Weesie R J, Merlin J C, de Groot H J, Britton G, Lugtenburg J, Jansen F J, Cornard J P
Laboratoire de Spectrochimie Infrarouge et Raman, CNRS UMR 8516, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Biospectroscopy. 1999;5(6):358-70. doi: 10.1002/(SICI)1520-6343(1999)5:6<358::AID-BSPY5>3.0.CO;2-1.
Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of alpha-crustacyanin reconstituted with specifically 13C-labeled astaxanthins at the positions 15, 15,15', 14,14', 13,13', 12,12', or 20,20' were recorded. This approach enabled us to obtain information about the effect of the ligand-protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the C==C stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250-1400 cm(-1) spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in alpha-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the pi-antibonding C==O orbitals and the HOMO-LUMO gap.
共振拉曼光谱和量子化学计算被用于研究虾青素在α-甲壳蓝蛋白（来自龙虾螯虾外壳的主要蓝色类胡萝卜素蛋白）中的电子吸收光谱所呈现的大的红移现象的分子起源。记录了用特定位置（15、15、15'、14、14'、13、13'、12、12'或20、20'）13C标记的虾青素重构的α-甲壳蓝蛋白的共振拉曼光谱。这种方法使我们能够获得关于配体-蛋白质相互作用对基态电子态下虾青素发色团几何结构影响的信息。每种标记化合物的C==C伸缩振动模式的下移幅度表明，多烯链中心部分的主要扰动是不均匀的。此外，1250 - 1400 cm(-1)光谱范围内的变化表明，虾青素多烯链在与蛋白质结合时几何结构发生了适度变化。半经验量子化学建模研究（奥斯汀方法1）表明，几何结构变化不能单独解释与蛋白质结合的虾青素从480 nm到632 nm的红移现象。计算结果与一种极化机制一致，该机制涉及虾青素发色团的两个酮官能团与质子或具有相当大小的正离子物种的另一种相互作用，并支持共振拉曼光谱和可见吸收光谱中观察到的变化。这些结果与基于固态核磁共振光谱对α-甲壳蓝蛋白发色团电荷密度研究得出的结论高度一致。从这些结果可知，显著的红移不仅可以用基态电子态构象的变化来解释，还可以用激发态电子态中的相互作用来解释，这种相互作用显著降低了π反键C==O轨道的能量和HOMO-LUMO能隙。
