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).