The beta-carotene radical cation and deprotonated neutral radicals were studied at the density functional theory (DFT) level using different density functionals and basis sets: B3LYP/3-21G, SVWN5/6-31G*, BPW91/DGDZVP2, and B3LYP/6-31G**. The geometries, total energies, spin distributions, and isotropic and anisotropic hyperfine coupling constants of these species were calculated. Deprotonation of the methyl group at the double bond of the cyclohexene ring of the carotenoid radical cation at 5 or 5' produces the most stable neutral radical because of retention of the pi-conjugated system while less stable deprotonation at 9 or 9' and 13 or 13' of the chain methyl groups causes significant distortion of the conjugation. The predicted methyl hyperfine coupling constants of 13-16 MHz of the neutral radicals are in good agreement with the previous electron nuclear double resonance (ENDOR) spectrum of photolyzed beta-carotene on a solid support. DFT calculations on the beta-carotene radical cation in a polar water environment showed that the polar environment does not cause significant changes in the proton hyperfine constants from those in the isolated gas-phase molecule. DFT calculated methyl proton hyperfine coupling constants of less than 7.2 MHz are in agreement with those reported for the radical cation in photosystem II (PS II) and those found in the absence of UV light for the radical cation on a silica alumina matrix.

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 beta-methyl proton hyperfine couplings to be 8 to 10 MHz for Car*+, in agreement with previously reported hyperfine couplings for carotenoid pi-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic alpha-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.

The carotenoid astaxanthin forms novel metal ion complexes with Ca2+, Zn2+ and Fe2+. MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca2+ and Zn2+ at low salt concentrations < 0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca2+ and by a factor of 7 for Zn2+ as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (> 0.2 mM) of salt 2:1 metal:astaxanthin complexes were formed in ethanol. In the presence of Ca2+ and Zn2+ salts the lifetime of the radical cation and dication formed electrochemically decreased relative to that of the carotenoid neutral radical. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical while proton loss at the C5, C9 or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41 or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13-methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.