External electric field driven ATP synthesis in chloroplasts: a slow, ATP synthase-dependent reaction

Abstract

The process of energy transduction in chloroplasts involves several sequential stages. Light induced charge separation triggers a series of redox reactions which lead to the formation of a transmembrane electric potential difference and a proton concentration gradient. In a well-coupled system these gradients drive the generation of ATP from ADP and pi. This final step of energy transduction and the mode by which it leads to ATP synthesis are not yet understood. One type of experimental approach aimed at the elucidation of the energy transduction process involves reduction of the number of factors which participate in it. Thus, an artificially imposed transmembrane proton concentration gradient has been demonstrated to activate ATP synthesis in systems in which electron transfer was completely blocked [l]. It was also shown that the ATP synthase enzyme complex could catalyze ADP phosphorylation when reconstituted into liposomes in absence of the components of the electron transport chain [2]. Recently a novel method has been presented in which formation of ATP from ADP and phosphate, catalyzed by thylakoid membranes can be driven by an external electric field [3]. In such a system not only electron transfer but also the formation of bulk to bulk ionic gradients is presumably absent during activation. As previously stated [4] this approach has clear kinetic advantages and has been shown to be effective in other energy transducing systems as well [5-71. A further development of this method involved the use of an enzymatic assay that allowed the continuous monitoring of ATP formation induced by exposure of thylakoid membranes to an external electric field [8]. Information regarding the time course of ATP synthesis has been thus obtained indicating that the external electric field induced ATP synthesis proceeds for relatively long periods (seconds) following the termination of the applied electric field pulse. Further studies of the unusual kinetics of external electric field driven ADP phosphorylation (EFP reaction) are presented in this report. They indicate the direct involvement of the CFt portion of the ATP synthase complex in the EFP reaction. The influence of inhibitors on the EFP reaction was investigated and compared to their effect on photophosphorylation, revealing important differences between the two.