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Steady-state and time-resolved absorption and fluorescence spectroscopic experiments have been carried out at room and cryogenic temperatures on aggregated and unaggregated monomeric and trimeric LHCII complexes isolated from spinach chloroplasts. Protein aggregation has been hypothesized to be one of the mechanistic factors controlling the dissipation of excess photo-excited state energy of chlorophyll during the process known as nonphotochemical quenching. The data obtained from the present experiments reveal the role of protein aggregation on the spectroscopic properties and dynamics of energy transfer and excited state deactivation of the protein-bound chlorophyll and carotenoid pigments.

Experimental and theoretical evidence is presented that supports the theory that the intramolecular charge transfer (ICT) state of peridinin is an evolved state formed via excited-state bond-order reversal and solvent reorganization in polar media. The ICT state evolves in

The spectroscopic properties and dynamics of the excited states of two different synthetic analogues of peridinin were investigated as a function of solvent polarity using steady-state absorption, fluorescence, and ultrafast time-resolved optical spectroscopy. The analogues are denoted S-1- and S-2-peridinin and differ from naturally-occurring peridinin in the location of the lactone ring and its associated carbonyl group, known to be obligatory for the observation of a solvent dependence of the lifetime of the S1 state of carotenoids. Relative to peridinin, S-1- and S-2-peridinin have their lactone rings two and four carbons more toward the center of the π-electron system of conjugated carbon-carbon double bonds, respectively. The present experimental results show that as the polarity of the solvent increases, the steady-state spectra of the molecules broaden, and the lowest excited state lifetime of S-1-peridinin changes from ~155 ps to ~17 ps which is similar to the magnitude of the effect reported for peridinin. The solvent-induced change in the lowest excited state lifetime of S-2-peridinin is much smaller and changes only from ~90 ps to ~67 ps as the solvent polarity is increased. These results are interpreted in terms of an intramolecular charge transfer (ICT) state that is formed readily in peridinin and S-1-peridinin, but not in S-2-peridinin. Quantum mechanical computations reveal the critical factors required for the formation of the ICT state and the associated solvent-modulated effects on the spectra and dynamics of these molecules and other carbonyl-containing carotenoids and polyenes. The factors are the magnitude and orientation of the ground and excited state dipole moments which must be suitable to generate sufficient mixing of the lowest two excited singlet states.

The peridinin-chlorophyll a-protein (PCP) is a light-harvesting pigment-protein complex found in many species of marine algae. It contains the highly substituted carotenoid peridinin and chlorophyll a, which together facilitate the transfer of absorbed solar energy to the photosynthetic reaction center. Photoexcited peridinin exhibits unorthodox spectroscopic and kinetic behavior for a carotenoid, including a strong dependence of the S(1) excited singlet state lifetime on solvent environment. This effect has been attributed to the presence of an intramolecular charge transfer (ICT) state in the molecule. The present work explores the effect of changing the extent of π-electron conjugation and attached functional groups on the nature of the ICT state of peridinin and how these factors affect the excited singlet and triplet state spectra and kinetics of the carotenoid. In this investigation three peridinin analogues denoted C-1-R-peridinin, C-1-peridinin, and D-1-peridinin were synthesized and studied using steady-state absorption and fluorescence techniques and ultrafast time-resolved transient absorption spectroscopy. The study explores the effect on the singlet and triplet state spectra and dynamics of removing the allene group from the peridinin structure and either replacing it with a rigid furanoid ring, replacing it with an epoxide group, or extending the polyene chain into the β-ionylidine ring.

Numerous femtosecond time-resolved optical spectroscopic experiments have reported that the lifetime of the low-lying S(1) state of carbonyl-containing polyenes and carotenoids decreases with increasing solvent polarity. The effect becomes even more pronounced as the number of double bonds in the conjugated π-electron system decreases. The effect has been attributed to an intramolecular charge transfer (ICT) state coupled to S(1), but it is still not clear what the precise molecular nature of this state is, and how it is able to modulate the spectral and dynamic properties of polyenes and carotenoids. In this work, we examine the nature of the ICT state in three substituted polyenes: crocetindial, which contains two terminal, symmetrically substituted carbonyl groups in conjugation with the π-electron system, 8,8'-diapocarotene-8'-ol-8-al, which has one terminal conjugated carbonyl group and one hydroxyl group, and 8,8'-diapocarotene-8,8'-diol, which has two terminal, symmetrically positioned, hydroxyl groups but no carbonyls. Femtosecond time-resolved optical spectroscopic experiments on these molecules reveal that only the asymmetrically substituted 8,8'-diapocarotene-8'-ol-8-al exhibits any substantial effect of solvent on the excited state spectra and dynamics. The data are interpreted using molecular orbital theory which shows that the ICT state develops via mixing of the low-lying S(1) (2(1)A(g)-like) and S(2) (1(1)B(u)-like) excited singlet states to form a resultant state that preferentially evolves in polar solvent and exhibits a very large (∼25 D) dipole moment. Molecular dynamics calculations demonstrate that the features of the ICT state are present in ∼20 fs.

This paper presents an ultrafast optical spectroscopic investigation of the excited state energies, lifetimes and spectra of specific geometric isomers of neoxanthin, violaxanthin, lutein, and zeaxanthin. All-trans- and 15,15'-cis-beta-carotene were also examined. The spectroscopy was done on molecules purified by HPLC frozen immediately to inhibit isomerization. The spectra were taken at 77 K to maintain the configurations and to provide better spectral resolution than seen at room temperature. The kinetics reveal that for all of the molecules except neoxanthin, the S(1) state lifetime of the cis-isomers is shorter than that of the all-trans isomers. The S(1) excited state energies of all the isomers were determined by recording S(1) --> S(2) transient absorption spectra. The results obtained in this manner at cryogenic temperatures provide an unprecedented level of precision in the measurement of the S(1) energies of these xanthophylls, which are critical components in light-harvesting pigment-protein complexes of green plants.

Azobenzene undergoes reversible cis--trans photoisomerization upon irradiation. Substituents often change the isomerization behavior of azobenzene, but not always in a predictive manner. The synthesis and properties of three azobenzene derivatives, AzoAMP-1, -2, and -3, are reported. AzoAMP-1 (2,2'-bis[N-(2-pyridyl)methyl]diaminoazobenzene), which possesses two aminomethylpyridine groups ortho to the azo group, exhibits minimal trans--cis photoisomerization and extremely rapid cis--trans thermal recovery. AzoAMP-1 adopts a planar conformation in the solid state and is much more emissive (Phi(fl) = 0.003) than azobenzene when frozen in a matrix of 1:1 diethylether/ethanol at 77 K. Two strong intramolecular hydrogen bonds between anilino protons and pyridyl and azo nitrogen atoms are responsible for these unusual properties. Computational data predict AzoAMP-1 should not isomerize following S(2)--S(0) excitation because of the presence of an energy barrier in the S(1) state. When potential energy curves are recalculated with methyl groups in place of anilino protons, the barrier to isomerization disappears. The dimethylated analogue AzoAMP-2 was independently synthesized, and the photoisomerization predicted by calculations was confirmed experimentally. AzoAMP-2, when irradiated at 460 nm, photoisomerizes with a quantum yield of 0.19 and has a much slower rate of thermal isomerization back to the trans form compared to that of AzoAMP-1. Its emission intensity at 77 K is comparable to that of azobenzene. Confirmation that the AzoAMP-1 and -2 retain excited state photochemistry analogous to azobenzene was provided by ultrafast transient absorption spectroscopy of both compounds in the visible spectral region. The isomerization of azobenzene occurs via a concerted inversion mechanism where both aryl rings must adopt a collinear arrangement prior to inversion. The hydrogen bonding in AzoAMP-1 prevents both aryl rings from adopting this conformation. To further probe the mechanism of isomerization, AzoAMP-3, which has only one anilinomethylpyridine substituent for hydrogen bonding, was prepared and characterized. AzoAMP-3 does not isomerize and exhibits emission (Phi(fl) = 0.0008) at 77 K. The hydrogen bonding motif in AzoAMP-1 and AzoAMP-3 provides the first example where inhibiting the concerted inversion pathway in an azobenzene prevents isomerization. These molecules provide important supporting evidence for the spectroscopic and computational studies aimed at elucidating the isomerization mechanism in azobenzene.

Carotenoids present in the photosynthetic light-harvesting reaction center (LHRC) complex from chlorosome lacking filamentous anoxygenic phototroph, Roseiflexus castenholzii were purified and characterized for their photochemical properties. The LHRC from anaerobically grown cells contains five different carotenoids, methoxy-keto-myxocoxanthin, gamma-carotene, and its three derivatives, whereas the LHRC from aerobically grown cells contains only three carotenoid pigments with methoxy-keto-myxocoxanthin being the dominant one. The spectroscopic properties and dynamics of excited singlet states of the carotenoids were studied by steady-state absorption, fluorescence and ultrafast time-resolved optical spectroscopy in organic solvent and in the intact LHRC complex. Time-resolved transient absorption spectroscopy performed in the near-infrared (NIR) on purified carotenoids combined with steady-state absorption spectroscopy led to the precise determination of values of the energies of the S(1)(2(1)A(g)(-)) excited state. Global and single wavelength fitting of the ultrafast spectral and temporal data sets of the carotenoids in solvents and in the LHRC revealed the pathways of de-excitation of the carotenoid excited states.

Steady-state and femtosecond time-resolved transient absorption spectra of diadinoxanthin and diatoxanthin were measured in the visible and near-infrared (NIR) regions at 293 K. The difference in energy between the visible S 0 → S 2 transitions from the steady-state absorption measurements and the S 1 → S 2 transitions observed in the transient absorption spectra in the NIR region yields precise values of the S 1 energies. The data are important for evaluating the mechanism by which excess energy is dissipated by algal systems that interconvert these xanthophylls in response to changes in photon flux levels in the marine environment.

The lifetime of the lowest excited singlet (S(1)) state of peridinin and many other carbonyl-containing carotenoids and polyenes has been reported to depend on the polarity of the solvent. This effect has been attributed to the presence of an intramolecular charge transfer (ICT) state in the manifold of excited states for these molecules. The nature of this ICT state has yet to be elucidated. In the present work, steady-state and ultrafast time-resolved optical spectroscopy have been performed on peridinin and three synthetic analogues, C(33)-peridinin, C(35)-peridinin, and C(39)-peridinin, which have different numbers of conjugated carbon-carbon double bonds. Otherwise, the molecules are structurally similar in that they possess the same functional groups. The trends in the positions of the steady-state and transient spectral profiles for this systematic series of molecules allow an assignment of the spectral features to transitions involving the S(0), S(1), S(2), and ICT states. A kinetics analysis reveals the lifetimes of the excited states and the dynamics of their excited state deactivation pathways. The most striking observation in the data is that the lifetime of the ICT state converges to the same value of 10.0 +/- 2.0 ps in the polar solvent, methanol, for all the peridinin analogues, regardless of the extent of pi-electron conjugation. This suggests that the ICT state is highly localized on the lactone ring, which is a common structural feature in all the molecules. The data further suggest that the S(1) and ICT states behave independently and that the ICT state is populated from both S(1) and S(2), the rate and efficiency from S(1) being dependent on the length of the pi-electron chain of the carotenoid and the solvent polarity.

Steady-state and ultrafast transient absorption spectra were obtained for a series of conformationally constrained, isomerically pure polyenes with 5-23 conjugated double bonds (N). These data and fluorescence spectra of the shorter polyenes reveal the N dependence of the energies of six (1)B(u)(+) and two (1)A(g)(-) excited states. The (1)B(u)(+) states converge to a common infinite polyene limit of 15,900 ± 100 cm(-1). The two excited (1)A(g)(-) states, however, exhibit a large (~9000 cm(-1)) energy difference in the infinite polyene limit, in contrast to the common value previously predicted by theory. EOM-CCSD ab initio and MNDO-PSDCI semiempirical MO theories account for the experimental transition energies and intensities. The complex, multistep dynamics of the 1(1)B(u)(+) → 2(1)A(g)(-) → 1(1)A(g)(-) excited state decay pathways as a function of N are compared with kinetic data from several natural and synthetic carotenoids. Distinctive transient absorption signals in the visible region, previously identified with S* states in carotenoids, also are observed for the longer polyenes. Analysis of the lifetimes of the 2(1)A(g)(-) states, using the energy gap law for nonradiative decay, reveals remarkable similarities in the N dependence of the 2(1)A(g)(-) decay kinetics of the carotenoid and polyene systems. These findings are important for understanding the mechanisms by which carotenoids carry out their roles as light-harvesting molecules and photoprotective agents in biological systems.

The spectroscopic properties and energy transfer dynamics of the protein-bound chlorophylls and xanthophylls in monomeric, major LHCII complexes, and minor Lhcb complexes from genetically altered Arabidopsis thaliana plants have been investigated using both steady-state and time-resolved absorption and fluorescence spectroscopic methods. The pigment-protein complexes that were studied contain Chl a, Chl b, and variable amounts of the xanthophylls, zeaxanthin (Z), violaxanthin (V), neoxanthin (N), and lutein (L). The complexes were derived from mutants of plants denoted npq1 (NVL), npq2lut2 (Z), aba4npq1lut2 (V), aba4npq1 (VL), npq1lut2 (NV), and npq2 (LZ). The data reveal specific singlet energy transfer routes and excited state spectra and dynamics that depend on the xanthophyll present in the complex.

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome - the light absorbing antenna complex - in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of Cba. tepidum grows slower and incorporates less BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays: (a) smaller cross-sectional radius and overall size; (b) simplified BChl c homologues with smaller side chains; (c) blue-shifted Qy absorption maxima and (d) a sigmoid-shaped circular dichroism (CD) spectra. Using a theoretical model we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis., 32 pages, 14 figures