title: Cation binding to halorhodopsin.
abstract: A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl(-) pump. It was recently demonstrated that the Natronomonas pharaonis halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn(2+) and Ca(2+) bind to the protein. Halorhodopsin has a high affinity for Mn(2+) ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn(2+) ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn(2+) ions, as well as Cl(-) ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl(-) anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration.
authors.affiliation: †Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Sansa
authors.initials: S
authors.lastname: Dutta
authors.affiliation: ‡Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Lev
authors.initials: L
authors.lastname: Weiner
authors.affiliation: †Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Mordechai
authors.initials: M
authors.lastname: Sheves
mesh_terms: D001667:Binding, Competitive; D002412:Cations; D002712:Chlorides / Q000737:chemistry; D025602:Halorhodopsins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000737:chemistry*; D008958:Models, Molecular; D019633:Natronobacterium / Q000737:chemistry; D011485:Protein Binding
chemical_list: D002412:Cations; D002712:Chlorides; D025602:Halorhodopsins; D008345:Manganese