Dietary bioflavonoids inhibit Escherichia coli ATP synthase in a differential manner

Abstract

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of 17 bioflavonoid compounds on purified F1 or membrane bound F1Fo E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC50 approximately 0.07 mM)>silymarin (IC50 approximately 0.11 mM)>baicalein (IC50 approximately 0.29 mM)>silibinin (IC50 approximately 0.34 mM)>rimantadin (IC50 approximately 2.0 mM)>amantidin (IC50 approximately 2.5 mM)>epicatechin (IC50 approximately 4.0 mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F1Fo membrane preparations and isolated purified F1. ATPase and growth assays suggested that the bioflavonoid compounds used in this study inhibited F1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

Figure 1

X-ray crystallographic structure of polyphenol binding site of ATP synthase. (A) Empty and (B) hypothetical binding of morin hydrate at the polyphenol binding pocket. Residues from α, β, and γ subunits involved in interaction with polyphenols are identified. In bovine two variants, Q274K and T277I, occur in the γ subunit and are identified in the figure. PDB file 2jj1 [16] with RasMol [55] was used to generate this figure.

Figure 2. Structures of bioflavonoids are shown in three groups

(I) exerting complete inhibition of F₁-ATPase activity, (II) exerting incomplete inhibition of F₁-ATPase activity, and (III) exerting insignificant inhibition of F₁-ATPase activity.

Figure 3. Complete inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase

Membranes or purified F₁ were preincubated for 60 min at 23°C with varied concentration of bioflavonoids shown in the figure and then aliquots added to 1 ml of assay buffer and ATPase activity determined. Details are given in Materials and Methods. Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 4. Incomplete inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase

Membranes or purified F₁ were preincubated for 60 min at 23°C with varied concentration of bioflavonoids identified in the figure and then aliquots added to 1 ml of assay buffer and ATPase activity determined. Details can be found Materials and Methods section. Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 5. Insignificant or no inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase

Membranes or purified F₁ were preincubated for 60 min at 23°C with varied concentration of bioflavonoids identified in the figure and then aliquots added to 1 ml of assay buffer and ATPase activity determined. Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 6. Results of Extra pulses of bioflavonoid compounds and reversal of inhibition by passing through centrifuge columns

(A), Membrane bound ATP synthase (Mbr) or purified F₁ (F₁) was inhibited with inhibitory concentrations of the bioflavonoid compounds shown in the figure for 60 min under conditions as described in Fig 3–4. Then a further pulse of identical inhibitory concentrations was added and incubation continued for 1 h before assay. The last digits represent the compound concentrations in [μM]. (B) Purified F₁ was incubated with inhibitory concentrations of bioflavonoid compounds for 60 min under conditions as described in Fig 2–4. Then the inhibited samples were passed twice through 1 ml centrifuge columns and ATPase activity was measured. The first bar is for purified F₁ with no compound (F1), followed by bars in presence of compounds.

Figure 6. Results of Extra pulses of bioflavonoid compounds and reversal of inhibition by passing through centrifuge columns

(A), Membrane bound ATP synthase (Mbr) or purified F₁ (F₁) was inhibited with inhibitory concentrations of the bioflavonoid compounds shown in the figure for 60 min under conditions as described in Fig 3–4. Then a further pulse of identical inhibitory concentrations was added and incubation continued for 1 h before assay. The last digits represent the compound concentrations in [μM]. (B) Purified F₁ was incubated with inhibitory concentrations of bioflavonoid compounds for 60 min under conditions as described in Fig 2–4. Then the inhibited samples were passed twice through 1 ml centrifuge columns and ATPase activity was measured. The first bar is for purified F₁ with no compound (F1), followed by bars in presence of compounds.