Cation Binding to Halorhodopsin

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl^– 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 Mn²⁺ and Ca²⁺ bind to the protein. Halorhodopsin has a high affinity for Mn²⁺ 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 Mn²⁺ ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn²⁺ 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

Conjugated Cofactor Enables Efficient Temperature-Independent Electronic Transport Across ∼6 nm Long Halorhodopsin

We observe temperature-independent electron transport, characteristic of tunneling across a ∼6 nm thick Halorhodopsin (phR) monolayer. phR contains both retinal and a carotenoid, bacterioruberin, as cofactors, in a trimeric protein-chromophore complex. This finding is unusual because for conjugated oligo-imine molecular wires a transition from temperature-independent to -dependent electron transport, ETp, was reported at ∼4 nm wire length. In the ∼6 nm long phR, the ∼4 nm 50-carbon conjugated bacterioruberin is bound parallel to the α-helices of the peptide backbone. This places bacterioruberin’s ends proximal to the two electrodes that contact the protein; thus, coupling to these electrodes may facilitate the activation-less current across the contacts. Oxidation of bacterioruberin eliminates its conjugation, causing the ETp to become temperature dependent (>180 K). Remarkably, even elimination of the retinal-protein covalent bond, with the fully conjugated bacterioruberin still present, leads to temperature-dependent ETp (>180 K). These results suggest that ETp via phR is cooperatively affected by both retinal and bacterioruberin cofactors

Spin-Controlled Photoluminescence in Hybrid Nanoparticles Purple Membrane System

Spin-dependent photoluminescence (PL) quenching of CdSe nanoparticles (NPs) has been explored in the hybrid system of CdSe NP purple membrane, wild-type bacteriorhodopsin (bR) thin film on a ferromagnetic (Ni-alloy) substrate. A significant change in the PL intensity from the CdSe NPs has been observed when spin-specific charge transfer occurs between the retinal and the magnetic substrate. This feature completely disappears in a bR apo membrane (wild-type bacteriorhodopsin in which the retinal protein covalent bond was cleaved), a bacteriorhodopsin mutant (D96N), and a bacteriorhodopsin bearing a locked retinal chromophore (isomerization of the crucial C13C14 retinal double bond was prevented by inserting a ring spanning this bond). The extent of spin-dependent PL quenching of the CdSe NPs depends on the absorption of the retinal, embedded in wild-type bacteriorhodopsin. Our result suggests that spin-dependent charge transfer between the retinal and the substrate controls the PL intensity from the NPs