Use of near-infrared Yb 3+ vibronic side band spectroscopy to probe protein metal-binding sites

Abstract

Near infrared Yb 3+ vibronic sideband spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This novel 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 Yb 3+ ion. Weak vibronic sidebands (VSB) whose energy shifts, with respect to the main 2 F 5 2 → 2 F 7 2 Yb 3+ zero-phonon electronic transition, represent the vibrational frequencies of the Yb 3+ ligands. As a model complex, we have studied the VSB spectrum of Yb 3+ complexed with Eriochrome Cyanine R (Yb:ECR), a ligand which possesses COO −, CO, and CO functional groups. VSB arising from the vibrational modes of these chemical groups are readily observed and well resolved. VSB fluorescence spectra of Yb 3+ reconstituted into the Fe 3+ binding sites of the iron-transporting protein human serum transferrin indicates that these two sites are not spectroscopically identical. The chemical nature of the ligands of the Yb 3+ in this protein, as deduced by the observed VSB frequencies, are entirely in agreement with the known crystal structure. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.