C.d.lo3 and linear dichroic'04 studies support this possibility.The polarized single-crystal absorption of FMN in flavodoxin indicates that both S1and Sz bands are polarized along the long molecular axis."' Defining the polarization angle 8 as the clockwise angle measured from the long axis (N-10- N-1 direction) the Sland S2 bands at 450 and 370 nm are then polarized at 16" and -4" respectively making At? of 20' for the angle between the two transition moments. The Kronig-Kramers transformation of specular reflection spectra of crystalline bis(l0-methylisoalloxazine)copper(II) perchlorate tetrahydrate yields the corresponding absorption spectra. lo6 Again S1(f =0.16) and Sz (f =0.15) transitions are found to be polarized along the long molecular axis with 8 = 26" and lo respectively and A8 of 25".The rotation of the two transition moments by 5-10' clockwise relative to those of the polarized single-crystal absorption of FMN may be attributed to substituent or crystal environmental effects. Considering this agreement between the two sets of polarization data is excellent. Thus earlier theoretical transition moment directions calculated from PPP (S1 along C-8-N-3 and S2 along C-7-C-2 with A8 = 25-30" depending on several parameters in the calculation)lol are experimentally confirmed. The polarization of both transitions is along the C-7-C-2 axis according to MIND0/3.1°7 Both fluorescence polarizationlO'*'Os and linear dichroi~m~~~ data are qualitatively consistent with the polarization directions from the single-crystal reflection and absorption studies and the PPP prediction.Polarization directions of S1and S2 bands are not significantly altered by substituting N-5 with carbon as in deazaflavin yielding A8 = 25" (from fluorescence polarization) and 27" (from PPP108). Higher-energy T -+ T* transitions of flavins in the 200-300 nm region are not well understood. It is likeIy that the U.V. band at 270 nm is composed of more than one 7r -+ T* transition. Thus specular reflectance spectroscopy of 10-methyl-isoalloxazine reveals bands at 284 nm (f = 0.2) 257 nm (f = 0.39) and 221 nm (f= 0.1l) whereas the solution spectrum shows only two peaks at 269 nm (f = 0.55) and 225 nm (f=0.11). Their polarization angles are 3' -19' and 42" respectively.Four possible n -+ T* transitions localized at N-1 C-2=0 C-4=0 and N-5 are not spectrally resolved as they are masked under the intense T -+ n* bands. It is likely that the lowest n -+ n* transition is localized at N-5 and that its low-intensity band is hidden under the second 7r -+ T* band at 370 nrn."' C.d. and specular reflection spectra of flavins do not reveal the location of an n -+ T*band indicating that such a transition if present is probably too weak (E <500) to be detectable. The n -* T* transition assigned to the solvent-sensitive shoulders of the 450 and 370 nm bands by various authors are not experimentally compatible and its loca- tion remains obscure. However it appears most likely that the lowest n -+ T* '02 (a)D.E. Edmondson and G. Tollin Biochemistry 1971 10 113; (6)G. Tollin ibid. 1968 7 1720. Io3 H. Harders S. Forster W. Voelter and A. Bacher Biochemistry 1974 13 3360. '04 J. Siodmiak and D. Frackowiak Photochem. and Photobiol. 1972 16 173. W. A. Eaton J. Hofrichter M. W. Makinen R. D. Anderson and M. L. Ludwig Biochemistry 1975 14 2146. M. W. Yu C. J. Fritchie A. F. Fucaloro and B. G. Anex J. Amer. Chem. SOC.,1976,98 6496. '" M. F. Teitell S. H. Suck and J. L. Fox J. Amer. Chem. Soc. 1978 in press. (a)M. Sun T. A. Moore and P.-S. Song J. Amer. Chem. SOC. 1972.94 1730; (6) M. Sun and P.-S. Song Biochemistry 1973 12 4663. 40 P.-s. Song transition occurs near the 370 nm band as dimethylalloxazines the tautomers of dimethylisoalloxazines with the S1tSo A,, at -380-390 nm are considerably less fluorescent than the latter suggesting that an n,r* state lies only slightly above the fluorescent r,v* state."' In this argument it is assumed that the tautomerism does not affect n +r* energy as much as it does r -+ r* energy.The fluorescence polarization degree in the red region of the first absorption band of alloxazine and lumichromes is >0.4.The angle between the two near-u.v. absorption bands (378 and 318 nm) of alloxazine is estimated to be about 30" from the fluorescence polarization data. This angle is somewhat larger than in iso-alloxazines. PPP also predicts a larger angle (430).lo8 The predominant configuration for the 580 nm band of flavin semiquinone arises from the same orbital pairs as in oxidized flavins i.e.HOMOjLEMO (half-However this band cannot be unambiguously identified as the S1 transition which may arise from an orbital promotion other than HOMO +LEMO.log Both experimental and theoretical investigations are called for before satisfactory spectroscopic assignments including polarizations can be developed for flavin semiquinone. Work derived from this laboratory has been supported by the Robert A. Welch Foundation (D-182). I also wish to thank Professors R. Birge R. Christofferson J. L. Fox and B. Kohler for making their preprints available. lo9 B. Grabe Acta Chem. Scand. 1974,28 363.