The complex of mitochondrial F1-ATPase with the natural inhibitor protein is unable to catalyze single-site ATP hydrolysis.

Interaction of mitochondrial F1-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [14C]nucleotides in F1-ATPase during steady-state [14C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.