Carotenoid Structures and Environments in Trimeric and Oligomeric Fucoxanthin Chlorophyll a/c₂ Proteins from Resonance Raman Spectroscopy

Resonance Raman (RR) spectroscopy is used to characterize the structures and environments of the carotenoid fucoxanthin (Fx), the primary light harvester in the membrane-intrinsic fucoxanthin chlorophyll a/c₂ proteins (FCP) from the diatom Cyclotella meneghiniana, thereby building on the findings from Stark spectroscopy and calculations (J. Phys. Chem. B 2008, 112 (37), 11838−11853). Solvent-dependent effects on the RR bands of isolated Fx and the xanthophyll-cycle carotenoid, diadinoxanthin (Ddx), are studied to better characterize the protein-specific environmental factors that affect their geometry and spectral signatures. In addition, excitation-wavelength-dependent (441.6−570 nm) changes in the RR bands of the ν₁ and ν₃ modes, as well as the conjugated C8 carbonyl stretch, allow the identification of 5–6 in both the trimeric (FCPa_trim) and oligomeric (FCPb_olig) forms of FCP. These Fx’s are broadly classified into two each of high (Fx_blue) and low (Fx_red) energy, and 1–2 of intermediate (Fx_green) energy that are allied to their location and function in the protein. The CC stretching frequencies (ν₁), which indicate conjugation over at least 7 double bonds, and the low intensity of the ν₄ C−H bending modes attest to their planar all-trans conformations both in the protein and in solution, with the protein-bound Fx_red’s exhibiting signs of nonlinearity. Additionally, red-edge excitation of Fx in solution, and in the FCPs, exhibits the effect of mixing between the two lowest-energy, 2¹A_g^*−-like and 1¹B_u^*+-like, excited states, which underpins the high light-harvesting and energy-transfer efficiency of the Fx_red’s. RR spectra also reveal differences between FCPa_trim and FCPb_olig complexes, such as the greater prevalence of Ddx in FCPb_olig. Importantly, the identification of 5–6 Fx’s per FCP monomer suggests that there may be more than the four Fx’s previously assumed per FCP monomer, or else there is definite heterogeneity in Fx structures and/or binding sites.