Direct determination of the S1 excited-state energies of xanthophylls by low-temperature fluorescence spectroscopy

The xanthophylls, violaxanthin, lutein, and zeaxanthin, associated with the antenna protein assembly of Photosystem II (PS II) play roles as light-harvesting pigments and protective agents in the photosynthetic apparatus of higher plants. The dissipation of excitation energy exceeding that needed for photosynthesis is thought to be regulated by an enzymatic process known as the xanthophyll cycle where violaxanthin and zeaxanthin are reversibly interconverted, but the role of the cycle in controlling the process in vivo is not clear. The two hypotheses are (i) direct quenching of chlorophyll excited states by the xanthophylls and (ii) indirect quenching via carotenoid-mediated changes in the structure of the light-harvesting complexes. These mechanisms depend on the structures and/or energetics of the xanthophyll pigments, which have not yet been fully elucidated. In this work, fluorescence spectroscopy at 77 K has been used to determine the energies of the S-1 excited states of violaxanthin, zeaxanthin, and the major xanthophyll component of green plants, lutein. High performance liquid chromatography (HPLC) was carried out just prior to the spectroscopic experiments to obtain isomerically pure samples devoid of fluorescent contaminants. The experiments at cryogenic temperatures provide enhanced resolution compared to room-temperature studies, reveal clearly the vibronic features of the fluorescence line shapes, and allow precise, direct assignments of the spectral origins and electronic-state energies of the molecules. The results are important for broadening our understanding of the mechanisms of light-harvesting and nonphotochemical dissipation of excess energy in plants.