Detection of a Yb(3+) binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Near infrared Yb(3+) vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb(3+) site is observed. The Yb(3+) binding site in bO is observed to consist of PO(2)(−) groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb(3+) in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca(2+) (or Mg(2+)) and lanthanides in phospholipid membrane proteins.