DIRECT VIBRATIONAL STRUCTURE OF PROTEIN METAL-BINDING SITES FROM NEAR-INFRARED YB3-BAND SPECTROSCOPY( VIBRONIC SIDE)

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to ob tain 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 mode l complexes of Yb3+ representing different ligand types were studied t o provide references for the VSB spectra of Yb3+-reconstituted metallo proteins. 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 w eak VSBs whose energy shifts, with respect to the main F-2(5/2) --> F- 2(7/2) Yb3+ electronic transition, represent the vibrational frequenci es of the Yb3+ ligands. The chemical nature of the ligands of the Yb3 in these proteins, as deduced by the observed VSB frequencies, is ent irely in agreement with their known crystal structures. For transferri n, replacement of the (CO32-)-C-12 metal counterion with (CO32-)-C-13 yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous pote ntial in elucidating the localized structure of metal binding sites in proteins.