ATP synthesis of Enterococcus hirae V-ATPase driven by sodium motive force

V-ATPases generally function as ion pumps driven by ATP hydrolysis in the cell, but their capability of ATP synthesis remains largely unexplored. Here we show ATP synthesis of Na+-transporting Enterococcus hirae V-ATPase (EhVoV1) driven by electrochemical potential gradient of Na+ across the membrane (sodium motive force, smf). We reconstituted EhVoV1 into liposome and performed a luciferin/luciferase-based assay to analyze ATP synthesis quantitatively. Our result demonstrates that EhVoV1 synthesizes ATP with a rate of 4.7 s-1 under high smf (269.3 mV). The Michaelis constants for ADP (21 {micro}M) and inorganic phosphate (2.1 mM) in ATP synthesis reaction were comparable to those for ATP synthases, suggesting similar substrate affinities among rotary ATPases regardless of their physiological functions. Both components of smf, Na+ concentration gradient across the membrane ({Delta}pNa) and membrane potential ({Delta}{psi}), contributed to ATP synthesis, with {Delta}pNa showing a slightly larger impact. At the equilibrium points where smf and Gibbs free energy of ATP synthesis are balanced, EhVoV1 showed reversible reactions between ATP synthesis and hydrolysis. The obtained Na+/ATP ratio (3.2 {+/-} 0.4) closely matched the value expected from the structural symmetry ratio between EhVo and EhV1 (10/3 = 3.3), indicating tight coupling between ATP synthesis/hydrolysis and Na+ transport. These results reveal inherent functional reversibility of EhVoV1. We propose that physiological function of EhVoV1 in vivo is determined by relatively small smf against large Gibbs free energy of ATP synthesis, in addition to the absence of inhibitory mechanisms of ATP hydrolysis which are known for ATP synthases.