USE OF NEAR-INFRARED YB3-BAND SPECTROSCOPY TO PROBE PROTEIN METAL-BINDING SITES( VIBRONIC SIDE)

Near infrared Yb3+ vibronic sideband spectroscopy is used to obtain st ructural information of metal binding sites in metalloproteins. This n ovel technique, using near-infrared excitation and detection (970-1200 nm) provides a selective well-resolved ''infrared-like'' vibrational spectrum of those ligands which are chelated to the Yb3+ ion. Weak vib ronic sidebands (VSB) whose energy shifts, with respect to the main F- 2(5/2) --> F-2(7/2) Yb3+ zero-phonon electronic transition, represent the vibrational frequencies of the Yb3+ ligands. As a model complex, w e have studied the VSB spectrum of Yb3+ complexed with Eriochrome Cyan ine R (Yb:ECR), a ligand which possesses COO-, C-O, and C=O functional groups. VSB arising from the vibrational modes of these chemical grou ps are readily observed and well resolved. VSB fluorescence spectra of Yb3+ reconstituted into the Fe3+ binding sites of the iron-transporti ng protein human serum transferrin indicates that these two sites are not spectroscopically identical. The chemical nature of the ligands of the Yb3+ in this protein, as deduced by the observed VSB frequencies, are entirely in agreement with the known crystal structure. This tech nique demonstrates enormous potential in elucidating the localized str ucture of metal binding sites in proteins.