A member
of the retinal protein family, halorhodopsin, acts as
an inward light-driven Cl<sup>–</sup> pump. It was recently
demonstrated that the <i>Natronomonas pharaonis</i> 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<sup>2+</sup> and Ca<sup>2+</sup> bind to the protein. Halorhodopsin
has a high affinity for Mn<sup>2+</sup> 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<sup>2+</sup> ions decreases upon removal
of the retinal chromophore from the protein or following bacterioruberin
oxidation. Our results also indicate that Mn<sup>2+</sup> ions, as
well as Cl<sup>–</sup> ions, first occupy binding sites other
than site I. The observed synergetic effect between cation and anion
binding suggests that while Cl<sup>–</sup> anions bind to halorhodopsin
at low concentrations, the occupancy of site I requires a high concentration