Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.
(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.
Weber
J
J
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Lee
R S
RS
Grell
E
E
Senior
A E
AE
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001313:Aurovertins / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001313:Aurovertins; C055520:linear-benzoadenosine diphosphate; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases