Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and P-i. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the gamma epsilon shaft that connects the alpha(3)beta(3) head and the membrane-anchored c(n) ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the gamma C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in gamma, arising from gamma...alpha(3)beta(3) interactions that cause resistance during gamma rotation within the apical bearing. Intriguingly, we find that destabilization of gamma occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.