Molecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase.

IF 1 is a natural inhibitor protein for mitochondrial F o F 1 ATP synthase that blocks catalysis and rotation of the F 1 by deeply inserting its N-terminal helices into F 1 . A unique feature of IF 1 is condition-dependent inhibition; although IF 1 inhibits ATP hydrolysis by F 1 , IF 1 inhibition is relieved under ATP synthesis conditions. To elucidate this condition-dependent inhibition mechanism, we have performed single-molecule manipulation experiments on IF 1 -inhibited bovine mitochondrial F 1 (bMF 1 ). The results show that IF 1 -inhibited F 1 is efficiently activated only when F 1 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 IF 1 inhibition. Investigation of mutant IF 1 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 F 1 .