Roselli C, Boussac A, Mattioli T A
Section de Bioénergétique, CEA, Gif-sur-Yvette, France.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12897-901. doi: 10.1073/pnas.91.26.12897.
Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.
近红外Yb3+振动边带（VSB）光谱法用于获取金属蛋白中金属结合位点的结构信息。该技术为那些与Yb3+离子螯合的配体提供了选择性的“类红外”振动光谱。研究了代表不同配体类型的各种Yb3+模型配合物的VSB光谱，为Yb(3+)重构金属蛋白的VSB光谱提供参考。用Yb3+取代了钙结合蛋白小白蛋白中的Ca2+和铁转运蛋白转铁蛋白中的Fe3+。重构到这两种蛋白中的Yb3+的荧光表现出微弱的VSB，相对于主要的2F5/2-->2F7/2 Yb3+电子跃迁，其能量位移代表了Yb3+配体的振动频率。根据观察到的VSB频率推断，这些蛋白中Yb3+配体的化学性质与它们已知的晶体结构完全一致。对于转铁蛋白，用13CO3(2-)取代12CO3(2-)金属抗衡离子产生了对应于碳酸根振动模式的VSB的预期同位素位移。该技术在阐明蛋白质中金属结合位点的局部结构方面显示出巨大潜力。
