IF1 is a natural inhibitor protein for mitochondrial FoF1 ATP synthase that blocks catalysis and rotation of the F1 by deeply inserting its N-terminal helices into F1. A unique feature of IF1 is condition-dependent inhibition; although IF1 inhibits ATP hydrolysis by F1, IF1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF1-inhibited bovine mitochondrial F1 (bMF1). The results show that IF1-inhibited F1 is efficiently activated only when F1 is rotated in the clockwise (ATP synthesis) direction, but not in the counterclockwise direction. The observed rotational-direction-dependent activation explains the condition-dependent mechanism of IF1 inhibition. Investigation of mutant IF1 with N-terminal truncations shows that the interaction with the γ subunit at the N-terminal regions is crucial for rotational-direction-dependent ejection, and the middle long helix is responsible for the inhibition of F1.
IF1 is a natural inhibitor of mitochondrial FoF1-ATP synthase, which blocks catalysis and rotation of the F1 motor. Here, the authors show the rotational-direction-dependence of activation from IF1 inhibition, with IF1 being readily dissociated when F1 rotates to the clockwise direction.