Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase.
In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.
Krebstakies
Thomas
T
Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Germany.
Zimmermann
Boris
B
Gräber
Peter
P
Altendorf
Karlheinz
K
Börsch
Michael
M
Greie
Jörg-Christian
JC
D025181:ATP Synthetase Complexes / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002232:Carbocyanines; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008081:Liposomes; D021122:Protein Subunits; D012235:Rhodamines; C085321:cyanine dye 5; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate; D025181:ATP Synthetase Complexes; D025243:Bacterial Proton-Translocating ATPases; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases