Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures

Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment–protein interactions responsible for the positions of the Q(y) bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Q(y) and Soret transition energies. Fluorescence excitation spectra were used to compute energy transfer efficiencies of the various complexes at room temperature and these were correlated with previously reported ultrafast, time-resolved optical spectroscopic dynamics data. The results illustrate the robust nature and value of the PCP complex, which maintains a high efficiency of antenna function even in the presence of non-native chlorophyll species, as an effective tool for elucidating the molecular details of photosynthetic light-harvesting

Spectroscopic properties of carotenoids in red and blue hues

Photosynthesis is a process by which plants, algae, and photosynthetic bacteria convert light into chemical energy. Light-harvesting is accomplished by antenna pigment-protein complexes absorbing light and transferring the excitation energy to reaction center pigment-protein complexes where charge separation occurs. The two classes of pigments involved in light-harvesting are chlorophylls and carotenoids. Peridinin-chlorophyll a-protein (PCP) from the dinoflagellate, Amphidinium carterae is valuable system for studying light-harvesting because its structure has been resolved by X-ray crystallography. In this thesis, two variants of the PCP complex, main-form (MFPCP) and high-salt (HSPCP), have been investigated using steady-state and ultrafast transient optical spectroscopy at room and cryogenic temperatures. The systematic difference in the structures and pigment compositions of the MFPCP and HSPCP allows exploration of the factors that control energy transfer. Both the MFPCP and HSPCP complexes exhibit very high efficiency (\u3e 95%) of peridinin-to-chlorophyll energy transfer. Spectroscopic studies of apo-PCP complexes reconstituted with peridinin and either chlorophyll a, chlorophyll b, chlorophyll d, 3-acetyl chlorophyll a and bacteriochlorophyll a were also carried out and found to exhibit very high energy transfer efficiencies. This further reveals the robust nature of the PCP complex; i.e. it can maintain a high energy transfer efficiency despite profound alterations in pigment composition. The factors controlling peridinin-to-chlorophyll energy transfer are the spectral overlap, distance between these pigments, and pigment-pigment and pigment-protein interactions. In this thesis, all of these factors are evaluated systematically. ^ In a complementary study, α-crustacyanin was investigated using steady-state and femtosecond transient absorption spectroscopy. α-crustacyanin is composed of eight β-crustacyanin and only binds the carotenoid, astaxanthin, which undergoes a large bathochromic spectral shift in the protein. The reason for the shift is described in this work. This system provides a means of understanding the intrinsic behavior of carotenoids in a protein environment in the absence of interactions with chlorophyls. Because the X-ray crystal structure of β-crustacyanin has been reported previously, this complex allows exploration of the relationship between structure, spectroscopic observables, and functions of protein-bound carotenoids.

Femtosecond time-resolved absorption spectoscopy of main-form and high-salt peridinin-chlorophyll a-proteins at low temperatures

Steady-state and femtosecond time-resolved optical methods have been used to compare the spectroscopic features and energy transfer dynamics of two systematically different light-harvesting complexes from the dinoflagellate 'Amphidinium carterae': main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a-proteins. Pigment analysis and X-ray diffraction structure determinations [Hofmann, E., Wrench, P. M., Sharples, F. P., Hiller, R. G., Welte, W., Diederichs, K. (1996) Science 272, 1788-1791; T. Schulte, F. P. Sharples, R. G. Hiller, and E. Hofmann, unpublished results] have revealed the composition and geometric arrangements of the protein-bound chromophores. The MFPCP contains eight peridinins and two chlorophyll (Chl) a, whereas the HSPCP has six peridinins and two Chl a, but both have very similar pigment orientations. Analysis of the absorption spectra has shown that the peridinins and Chls absorb at different wavelengths in the two complexes. Also, in the HSPCP complex, the Qy transitions of the Chls are split into two well-resolved bands. Quantum computations by modified neglect of differential overlap with partial single and double configuration interaction (MNDO-PSDCI) methods have revealed that charged amino acid residues within 8 Å of the pigment molecules are responsible for the observed spectral shifts. Femtosecond time-resolved optical spectroscopic kinetic data from both complexes show ultrafast (<130 fs) and slower (~2 ps) pathways for energy transfer from the peridinin excited singlet states to Chl. The Chl-to-Chl energy transfer rate constant for both complexes was measured and is discussed in terms of the Förster mechanism. It was found that, upon direct Chl excitation, the Chl-to-Chl energy transfer rate constant for MFPCP was a factor of 4.2 larger than for HSPCP. It is suggested that this difference arises from a combination of factors including distance between Chls, spectral overlap, and the presence of two additional peridinins in MFPCP that act as polarizable units enhancing the rate of Chl-to-Chl energy transfer. The study has revealed specific pigment-protein interactions that control the spectroscopic features and energy transfer dynamics of these light-harvesting complexes.12 page(s