238 BiochimicaetBiophysicaActa 807 (1985) 238-244 Elsevier BBA41740 Inhibition of Escherichia coli H +-ATPase by venturicidin, oligomycin and ossamycin David S. Perlin *, Lisa R. Latchney and Alan E. Senior Department of Biochemistry, P.O. Box 607, University of Rochester Medical Center, Rochester, N Y 14642 (U.S.A.) (ReceivedNovember7th, 1984) Key words: Venturicidin; Oligomycin; Ossamycin;Proton translocation:H+-ATPase; (E. coli) The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coil H +-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H +-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclobexyl[14C]carbodiimide and the F 0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coil F o or FI; rather, it was found to uncouple ATP hydrolysis from proton transport. Introduction The H+-ATPase (F1F0) of Escherichia coli membranes catalyses ATP synthesis and the for- mation of ATP-driven proton electrochemical gradients, and resembles analogous enzymes from mitochondria, chloroplasts and other bacteria [1,2]. Experiments with specific inhibitors have provided important information about the partial reactions involved in ATP synthesis and ATP-driven proton translocation. The antibiotics oligomycin, venturi- cidin and ossamycin inhibit the mitochondrial H÷-ATPase by interacting with the F0-sector to block proton translocation [3]. These antibiotics are complex hydrophobic compounds whose chemical properties and structures have been well-defined [4]. Oligomycin is reported to be quite specific for * To whomcorrespondenceshouldbe addressed. Abbreviations: DCCD, N,N'-dicyclohexyl[14C]carbodiimide; DMSO, dimethyl sulfoxide. H +-ATPases from mitochondria or certain photo- synthetic bacteria [4], and has been reported not to inhibit H+-ATPases from chloroplasts [4], E. coli [5] or other bacteria [4]. Venturicidin and ossamy- cin strongly inhibit mitochondrial H+-ATPase [3,4]; venturicidin has also been reported to inhibit H+-ATPase from P. denitrificans [6]. How- ever, the mechanism of inhibition of proton trans- port by these antibiotics remains obscure due, in part, to the complex and unresolved structure of the mitochondrial F0-sector. In E. coil, the F0-sec- tor appears simpler and better characterized, con- sisting of only three different subunits, each of which has been sequenced [1,2]. Several authors have predicted secondary and tertiary structures of E. coli F0-subunits [7-9]. Therefore, it seemed of value to investigate the action of these antibiotics on the E. coli enzyme. In this report we describe for the first time the effects of venturicidin and ossamycin on the H ÷- ATPase from E. coli, together with a reexamina- tion of the effects of oligomycin. 0005-2728/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)