Phosphorylation in a simple system of lipids and chloroplast ATP synthetase driven by pulsed ionising radiation

Abstract

WE report here the observation of an ADP-dependent phosphorylation reaction in an artificial lipid vesicle system driven by pulsed ionising radiation. This reaction is completely dependent on the only enzyme incorporated into the phospholipid vesicle bilayer, the ATP synthetase (CF₀–CF₁) complex from spinach chloroplasts. Previous attempts to demonstrate directly an obligatory coupling between the proton pumping and phosphorylation activities of an ATP synthetase enzyme in resolved, reconstituted vesicle systems have required the presence of additional protein components^1–3 to generate the transmembrane pH gradient and/or membrane potential postulated by the chemiosmotic hypothesis⁴ as the driving force for the ATP synthesis reaction: ADP+P_i+nH⁺_in ⇌ ATP+H₂O+nH⁺_out Pulsed high-energy radiolysis of water produces, in a very brief time increment, significant quantities of protons along with the highly reactive transient species e⁻_aq, OH, and H (δ[H⁺]∼ 10⁻⁵ M in 50 ns)⁵. Assuming a uniform production of protons throughout the aqueous media, differential buffering between the inner and outer aqueous compartments of phospholipid vesicles will result in the effectively instantaneous formation of a transmembrane proton gradient. We have applied the pulse radiolysis technique to drive the ATP synthetase activity of phospholipid vesicles reconstituted with the chloroplast CF₀–CF₁ enzyme complex.