Cation Binding to Xanthorhodopsin: Electron Paramagnetic Resonance and Magnetic Studies

Xanthorhodopsin (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light-harvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR are dramatically affected due to the protonation of two different residues. It is still unclear whether xR can bind cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn²⁺ and Ca²⁺, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn²⁺ to a high-affinity binding site followed by binding of ∼40 equiv in cooperative manner and ∼100 equiv of Mn²⁺ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn²⁺ cations to xR is due to the formation of Mn²⁺ clusters. Our data demonstrate that Ca²⁺ cations bind to DI-xR with a lower affinity than Mn²⁺, supporting the assumption that binding of Mn²⁺ occurs through cluster formation, because Ca²⁺ cations cannot form clusters in contrast to Mn²⁺.