Pulsed EPR and DFT Characterization of Radicals Produced by Photo-Oxidation of Zeaxanthin and Violaxanthin on Silica−Alumina

Abstract

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica−alumina produces the carotenoid radical cations (Car^•+) and also the neutral carotenoid radicals (#Car^•) as a result of proton loss (indicated by #) from the C4(4‘) methylene position or one of the methyl groups at position C5(5‘), C9(9‘), or C13(13‘), except for violaxanthin where the epoxide at positions C5(5‘)−C6(6‘) raises the energy barrier for proton loss, and the neutral radicals #Car^•(4) and #Car^•(5) are not observed. DFT calculations predict the largest isotropic β-methyl proton hyperfine couplings to be 8 to 10 MHz for Car^•+, in agreement with previously reported hyperfine couplings for carotenoid π-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic α-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.