Kinetic analysis of the inhibition mechanism of bovine mitochondrial F1-ATPase inhibitory protein using biochemical assay.

ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N-terminus into the rotor-stator interface of F1-ATPase. Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analyzed the kinetics of IF1-mediated inhibition under a wide range of [ATP]s and [IF1]s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with [IF1]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N-terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on the present findings, we propose an IF1-mediated inhibition scheme.