Kinetic analysis of the inhibition mechanism of bovine mitochondrial F(1)-ATPase inhibitory protein using biochemical assay

ATPase inhibitory factor 1 (IF(1)) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F(1)-ATPase, by inserting its N-terminus into the rotor–stator interface of F(1)-ATPase. Although previous studies have proposed a two-step model for IF(1)-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF(1)-mediated inhibition under a wide range of [ATP]s and [IF(1)]s, using bovine mitochondrial IF(1) and F(1)-ATPase. Typical hyperbolic curves of inhibition rates with [IF(1)]s were observed at all [ATP]s tested, suggesting a two-step mechanism: the initial association of IF(1) to F(1)-ATPase and the locking process, where IF(1) 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 F(1)-ATPase, this result means that IF(1) associates with F(1)-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 F(1)-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF(1) in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF(1)-mediated inhibition scheme.