STUDIES OF CATION-BINDING IN ZNCL2-REGENERATED BACTERIORHODOPSIN BY X-RAY-ABSORPTION FINE-STRUCTURES - EFFECTS OF REMOVING WATER-MOLECULES AND ADDING CL- IONS

The binding of Zn2+ in Zn2+-regenerated bacteriorhodopsin (bR) was stu died under various conditions by x-ray absorption fine structures (XAF S). The 0.9:1 and 2:1 Zn2+:bR samples gave similar XAFS spectra, sugge sting 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 o r nitrogen ligands. Upon drying, two ligands are lost, suggesting the existence of two weakly bound water ligands near the cation-binding si te in bacteriorhodopsin. When excess Cl- ions were present before dryi ng in the Zn2+-regenerated bR samples, it was found that two of the li gands 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 dryi ng or by exchanging with Cl- ions. Type II ligands cannot be removed b y drying, but can be replaced by Cl- ligands. Type III ligands are wea kly bound to the metal cation and are most likely water molecules that can be removed by evaporation under vacuum or by drying with anhydrou s CaSO4. The results are discussed in terms of the possible structure of the strongly binding site of Zn2+ in bR.