Binding of Phytopolyphenol Piceatannol Disrupts β/γ Subunit Interactions and Rate-limiting Step of Steady-state Rotational Catalysis in Escherichia coli F1-ATPase

• Published in: The Journal of biological chemistry 2012-06, Vol.287 (27), p.22771-22780
• Main Authors: Sekiya, Mizuki, Nakamoto, Robert K., Nakanishi-Matsui, Mayumi, Futai, Masamitsu
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antioxidants - metabolism; Antioxidants - pharmacology; ATP Synthase; Binding Sites - drug effects; Binding Sites - physiology; Bioenergetics; Catalysis; Enzyme Activation - drug effects; Enzyme Activation - physiology; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; F1F0-ATPase; Molecular Sequence Data; Mutagenesis - physiology; Phytopolyphenol; Polyphenols - chemistry; Polyphenols - metabolism; Polyphenols - pharmacology; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Proton-Translocating ATPases - chemistry; Proton-Translocating ATPases - genetics; Proton-Translocating ATPases - metabolism; Quercetin - metabolism; Quercetin - pharmacology; Single Molecule Biophysics; Small Molecules; Stilbenes - metabolism; Stilbenes - pharmacology; Temperature; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.374868

In observations of single molecule behavior under Vmax conditions with minimal load, the F1 sector of the ATP synthase (F-ATPase) rotates through continuous cycles of catalytic dwells (∼0.2 ms) and 120° rotation steps (∼0.6 ms). We previously established that the rate-limiting transition step occurs during the catalytic dwell at the initiation of the 120° rotation. Here, we use the phytopolyphenol, piceatannol, which binds to a pocket formed by contributions from α and β stator subunits and the carboxyl-terminal region of the rotor γ subunit. Piceatannol did not interfere with the movement through the 120° rotation step, but caused increased duration of the catalytic dwell. The duration time of the intrinsic inhibited state of F1 also became significantly longer with piceatannol. All of the beads rotated at a lower rate in the presence of saturating piceatannol, indicating that the inhibitor stays bound throughout the rotational catalytic cycle. The Arrhenius plot of the temperature dependence of the reciprocal of the duration of the catalytic dwell (catalytic rate) indicated significantly increased activation energy of the rate-limiting step to trigger the 120° rotation. The activation energy was further increased by combination of piceatannol and substitution of γ subunit Met23 with Lys, indicating that the inhibitor and the β/γ interface mutation affect the same transition step, even though they perturb physically separated rotor-stator interactions. Background: The β/γ subunit interactions are critical for rotational catalysis in ATP synthase. Results: Piceatannol increases the activation energy for the rate-limiting transition state. Conclusion: Piceatannol binding and a β/γ subunit interface mutation, although the sites are physically separated, affect the same rate-limiting transition-state step. Significance: Multiple rotor-stator interactions contribute to formation of the transition state.