No abstract

a-Crustacyanin, the 320 kDa astaxanthin-protein from the carapace of the lobster, Homarus gammarus, is the best known of the blue-purple carotenoproteins found in marine invertebrate animals. Reconstituted a-crustacyanin complexes have been prepared from a range of natural and synthetic carotenoids. Only normal C~O carotenoids in the all-E configuration fit into the binding site, though some variation in the ring size, shape and methylation pattern is tolerated. The C(20) and C(20') methyl groups must be present; presumably these are involved in essential steric interactions. The main structural requirement is the presence of keto groups at C(4) and C(4'); these must be conjugated with the main polyene chain. Circular dichroism shows that the carotenoid chromophore experiences a chiral twist, but this is not a major factor in the spectral shift, and that the two astaxanthin molecules in the P-crustacyanin dimer are close together and show some interaction in the excited state. Resonance Raman and NMR spectroscopy of complexes containing I3C-labelled astaxanthins shows that the blue colour can be attributed to perturbation of the ground-state electronic structure of the carotenoid, caused by polarization of the chromophore. The results are consistent with protonation of the C(4) and C(4') keto groups, but the magnitude of the polarization effect is not the same in the two halves of the molecule.

No abstract

Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation.