ULTRAFAST ENERGY TRANSFER FROM RHODOPIN ... - Springer Link

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Alisdair N. Macphersonl , Juan B. Arellanol ,2, Niall J. Frase~,. Richard J. Cogdell2 and Tomas Gillbrol. IDepartmeilt of Physical Chemistry, University ofUmea, ...
ULTRAFAST ENERGY TRANSFER FROM RHODOPIN GLUCOSIDE IN THE LIGHT HARVESTING COMPLEXES OF RPS. ACIDOPHlLA. Alisdair N. Macpherson l , Juan B. Arellano l ,2, Niall J. Frase~, Richard J. Cogdell2 and Tomas Gillbro l IDepartmeilt of Physical Chemistry, University ofUmea, S-901 87 Umea, Sweden, 2Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow, G12 8QQ, U.K.

Key words: carotenoids, excited state dynamics, fluorescence lifetimes, LH2, LH1, ultrafast spectroscopy. 1. Introduction

The light-harvesting antenna of purple bacteria are ideal complexes in which to study the energy transfer function of carotenoids. Light-harvesting complexes which accumulate a single type of carotenoid can be readily isolated and the carotenoid can be selectively excited in the blue-green spectral region. Furthermore, nature assembles a variety of purple bacteria antenna complexes, with a range of energy transfer efficiencies, by packing bacteriochlorophylls into spectroscopically distinct ring-like structures in close proximity to a range of carotenoids with different conjugation lengths (l). The details of the singlet-singlet energy transfer mechanism and how factors such as energy levels, lifetimes and geometrical parameters (distances and orientations) affect the efficiency of energy transfer are not clearly understood. Complicating matters is that carotenoids have two low-lying excited singlet states, the strongly-allowed S2 (Bu+) state and the lower-lying SI (2Ag-) state, from which energy transfer could occur (2,3). To determine the involvement of each of these states in light-harvesting, we have measured the kinetics of the excited singlet states of the carotenoids both in vivo and in organic solvents by ultrafast transient absorption and fluorescence upconversion techniques.

2. Material and Methods The LH2 and LHIIRC complexes of Rhodopseudomonas acidophila (strain 10050) were isolated by sucrose density centrifugation following solubilisation of the membranes with 2% LDAO. Measurements were made in 20 mM Tris, pH 8.0 containing 0.1 % LDAO and 25mM ascorbate was added for the LH1IRC complex. The B850 complex was prepared by incubating the native LH2 with 1% TBG-lO detergent at pH 4.7 and the dissociated B800 bacteriochlorophylls were removeq by column chromatography prior to detergent exchange (N. J Fraser, R. J. Cogdell, B. Ucker and H. Scheer, unpublished). Rhodopin glucoside was extracted from whole cells and was purified by stepwise elution from a silica gel column followed by thin layer chromatography. However, we could not obtain rhodopin glucoside that was sufficiently pure for steady-state fluorescence 9 G. Garab (ed.), Photosynthesis: Mechanisms and Effects, Vol. I, 9-14. © 1998 Kluwer Academic Publishers.

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emission measurements by this procedure. A very simple method was discovered to rapidly obtain this highly polar carotenoid with a high (fluorescence) purity. Methanol was added to LH2 to a concentration of 85%, the solution was loaded into a syringe and filtered through a 0.2 J.Lm PTFE syringe filter (Millex-FGS), trapping the carotenoid. After repeated washing with 85% methanol, the carotenoid was liberated with acetone and dried under nitrogen. A fluorescence upconversion spectrometer with a ca. 170 fs response function (4) was used to accurately determine both the decay kinetics of the S2 state of the carotenoids and the rise kinetics of the bacteriochlorophyll emission. The carotenoid was excited at 82 MHz in the 490-500 nm region by frequency doubling the ca. 60 fs IR pulses produced by a Ti:sapphire laser. The lifetime of the SI state of the carotenoid was determined near the excited state absorption maximum, with excitation pulses at 485490 nm from a regeneratively amplified 5 kHz Ti:sapphire / OPA laser system and white light probe pulses. The instrumental response function was ca. 180 fs (FWHM).

3. Results and Discussion The absorption spectra of the native LH2 (B800-850) and the B850 (B800-free) complexes from Rps. acidophUa (strain 10050) normalised in the carotenoid absorption region are shown in Figure 1. Also shown is the absorption spectrum of rhodopin glucoside dissolved in benzyl alcohol. Detergent treatment and removal of the B800 band results in a 4 nm blue shift of the carotenoid maxima and in some loss of fine structure. Benzyl alcohol was the solvent that had absorption maxima and a fine structure ratio closest (ca. 3 nm shifted to the red) to the LH2 protein. It is therefore used in this study as a reference system (for the protein environment around the carotenoid) in which no energy transfer occurs. 0.2

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Figure 1. Absorption (normalised) and corrected fluorescence emission spectra of rhodopin glucoside in the B850 (dashed) and LH2 (solid) protein complexes of Rps. acidophila and benzyl alcohol (dotted). The fluorescence excitation wavelength was 485 nm and the background Raman signal was subtracted. The intensity scale of the emission in benzyl alcohol is not the same as for the two protein complexes.

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An attempt was made to observe fluorescence emission from the S2 state of rhodopin glucoside in the protein complexes. Extremely weak emission bands were observed peaking close to 570 nm (Figure 1), in addition to interfering emission from strongly fluorescent bacteriochlorophyll breakdown products (5). The emission was stronger for the B850 complex, consistent with the longer lifetime of the carotenoid S2 state in this complex (see below). Emission was also found in the same region for rhodopin glucoside in benzyl alcohol (Figure 1) and from the B800-820 complex of Rps. acidophila (strain 7750) (6), but the bandwidth is notably larger. The time resolved fluorescence decay kinetics of the S2 state of rhodopin glucoside in benzyl alcohol, LH2 and the B850 complex in this wavelength region are shown in Figure 2. It is clear that the S2lifetime is significantly shorter in the native LH2 (61 fs, 98.9%) and B850 (76 fs, 99.5%) 1.0 protein complexes than in benzyl Similar alcohol (132 fs, 99.4%). lifetimes have been reported for the 0.8 LHI and LH2 complexes of Rb. ~ Ul sphaeroides 2.4.1 (7) and for the c: 0.6 of Rps. B800-820 complex ~ acidophila (strain 7750) (6). The S2 il 0.4 lifetime of carotenoids depends upon ~ Ul 0.2 both the polarity and the polarisability of the environment (4) and may also be affected by distortions from planarity. For rhodopin glucoside, 0.75 -0.25 0.00 0.25 0.50 the dependence of the S2 lifetime on time (ps) solvent is actually not very large and the S2 lifetime is shortest in solvents with a low refractive index (ca. 105 fs Figure 2. Decay kinetics of the rhodopin glucoside S2 state emission excited at in ethanol). This behaviour is opposite to that reported for fJ489 nm and measured with parallel polarisation at 578 nm in benzyl alcohol, carotene and spheroidene (4,7) and indicates that the change in energy B850 and LH2. A gaussian response gap between the S2 and S I states with function of 170 fs is also shown. solvent is not the only parameter affecting the lifetime. .-----,r=---,-----r--~

Having established that the lifetime of the S2 state of rhodopin glucoside is shorter in the protein complexes than in simple solvents, we determined if this quenching was at least partly the result of energy transfer, by monitoring the isotropic rise kinetics of the bacteriochlorophyll emission following excitation of the carotenoid. For the B850 (B800-free) complex, only a single ultrafast rise component was observed in the range 795-900 nm. For the native LH2, the formation of the B850 emission at 872 nm is clearly biexponential (Figure 3). The dominant fast component has an identical lifetime in both complexes and can be attributed to energy transfer from the S2 state of the carotenoid. The slower rise component (820 fs) observed in LH2 is similar to the B800 to B850 transfer time (8). We cannot rule out the possibility of there being an additional slower rise component with a small amplitude. This could be masked by the ca. 8 ps decay component, determined from longer time-range scans, resulting from singlettriplet annhilation (9).

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