Ronald Gebhard

Low-lying electronic states of carotenoids

Four all-trans carotenoids, spheroidene, 3,4-dihydrospheroidene, 3,4,5,6-tetrahydrospheroidene, and 3,4,7,8-tetrahydrospheroidene, have been purified using HPLC techniques and analyzed using absorption, fluorescence and fluorescence excitation spectroscopy of room temperature solutions. This series of molecules, for which the extent of pi-electron conjugation decreases from 10 to seven carbon-carbon double bonds, exhibits a systematic crossover from S2----S0 (1(1)Bu----1(1)Ag) to S1----S0 (2(1)Ag----1(1)Ag) emission with decreasing chain length. Extrapolation of the S1----S0 transition energies indicates that the 2(1)Ag states of longer carotenoids have considerably lower energies than previously thought. The energies of the S1 states of spheroidenes and other long carotenoids are correlated with the S1 energies of their chlorophyll partners in antenna complexes of photosynthetic systems. Implications for energy transfer in photosynthetic antenna are discussed.

MAGIC ANGLE SPINNING NMR STUDIES ON THE METARHODOPSIN II INTERMEDIATE OF BOVINE RHODOPSIN: EVIDENCE FOR AN UNPROTONATED SCHIFF BASE

Abstract— Magic angle spinning (MAS)W‐NMR spectra of the metarhodopsin II intermediate h been obtained using bovine rhodopsin regenerated with retinal 13C‐labeled at the C‐13 and C positions to investigate the protonation state of the retinal Schiff base linkage. The 13C‐labe rhodopsin was reconstituted into 1,2‐dipalmitoleoylphosphatidylcholine bilayers to increase the amo of meta II trapped at low temperature. Both the 13C‐15 (159.2 ppm) and 13C‐13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the “C‐15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70–90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules the possibility that meta II non‐covalently binds retinal or is a carbinolamine intermediate and provi convincing evidence that Schiff base deprotonation occurs in the meta I‐meta II transition, an ev that is likely to be important in triggering the activation of transducin.

The dynamics of the S1 excited states of carotenoids

Abstract Transient absorption spectroscopy was used to measure the S 1 dynamics of four all-trans-carotenoids: 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene and spheroidene. After excitation into the S 2 state and subsequent relaxation to the S 1 state, the S 1 → S n electronic absorption bands in the region 460–520 nm were probed and observed to decay with single-exponential kinetics: τ = 407 ± 23 ps for 3,4,7,8-tetrahydrospheroidene, 85 ± 5 ps for 3,4,5,6-tetrahydrospheroidene, 25.4 ± 0.9 ps for 3,4-dihydrospheroidene and 8.7 ± 0.1 ps for spheroidene. These data are discussed in three contexts: (1) as a test of the adherence of carotenoids to the energy gap law; (2) as a means of determining the S 1 state energy of spheroidene, which appears to be at 14100 cm −1 ; and (3) in terms of the constraints placed on the efficiencies of carotenoid-to-chlorophyll energy transfer in photosynthetic systems.

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 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.

Carotenoid-to-bacteriochlorophyll singlet energy transfer in carotenoid-incorporated B850 light-harvesting complexes of Rhodobacter sphaeroides R-26.1.

Four carotenoids, 3,4,7,8‐tetrahydrospheroidene, 3,4,5,6‐tetrahydrospheroidene, 3,4‐dihydrospheroidene and spheroidene, have been incorporated into the B850 light‐harvesting complex of the carotenoidless mutant, photosynthetic bacterium, Rhodobacter sphaeroides R‐26.1. The extent of π‐electron conjugation in these molecules increases from 7 to 10 carbon‐carbon double bonds. Carotenoid‐to‐bacteriochlorophyll singlet state energy transfer efficiencies were measured using steady‐state fluorescence excitation spectroscopy to be 54 ± 2%, 66 ± 4%, 71 ± 6% and 56 ± 3% for the carotenoid series. These results are discussed with respect to the position of the energy levels and the magnitude of spectral overlap between the S, (2′AJ state emission from the isolated carotenoids and the bacteriochlorophyll absorption of the native complex. These studies provide a systematic approach to exploring the effect of excited state energies, spectral overlap and excited state lifetimes on the efficiencies of carotenoid‐to‐bacteriochlorophyll singlet energy transfer in photosynthetic systems.