Okuni, Y. | Search bioRxiv

bioRxiv2024

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.

biophysics↗

bioRxiv2022

Direct Observation of Stepping Rotation of V-ATPase Reveals Rigid Coupling between Vo and V1 Motors

V-ATPases are rotary motor proteins which convert chemical energy of ATP into electrochemical potential of ions across the cell membrane. V-ATPases consist of two rotary motors, Vo and V1, and Enterococcus hirae V-ATPase (EhVoV1) actively transports Na+ in Vo (EhVo) by using torque generated by ATP hydrolysis in V1 (EhV1). Here, we observed ATP-driven stepping rotation of detergent-solubilized EhVoV1 wild-type, aE634A, and BR350K mutants under the various Na+ and ATP concentrations ([Na+] and [ATP], respectively) by using a 40-nm gold nanoparticle as a low-load probe. When [Na+] was low and [ATP] was high, under the condition that only Na+ binding to EhVo is the rate-limiting, wild-type and aE634A exhibited 10-pausing positions reflecting 10-fold symmetry of the EhVo rotor and almost no backward steps. Duration time before forward steps was inversely proportional to [Na+], confirming that Na+ binding triggers the steps. When both [ATP] and [Na+] were low, under the condition that both Na+ and ATP bindings are rate-limiting, aE634A exhibited 13-pausing positions reflecting 10- and 3-fold symmetries of EhVo and EhV1, respectively. Distribution of duration time before forward step was well fitted by a sum of two exponential decay functions with distinct time constants. Furthermore, frequent backward steps smaller than 36{degrees} were observed. Small backward steps were also observed during long, three ATP cleavage pauses of BR350K. These results indicate that EhVo and EhV1 do not share pausing positions and Na+ and ATP bindings occur at different angles, and the coupling between EhVo and EhV1 is not elastic but rigid. Significance StatementV-ATPases are ion pumps consisting of two rotary motor proteins Vo and V1, and actively transport ions across the cell membrane by using chemical energy of ATP. To understand how V-ATPases transduce the energy in the presence of structural symmetry mismatch between Vo and V1, we simultaneously visualized rotational pauses and forward and backward steps of Vo and V1 coupled with ion transport and ATP hydrolysis reaction, respectively. Our results indicate rigid coupling of a V-ATPase which has multiple peripheral stalks, in contrast to elastic coupling of F-ATPases with only one peripheral stalk, which work as ATP synthase. Our high-speed/high-precision single-molecule imaging of rotary ATPases in action will pave the way for a comprehensive understanding of their energy transduction mechanisms.

biophysics↗