Reconstitution of the heliobacterial photochemical reaction center and cytochrome c ₅₅₃ into a proteoliposome system

The heliobacterial reaction center (HbRC) is the simplest known photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its electron donor is a cytochrome (cyt) c₅₅₃ attached to the membrane via a covalent linkage with a diacylglycerol. We have reconstituted purified HbRC into liposomes mimicking the phospholipid composition of heliobacterial membranes. We also incorporated a lipid with a headgroup containing Ni(II):nitrilotriacetate (NTA) to provide a binding site for the soluble version of the heliobacterial cyt c₅₅₃ in which the N-terminal membrane attachment site is replaced by a hexahistidine tag. The HbRC was inserted into the liposomes with the donor side preferentially exposed to the exterior; this bias increased to nearly 100% with higher concentrations (≥ 10 mol%) of the Ni(II)-NTA lipid in the membrane, and is most likely due to the net negative charge of the surface of the membrane. The HbRC in proteoliposomes without the Ni(II)-NTA lipid exhibited normal charge separation and subsequent charge recombination of the P₈₀₀ ⁺F_X ⁻ state in 15 ms; however, the oxidized primary donor (P₈₀₀ ⁺) was not significantly reduced by added H₆-cyt c₅₅₃. In contrast, with proteoliposomes containing the Ni(II)-NTA lipid, addition of H₆-cyt c₅₅₃ resulted in a new kinetic component resulting from fast reduction (2–5 ms) of P₈₀₀ ⁺ by H₆-cyt c₅₅₃. The contribution of this kinetic component varied with the concentration of added H₆-cyt c₅₅₃ and could represent 80% or more of the total P₈₀₀ ⁺ decay. Thus, the HbRC and its interaction with its native electron donor have been reconstituted into an artificial membrane system.