Pigment organisation in the membrane-intrinsic major light-harvesting complex of Amphidinium carterae: structural characterisation of the peridinins and chlorophylls a and c₂ by resonance Raman spectroscopy and from sequence analysis

The structures and environments of the protein-bound peridinins (Pers) and chlorophylls (Chls) a/c₂ in the membrane-intrinsic major light-harvesting complex of the dinoflagellate Amphidinium carterae (LHC_Amph) are characterised using resonance Raman (RR) spectroscopy with 11 excitation wavelengths, at 77 K. The excitation-dependent variation in the CC stretching mode (ν₁) suggests the presence of three Pers with conjugation lengths over 8 double bonds (dBs), and one diadinoxanthin, between 413.7 and 528.7 nm. Two Per_red species are revealed on excitation at 550 and 560 nm. These Per_red species exhibit anomalously low ν₁ values, together with notable resonant enhancement of lactone ring-breathing and -deformation modes. To discern protein-specific effects, the RR spectra are compared to that of Per in polar (acetonitrile), polarisable (toluene) and polar-protic (ethanol) solvents. Resonantly enhanced lactone, ring-breathing (942 cm^{- 1}) and ring-deformation (~ 650 cm^{- 1}), modes are identified both in solution, and in the protein, and discussed in the context of the mixing of the S₁ and S₂ states, and formation of the intramolecular charge-transfer (ICT) state. In the Chl-absorbing region, two sets of Chl c₂'s, and (at least) six Chl a's can be differentiated. With a pigment ratio of 5-6 (Chl a):2 (Chl c₂):5-6 (Per):1 Ddx determined from the fit to the RT absorption and 77 K RR spectra, sequence comparison of LHC_Amp to LHCII, and the diatom LHC, fucoxanthin-chlorophyll-a/c-protein (FCP), a template for the conserved pigment binding sites is proposed, to fill the paucity of structural information in the absence of a crystal structure for LHC_Amph.