Studies of cation binding in ZnCl2-regenerated bacteriorhodopsin by x-ray absorption fine structures: effects of removing water molecules and adding Cl- ions

Abstract

The binding of Zn2+ in Zn2+-regenerated bacteriorhodopsin (bR) was studied under various conditions by x-ray absorption fine structures (XAFS). The 0.9:1 and 2:1 Zn2+:bR samples gave similar XAFS spectra, suggesting that Zn2+ might have only one strong binding site in bR. It was found that in aqueous bR solution, Zn2+ has an average of six oxygen or nitrogen ligands. Upon drying, two ligands are lost, suggesting the existence of two weakly bound water ligands near the cation-binding site in bacteriorhodopsin. When excess Cl- ions were present before drying in the Zn2+-regenerated bR samples, it was found that two of the ligands were replaced by Cl- ions in the dried film, whereas two remain unchanged. The above observations suggest that Zn2+ has three types of ligands in regenerated bR (referred to as types I, II, and III). Type I ligands are strongly bound. These ligands cannot be removed by drying or by exchanging with Cl- ions. Type II ligands cannot be removed by drying, but can be replaced by Cl- ligands. Type III ligands are weakly bound to the metal cation and are most likely water molecules that can be removed by evaporation under vacuum or by drying with anhydrous CaSO4. The results are discussed in terms of the possible structure of the strongly binding site of Zn2+ in bR.

Location of a Cation-Binding Site in the Loop between Helices F and G of Bacteriorhodopsin as Studied by 13C NMR

“…The structural model of the cation-binding site includes only oxygens of carbonyl groups in the backbone and water molecules as the ligands of the bound cation. This kind of cation-binding site is consistent with the structures of the cation-binding sites proposed by x-ray absorption studies, which are suggested to include oxygen atoms as ligands (Zhang et al, 1997;Engelhard et al, 1987;Sepulcre et al, 1996).…”

X‐ray absorption and molecular dynamics study of cation binding sites in the purple membrane

“…Thus, Tuzi et al using 13 C NMR techniques proposed the existence of a cation binding site located on the loop between helices F and G,14 Eliash et al have shown a Mn 2+ ‐specific binding site on the extracellular part of the membrane,15 and Sanz et al established Glu194 and Glu204 as residues involved in cation binding 16. In addition, a high‐affinity binding site located near the retinal pocket had also been suggested by several authors 17–24…”