Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation.

FF ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acid/alkaline). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2.A1 F ATPase (TA2F) with two forms of single molecule analysis, a magnetic bead duplex and a gold nanoparticle. TA2F rotated in a counterclockwise direction in both systems, adhering to Michaelis-Menten kinetics with a maximum rotation rate (V) of 112.4 revolutions/s. TA2F displayed 120° unitary steps coupled with ATP hydrolysis. Torque measurements revealed the highest torque (52.4 piconewtons) derived from an F molecule using fluctuation theorem. The implications of high torque in terms of extreme environment adaptation are discussed.