The resonance Raman spectrum of the 11-cis retinal protonated Schiff base chromophore in rhodopsin exhibits low-frequency normal modes at 93, 131, 246, 260, 320, 446, and 568 cm -1 . Their relatively strong Raman activities reveal that the photoexcited chromophore undergoes rapid nuclear motion along torsional coordinates that may be involved in the 200-fs isomerization about the C 11 dC 12 bond. Resonance Raman spectra of rhodopsins regenerated with isotopically labeled retinal derivatives and demethyl retinal analogues were obtained in order to determine the vibrational character of these low-frequency modes and to assign the C 11 dC 12 torsional mode. 13 C substitutions of atoms in the C 12 -C 13 or C 13 dC 14 bond cause the 568-cm -1 mode to shift by ∼8 cm -1 , and deuteration of the C 11 dC 12 bond downshifts the 568-and 260-cm -1 modes by ∼35 and 5 cm -1 , respectively. The magnitudes of these shifts are consistent with those calculated for modes containing significant C 11 dC 12 torsional character. Thus, we assign the 568-cm -1 mode to a localized C 11 dC 12 torsion and the 260-cm -1 mode to a more delocalized torsional vibration involving coordinates from C 10 to C 13 . Consistent with these assignments, these two modes are not Raman active in 13-demethyl, 11-cis rhodopsin which has a planar C 10 ‚‚‚C 13 geometry. Furthermore, the relative Raman scattering strengths of the 260-and 568-cm -1 modes are ∼2-fold higher with preresonant excitation. These data quantitate the instantaneous torsional dynamics of the chromophore about its C 11 dC 12 bond on the S 1 surface and indicate that the isomerization process is facilitated by vibronic coupling of the S 1 and S 2 surfaces via C 11 dC 12 torsional distortion, which reduces the excited-state barrier along the reaction trajectory. We have also examined the low-frequency Raman spectrum of the trans primary photoproduct, bathorhodopsin, and discuss the relevance of its low-frequency torsional modes at ∼54, 92, 128, 151, 262, 276, 324, and 376 cm -1 to the observed femtosecond photochemical dynamics.

The role of intramolecular steric interactions in the isomerization of the 11-cis-retinal chromophore in the photoreceptor protein rhodopsin is examined with resonance Raman and CD spectroscopy combined with quantum yield experiments. The resonance Raman spectra and CD spectra of 13-demethylrhodopsin indicate that its chromophore, an analog in which the nonbonded interaction between the 10-H and the 13-CH3 groups is removed, is less distorted in the C10...C13 region than the native chromophore. The reduced torsional and hydrogen-out-of-plane resonance Raman intensities further indicate that the excited state potential energy surface has a much shallower slope along the isomerization coordinate. This is consistent with the decrease in quantum yield from 0.67 in rhodopsin to 0.47 in 13-demethylrhodopsin. The resonance Raman intensities show that the steric twist is reintroduced by addition of a methyl group at the C10 position. However, the quantum yield of 10-methyl-13-demethylrhodopsin is found to be only 0.35. This is attributed to nonisomorphous protein-analog interactions. The nonbonded interaction between the 10-hydrogen and the 13-methyl group in 11-cis-retinal makes this isomer particularly effective as the light-sensing chromophore in all visual pigments.

The spectroscopic properties of spheroidene and a series of spheroidene analogs with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation, and time-resolved absorption spectroscopy. The spheroidene analogs studied here were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene and taken together with data from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene already published (DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J.; Farhoosh, R.; Frank, H. A. Biochim. Biophys. Acta 1992, 1102, 107) provide a systematic series of molecules for understanding the molecular features that control energy transfer to bacteriochlorophyll in photosynthetic bacterial light-harvesting complexes. All of the molecules were purified by high-pressure liquid chromatographic techniques prior to the spectroscopic experiments. The absorption spectra of the molecules were observed to red-shift with increasing extent of π-electron conjugation. The room temperature fluorescence data show a systematic crossover from dominant S1 → S0 (2Ag → 11Ag) emission to dominant S2 → S0 (11Bu → 11Ag) with increasing extent of conjugation. The S2 fluorescence quantum yields of all the carotenoids in the series were measured here and indicate that 3,4-dihydrospheroidene with nine carbon−carbon double bonds has an S2 quantum yield of (2.7 ± 0.3) × 10-4 which is the highest value in the series. The lifetimes of the S1 states of the molecules were determined from time-resolved transient absorption spectroscopy and found to decrease as the conjugated chain length increases. The transient data are discussed in terms of the energy gap law for radiationless transitions which allows a prediction of the S1 energies of the molecules. The implications of these results for the process of light harvesting by carotenoids in photosynthesis are discussed.

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.

A series of apocarotenes with 5 to 11 conjugated double bonds were synthesized and all-trans isomers were isolated using HPLC techniques. Absorption, fluorescence, and fluorescence excitation spectra were obtained in 77 K glasses. As previously noted for other polyenes and carotenoids, fluorescence spectra of the apocarotenes exhibit a systematic crossover from S1(2Ag) → S0(11Ag) to S2(21Ag) → S0(11Ag) emissions and a sharp decrease in fluorescence yields with increasing conjugation. The apocarotene spectra have sufficient resolution to accurately locate the dominant vibronic bands of the S0(11Ag) → S2(11Bu) and S1(21Ag) → S0(11Ag) transitions, thus leading to an accurate catalog of S1 and S2 electronic energies as a function of conjugation length. We also have obtained the low-temperature absorption and fluorescence spectra of several model polyenes and diapocarotenes. Comparisons between these series allow a systematic exploration of the influence of terminal cyclohexenyl rings on the energies of carotenoid S1 and S2 states. In addition, these preliminary studies indicate that the nature of the terminal double bond has a significant influence on nonradiative decay processes in longer carotenoid systems. Implications regarding the use of energy gap law extrapolations to estimate the 21Ag energies of long carotenoids are discussed.

Spheroidene and a series of spheroidene analogues with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh, R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57, 49. Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J.; Frank, H. A. Photosynth. Res. 1994, 42, 157), provide a systematic series of molecules for understanding the molecular features that determine the mechanism of energy transfer from carotenoids to BChl in photosynthetic bacterial light-harvesting complexes. The data support the hypothesis that only carotenoids having 10 or less carbon−carbon double bonds transfer energy via their 2Ag (S1) states to BChl to any significant degree. Energy transfer via the 11Bu (S2) state of the carotenoid becomes more important than the S1 route as the number of conjugated carbon−carbon double bonds increases. The results also suggest that the S2 state associated with the Q x transition of the B850 BChl is the most likely acceptor state for energy transfer originating from both the 21Ag (S1) and 11Bu (S2) states of all carotenoids.

The spectroscopic properties of open-chain, all-trans-C 30 carotenoids having seven, eight and nine π-electron conjugated carbon-carbon double bonds were studied using steady-state absorption, fluorescence, fluorescence excitation and time-resolved absorption spectroscopy. These diapocarotenes were purified by high performance liquid chromatography (HPLC) prior to the spectroscopic experiments. The fluorescence data show a systematic crossover from dominant S 1 f S 0 (2 1 A g f 1 1 A g ) emission to dominant S 2 f S 0 (1 1 B u f 1 1 A g ) with increasing extent of conjugation. The low temperatures facilitated the determination of the spectral origins of the S 1 f S 0 (2 1 A g f 1 1 A g ) emissions, which were assigned by Gaussian deconvolution of the experimental line shapes. The lifetimes of the S 1 states of the molecules were measured by transient absorption spectroscopy and were found to decrease as the conjugated chain length increases. The energy gap law for radiationless transitions is used to correlate the S 1 energies with the dynamics. These molecules provide a systematic series for understanding the structural features that control the photochemical properties of open-chain, diapocarotenoids. The implications of these results on the roles of carotenoids in photosynthetic organisms are discussed.

Steady-state absorption and femtosecond time-resolved optical spectroscopic studies have been carried out on all-trans-beta-carotene, 15,15'-cis-beta-carotene, all-trans-spheroidene, and 13,14-locked-cis-spheroidene. We examine in detail the effect of isomer geometry on the spectroscopic properties and photophysics of the low-lying S(1) (2(1)A(g)(-)) and S(2) (1(1)B(u)(+)) excited states of these molecules. The experiments on 13,14-locked-cis-spheroidene, a molecule incapable of undergoing cis-to-trans isomerization, provide a unique opportunity to examine the role of isomer geometry in controlling excited-state deactivation of carotenoids. The kinetic results have been obtained using both single wavelength transient absorption measurements and global fitting procedures. The overall scheme for the deactivation of these molecules after S(0) --> S(2) photon absorption is decay of S(2) to a vibrationally hot S(1) state, followed by vibrational relaxation within S(1), and finally, S(1) --> S(0) internal conversion back to the ground state. Changes in isomer geometry are shown to lead to small but noticeable alterations in the spectroscopic and kinetic behavior of the molecules. The effects are interpreted in terms of minor alterations in excited-state energy and vibrational coupling upon isomerization that bring about changes in the spectroscopic and kinetic behavior of this biologically important class of pigments.