Veeradej Chynwat

Photophysics of the carotenoids associated with the xanthophyll cycle in photosynthesis

Green plants use the xanthophyll cycle to regulate the flow of energy to chlorophylla within photosynthetic proteins. Under conditions of low light intensity violaxanthin, a carotenoid possessing nine conjugated double bonds, functions as an antenna pigment by transferring energy from its lowest excited singlet state to that of chlorophylla within light-harvesting proteins. When the light intensity increases, violaxanthin is biochemically transformed into zeaxanthin, a carotenoid that possesses eleven conjugated double bonds. The results presented here show that extension of the ⧄ conjugation of the polyene lowers the energy of the lowest excited singlet state of the carotenoid below that of chlorophylla. As a consequence zeaxanthin can act as a trap for the excess excitation energy on chlorophylla pigments within the protein, thus regulating the flow of energy within photosynthetic light-harvesting proteins.

The application of the energy gap law to the S1 energies and dynamics of carotenoids

Abstract The energy gap law for radiationless transitions set forth by Englman and Jortner (Mol. Phys. 18 (1970) 145) has been evaluated for use in deducing the S 1 energies of carotenoids. A simultaneous knowledge of the dynamics and energies of the S 1 states of carotenoids are available for only a few of these molecules owing to the lack of detectable S 1 state fluorescence in many cases. All the available data where the S 1 dynamics and energies of carotenoids are simultaneously known were fit by the energy gap law expression in its full exponential form. The parameters derived from the computer optimization suggest that the weak coupling limit form of the energy gap law is valid for describing the relationship between the dynamics and energetics of carotenoid molecules. The optimal fitting parameters were then used in conjunction with the energy gap law expression to calculate the S 1 energies of several biologically important carotenoids from the lifetimes of their S 1 states. Particularly important is the S 1 energy of β-carotene determined in this analysis to be 14 100 cm −1 .

On the photophysics and photochemical properties of carotenoids and their role as light-harvesting pigments in photosynthesis

In photosynthetic organisms, carotenoids have been implicated in several diverse roles. Yet, owing to profound technical difficulties encountered in attempting to examine the electronic state energies and dynamics of carotenoids both in vitro and in vivo, several questions remain, and much of the data and interpretations of the results are controversial. This paper will discuss some of these questions and controversies, the resolution of which is important in unraveling the manner in which carotenoids function in photosynthetic systems.

The lifetimes and energies of the first excited singlet states of diadinoxanthin and diatoxanthin: the role of these molecules in excess energy dissipation in algae

The lifetimes of the first excited singlet states (2(1)A(g)) of diadinoxanthin and diatoxanthin, carotenoids involved in the xanthophyll cycle in some genera of algae, have been measured by femtosecond time-resolved optical spectroscopy to be 22.8 +/- 0.1 ps and 13.3 +/- 0.1 ps, respectively. Using the energy gap law for radiationless transitions set forth by Englman and Jortner (Mol. Phys. 18 (1970) 145-164), these lifetimes correspond to S1 excited state energies of 15210 cm-1 for diadinoxanthin and 14620 cm-1 for diatoxanthin. The lowest excited singlet state energy of Chl a has an energy of 14700 cm-1. The fact that the S1 state energy of diadinoxanthin lies above that of Chl a, whereas the S1 state energy of diatoxanthin lies below that of Chl a, suggests that the xanthophyll cycle involving the enzymatic interconversion of diadinoxanthin and diatoxanthin may play a role in regulating energy flow between these molecules and Chl a in many species of algae, essentially fulfilling a role identical to that proposed for violaxanthin and zeaxanthin in higher plants and green algae (Frank et al. (1994) Photosyn. Res. 41, 389-395).

Carotenoid triplet state formation in Rhodobacter sphaeroides R-26 reaction centers exchanged with modified bacteriochlorophyll pigments and reconstituted with spheroidene.

Triplet state electron paramagnetic resonance (EPR) experiments have been carried out at X-band on Rb. sphaeroides R-26 reaction centers that have been reconstituted with the carotenoid, spheroidene, and exchanged with 132-OH-Zn-bacteriochlorophyll a and [3-vinyl]-132-OH-bacteriochlorophyll a at the monomeric, ‘accessory’ bacteriochlorophyll sites BA,B or with pheophytin a at the bacteriopheophytin sites HA,B. The primary donor and carotenoid triplet state EPR signals in the temperature range 95–150 K are compared and contrasted with those from native Rb. sphaeroides wild type and Rb. sphaeroides R-26 reaction centers reconstituted with spheroidene. The temperature dependencies of the EPR signals are strikingly different for the various samples. The data prove that triplet energy transfer from the primary donor to the carotenoid is mediated by the monomeric, BChlB molecule. Furthermore, the data show that triplet energy transfer from the primary donor to the carotenoid is an activated process, the efficiency of which correlates with the estimated triplet state energies of the modified pigments.

The spectroscopic and photochemical properties of locked-15,15′-cis-spheroidene in solution and incorporated into the reaction center of Rhodobacter sphaeroides R-26.1

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15′-cis-spheroidene, were studied by absorption, fluorescence, circular dichroism, fast transient absorption and electron spin resonance spectroscopies in solution and after incorporation into the reaction center of Rhodobacter (Rb.) sphaeroides R-26.1. HPLC purification of the synthetic molecule reveals the presence of several di-cis geometric isomers in addition to the mono-cis isomer of locked-15,15′-cis-spheroidene. In solution, the absorption spectrum of the purified mono-cis sample was red-shifted and showed a large cis-peak at 351 nm compared to unlocked all-trans spheroidene. Molecular modeling and semi-empirical calculations reveal how geometric isomerization and structural factors affect the room temperature spectra. The spectroscopic studies of the purified locked-15,15′-mono-cis molecule in solution reveal a more stable manifold of excited states compared to the unlocked spheroidene. Reaction centers of Rb. sphaeroides R-26.1 in which the locked-15,15′-cis-spheroidene was incorporated show no difference in either the spectroscopic properties or photochemistry compared to reaction centers in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 reaction centers which naturally contain spheroidene. The data suggest that the natural selection of a cis-isomer of spheroidene for incorporation into native reaction centers of Rb. sphaeroides wild type strain 2.4.1 is more determined by the structure or assembly of the reaction center protein than by any special quality of the cis-isomer of the carotenoid that would affect its ability to participate in triplet energy transfer or carry out photoprotection.

Triplet Energy Transfer between the Primary Donor and Carotenoids in Rhodobacter sphaeroides R-26.1 Reaction Centers Incorporated with Spheroidene Analogs Having Different Extents of π-Electron Conjugation

Abstract— Three carotenoids, spheroidene, 3,4‐dihydrospheroidene and 3,4,5,6‐tetrahydrospheroidene, having 8, 9 and 10 conjugated carbon‐carbon double bonds, respectively, were incorporated into Rhodobacter (Rb.) sphaeroides R‐26.1 reaction centers. The extents of binding were found to be 95±5% for spheroidene, 65±5% for 3,4‐dihydrospheroidene and 60±10% for 3,4,5,6‐tetrahydrospheroidene. The dynamics of the triplet states of the primary donor and carotenoid were measured at room temperature by flash absorption spectroscopy. The carotenoid, spheroidene, was observed to quench the primary donor triplet state. The triplet state of spheroidene that was formed subsequently decayed to the ground state with a lifetime of 7.0±0.5 μs. The primary donor triplet lifetime in the Rb. sphaeroides R‐26.1 reaction centers lacking carotenoids was 60±5 μs. Quenching of the primary donor triplet state by the carotenoid was not observed in the Rb. sphaeroides R‐26.1 reaction centers containing 3,4‐dihydrospheroidene nor in the R‐26.1 reaction centers containing 3,4,5,6‐tetrahydrospheroidene. Triplet‐state electron paramagnetic resonance was also carried out on the samples. The experiments revealed carotenoid triple‐state signals in the Rb. sphaeroides R‐26.1 reaction centers incorporated with spheroidene, indicating that the primary donor triplet is quenched by the carotenoid. No carotenoid signals were observed from Rb. sphaeroides R‐26.1 reaction centers incorporating 3,4‐dihydrospheroidene nor in reaction centers incorporating 3,4,5,6‐tetrahydrospheroidene. Circular dichroism, steady‐state absorbance band shifts accompanying the primary photochemistry in the reaction center and singlet energy transfer from the carotenoid to the primary donor confirm that the carotenoids are bound in the reaction centers and interacting with the primary donor. These studies provide a systematic approach to exploring the effects of carotenoid structure and excited state energy on triplet transfer between the primary donor and carotenoids in reaction centers from photosynthetic bacteria.

Triplet state energy transfer between the primary donor and the carotenoid in Rhodobacter sphaeroides R-26.1 reaction centers exchanged with modified bacteriochlorophyll pigments and reconstituted with spheroidene.

Abstract— The dynamics of triplet energy transfer between the primary donor and the carotenoid were measured on several photosynthetic bacterial reaction center preparations from Rhodobacter sphaeroides: (a) wild‐type strain 2.4.1, (b) strain R‐26.1, (c) strain R‐26.1 exchanged with 132‐hy‐droxy‐[Zn]‐bacteriochlorophyll at the accessory bacteriochlorophyll (BChl) sites and reconstituted with spheroidene and (d) strain R‐26.1 exchanged with P‐vinyl]‐132‐hydroxy‐bacteriochlorophyll at the accessory BChl sites and reconstituted with spheroidene. The rise and decay times of the primary donor and carotenoid triplet‐triplet absorption signals were monitored in the visible wavelength region between 538 and 555 run as a function of temperature from 4 to 300 K. For the samples containing carotenoids, all of the decay times correspond well to the previously observed times for spheroidene (5 ± 2 us). The rise times of the carotenoid triplets were found in all cases to be biexponen‐tial and comprised of a strongly temperature‐dependent component and a temperature‐independent component. From a comparison of the behavior of the carotenoid‐con‐taining samples with that from the reaction center of the carotenoidless mutant Rb. sphaeroides R‐26.1, the temperature‐independent component has been assigned to the buildup of the primary donor triplet state resulting from charge recombination in the reaction center. Arrhenius plots of the buildup of the carotenoid triplet states were used to determine the activation energies for triplet energy transfer from the primary donor to the carotenoid. A model for the process of triplet energy transfer that is consistent with the data suggests that the activation barrier is strongly dependent on the triplet state energy of the accessory BChl pigment, BChlB.

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 μs for the B800-850 complex, 14±2 μs for the B850 complex incorporated with spheroidene, and 19±2 μs for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 μs. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.

Protein Modifications Affecting Triplet Energy Transfer in Bacterial Photosynthetic Reaction Centers

The efficiency of triplet energy transfer from the special pair (P) to the carotenoid (C) in photosynthetic reaction centers (RCs) from a large family of mutant strains has been investigated. The mutants carry substitutions at positions L181 and/or M208 near chlorophyll-based cofactors on the inactive and active sides of the complex, respectively. Light-modulated electron paramagnetic resonance at 10 K, where triplet energy transfer is thermally prohibited, reveals that the mutations do not perturb the electronic distribution of P. At temperatures > or = 70 K, we observe reduced signals from the carotenoid in most of the RCs with L181 substitutions. In particular, triplet transfer efficiency is reduced in all RCs in which a lysine at L181 donates a sixth ligand to the monomeric bacteriochlorophyll B(B). Replacement of the native Tyr at M208 on the active side of the complex with several polar residues increased transfer efficiency. The difference in the efficiencies of transfer in the RCs demonstrates the ability of the protein environment to influence the electronic overlap of the chromophores and thus the thermal barrier for triplet energy transfer.