Mechanism of inhibition of bovine F1-ATPase by resveratrol and related polyphenols

Abstract

The structures of F(1)-ATPase from bovine heart mitochondria inhibited with the dietary phytopolyphenol, resveratrol, and with the related polyphenols quercetin and piceatannol have been determined at 2.3-, 2.4- and 2.7-A resolution, respectively. The inhibitors bind to a common site in the inside surface of an annulus made from loops in the three alpha- and three beta-subunits beneath the "crown" of beta-strands in their N-terminal domains. This region of F(1)-ATPase forms a bearing to allow the rotation of the tip of the gamma-subunit inside the annulus during catalysis. The binding site is a hydrophobic pocket between the C-terminal tip of the gamma-subunit and the beta(TP) subunit, and the inhibitors are bound via H-bonds mostly to their hydroxyl moieties mediated by bound water molecules and by hydrophobic interactions. There are no equivalent sites between the gamma-subunit and either the beta(DP) or the beta(E) subunit. The inhibitors probably prevent both the synthetic and hydrolytic activities of the enzyme by blocking both senses of rotation of the gamma-subunit. The beneficial effects of dietary resveratrol may derive in part by preventing mitochondrial ATP synthesis in tumor cells, thereby inducing apoptosis.

Fig. 1.

Structures of polyphenol inhibitors of bovine F₁-ATPase. (I) Resveratrol. (II) Piceatannol. (III) Quercetin.

Fig. 2.

The site of binding of resveratrol in bovine F₁-ATPase. The α-, β- and γ-subunits are red, yellow, and blue, respectively, and resveratrol is green. (A) Ribbon view of F₁-ATPase upwards from the mitochondrial membrane along the central axis of the γ-subunit, showing the inhibitor in solid representation bound between the γ- and β_TP-subunits. (B) Side view of a solid representation of resveratrol bound in a pocket in F₁-ATPase between the γ- and β_TP-subunits. For clarity, the β_DP and β_E subunits and the three α-subunits have been removed. The pocket is in the “bearing” consisting of the sleeve provided by the N-terminal regions of α- and β-subunits in the “crown” domain of F₁-ATPase and the α-helical tip of the C-terminal region of the γ-subunit. (C) Side view in stereo showing interactions of resveratrol with side chains in the binding pocket shown in stick representation with oxygen and nitrogen atoms in binding-site residues in red and dark blue, respectively. The residues shown are either within 4 Å of the inhibitor and form hydrophobic interactions, or they are linked to it via hydrogen bond networks (dotted lines) involving water molecules (light blue spheres), and by a hydrogen bond from the amido group of Val-279 to the π-electrons of the m-dihydroxyphenyl moiety of resveratrol (orange dotted line). (D and E) view of the binding pocket and bound resveratrol (with red oxygen atoms) in solid representation. D is the same view as in C, and in E, bound resveratrol and its binding pocket are viewed along the axis of γ-subunit, upwards from the mitochondrial membrane.

Fig. 3.

Comparison of the modes of binding to bovine F₁-ATPase of piceatannol and quercetin with that of resveratrol. (A) Major binding modes of resveratrol (green) and piceatannol (gray). (B) Major binding mode of resveratrol (green) and cis-quercetin (gray).