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

Abstract

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 Qy bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Qy 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.

Fig. 1

Structure of the pigments in a monomeric subunit of the MFPCP complex. The coordinates were taken from Protein Data Bank with 1PPR code. Also shown are the structures of peridinin and the chlorophylls used in this study

Fig. 2

Absorption spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a. The spectra were taken at 10 K and were normalized at 500 nm

Fig. 3

Gaussian deconvolutions of the 10K absorption spectra in the Q_y region of purified Chls in 2-MTHF solvent. The fit (dashed line) is the sum of two Gaussian components built on a broad background. The data for the Gaussian deconvolution of the Chl a spectrum was taken from a previous study (Ilagan et al. 2004)

Fig. 4

Gaussian deconvolutions of the 10K absorption spectra in the Q_y region of PCP complexes reconstituted with peridinin and: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a

Fig. 5

Reconstruction of the 10K absorption spectra of the reconstituted PCP complexes in the 375–600nm region. The analysis was carried out by linearly combining individual 10 K absorption spectra of peridinin and Chl taken in 2-MTHF solvent as described in the text

Fig. 6

Fluorescence spectra of the PCP complexes reconstituted with peridinin and either Chl a, Chl b, Chl d, aChl a (3-acetyl-Chl a) or BChl a taken at 77 K. The spectra were normalized to their λ_max values

Fig. 7

Overlay of the room temperature fluorescence excitation (ex) and 1-T (where T is transmittance) spectra of reconstituted PCP complexes containing: (A) Chl a; (B) Chl b; (C) Chl d; (D) aChl a (3-acetyl-Chl a); and (E) BChl a. The intensity of each fluorescence excitation spectrum was normalized to correspond to the intensity of its corresponding 1-T spectrum in the Q_y region

Fig. 8

A view of the structure of the MFPCP complex focusing on the Ring I of Chl 601. Per 612, Per 614 and Leu 204 are in close proximity to the Ring I of Chl 601. The coordinates of the structure were taken from Protein Data Bank with 1PPR code

Fig. 9

Schematic diagram of energy levels and energy transfer pathways between peridinin and Chl in the PCP complexes. k_ET1 and k_ET2 (solid lines) are rate constants for energy transfer from the S₂ and S₁/ICT states of peridinin to Chl. k_IC2 and k_IC1 (dashed lines) are rate constants for internal conversion from S₂ to S₁/ICT and S₁/ICT to S₀, respectively