Mechanistic basis for differential inhibition of the F₁F_o‐ATPase by aurovertin

Abstract

The mitochondrial F 1 F o -ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F 1 F o -ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F 1 domain and inhibits F 1 F o -ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F 1 F o -ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F 1 F 0 -ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F 1 F o -ATPase catalyzes ATP synthesis and hydrolysis.

Three-color Förster resonance energy transfer within single F[sub O]F[sub 1]-ATP synthases: monitoring elastic deformations of the rotary double motor in real time

“…A custom-made confocal microscope setup based on an Olympus IX 71 was used 40,41 for the triple-FRET measurements with single F O F 1 -ATP synthases. Three different lasers were applied: two fiber coupled picosecond pulsed lasers with arbitrary repetition rates up to 80 MHz at 488 nm (PicoTa 490, Picoquant) and 635 nm (LDH-P-635B, Picoquant) were used, and the third, continuous wave laser at 532 nm (compass 315, Coherent) was switched by a fast acusto optical modulator (AOM type 335-192, Crystal Technologies).…”

Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy

“…Samples were then incubated for 5 min at 37°C with the conditions as follows: "apo," 5 mM The home-built microscope with expanded confocal detection volume of about 10 fl was described previously (31,32). Two lasers in duty cycle-optimized alternating fashion were used to excite the donor and acceptor (33).…”

Yellow Pigment Aurovertins Mediate Interactions between the Pathogenic Fungus Pochonia chlamydosporia and Its Nematode Host

“…44 A previous study revealed that aurovertin inhibited ATPase considerably in the absence of F O and found that some protective action occurred in the presence of F O . 45 Furthermore, the gene encoding OSCP is involved in controlling respiration and regulates growth rate and body size, aging, and rates of behaviors such as pharyngeal pumping, defecation, and locomotion. Our results that the RNAi mutants in R53.4 or hpo-18 caused the nematodes to be more sensitive to aurovertin D were consistent with previous reports.…”