The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F₁F_o-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14−47. The immediately adjacent sequences 10−13 and 48−56 help to stabilize the complex between F₁F_o-ATPase and the inhibitor protein, and residues 1−9 and 57−84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly α-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10−50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they form a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular α-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10−40 and 23−84 of the inhibitor protein can bind to F₁F_o-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22−46 and 44−84 do not compete with the inhibitor subunit for its binding site on F₁F_o-ATPase.

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