title: Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics.
abstract: BACKGROUND
  The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.
  
  
  METHODS
  The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide-enzyme interaction.
  
  
  RESULTS
  When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT≥1μM. The TBT-driven enzyme desensitization was reversed by DTE.
  
  
  CONCLUSIONS
  The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.
  
  
  CONCLUSIONS
  The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Nesci
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.
authors.forename: Vittoria
authors.initials: V
authors.lastname: Ventrella
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Trombetti
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Pirini
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, via Tolara di Sopra 50, 40064 Ozzano Emilia, Bologna, Italy. Electronic address: alessandra.pagliarani@unibo.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pagliarani
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D008928:Mitochondria / Q000201:enzymology*; D009840:Oligomycins / Q000494:pharmacology*; D010084:Oxidation-Reduction; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013552:Swine; D014220:Trialkyltin Compounds / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D009840:Oligomycins; D013438:Sulfhydryl Compounds; D014220:Trialkyltin Compounds; C011559:tributyltin; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol
keywords: Macrolide binding region; Mitochondrial F(1)F(O)-ATPase; Oligomycin; Thiol oxidation; Tributyltin; Uncompetitive inhibition