The mechanism of the colour shift of astaxanthin in alpha-crustacyanin as investigated by C-13 MAS NMR and specific isotope enrichment

By selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in alpha-crustacyanin, the major blue astaxanthin binding carotenoprotein complex from the carapace of the lobster Homarus gammarus, have been analysed. C-13 Magic Angle Spinning (MAS) NMR spectra were obtained after reconstitution with astaxanthins labelled in the centre of the molecule or at the two keto groups. The MAS data reveal electrostatic polarizations of the conjugated chain. In addition, solid state NMR results for pure unlabelled astaxanthin can be compared with natural abundance C-13 MAS data for canthaxanthin and beta-carotene, to address the effect of the ring functionalities on the electronic properties of the polyene chain. Quantum chemical calculations were performed to reconcile the MAS data with one of several simple and straightforward mechanisms for the colour shift. The results point towards a colour shift mechanism in which the astaxanthin may be doubly charged, possibly by a double protonation of the two ring keto groups.