The ⑀ subunit in F 0 F 1 -ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed ⑀ cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P.
In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.
The a subunit of F(1)F(o) (F(1)F(o)-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H(+)-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F(o) a-less F(1)F(o) complex in Escherichia coli membrane vesicles and liposomes. The resulting F(1)F(o) has a H(+)-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F(1)F(o). By using this procedure, we analysed five mutations of F(1)F(o), where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(o) a subunits were successfully incorporated into F(1)F(o), showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F(1)F(o). The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H(+)-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.
In order to get insight into the origin of apparent negative cooperativity observed for F(1)-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F(1)-ATPase, alpha((W463F)3)beta((Y341W)3)gamma and alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma. For alpha((W463F)3)beta((Y341W)3)gamma, apparent K(m)'s of ATPase kinetics (4.0 and 233 microM) did not agree with apparent K(m)'s deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 microM). On the other hand, in case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, which lacks noncatalytic nucleotide binding sites, the apparent K(m) of ATPase activity (10 microM) roughly agreed with the highest K(m) of fluorescence measurements (27 microM). The results indicate that in case of alpha((W463F)3)beta((Y341W)3)gamma, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent K(m) of ATPase activity does not represent the ATP binding to a catalytic site. In case of alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma, the K(m) of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by alpha((K175A/T176A/W463F)3)beta((Y341W)3)gamma was rather linear compared with that of alpha((W463F)3)beta((Y341W)3)gamma, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F(1)-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed K(m)'s of 100-300 microM and 1-30 microM range for wild-type F(1)-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.
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