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Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coli FoF1-ATP Synthase*

Open AccessPublished:May 01, 2009DOI:https://doi.org/10.1074/jbc.M109.003798
      The ϵ subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal α-helices of ϵ, and the “extended” but not “hairpin-folded” state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of ϵ has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the ϵ subunit lacked the C-terminal domain (FoF1ϵΔC), and ATP synthesis driven by acid-base transition (ΔpH) and the K+-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (FoF1ϵWT). The turnover numbers (kcat) of FoF1ϵWT were severalfold lower than those of FoF1ϵΔC. FoF1ϵWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1ϵWT and FoF1ϵΔC on various combinations of ΔpH and ΔΨ was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of ϵ. Solubilized FoF1ϵWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1ϵΔC. These results suggest that the C-terminal domain of the ϵ subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit.
      FoF1-ATP synthase (FoF1)
      The abbreviations used are: FoF1
      FoF1-ATP synthase
      F1
      F1-ATPase
      EFoF1
      FoF1 from E. coli
      EF1
      F1 from E. coli
      TFoF1
      FoF1 from thermophilic Bacillus PS3
      TF1
      F1 from thermophilic Bacillus PS3
      FoF1ϵΔC
      FoF1 lacking the two α-helices at the C terminus of the ϵ subunit
      FoF1ϵWT
      wild-type FoF1
      C12E8
      octaethylene glycol mono-n-dodecyl ether
      PAB
      4-aminobenzamidine dihydrochloride
      TNP-ATP
      2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate.
      3The abbreviations used are: FoF1
      FoF1-ATP synthase
      F1
      F1-ATPase
      EFoF1
      FoF1 from E. coli
      EF1
      F1 from E. coli
      TFoF1
      FoF1 from thermophilic Bacillus PS3
      TF1
      F1 from thermophilic Bacillus PS3
      FoF1ϵΔC
      FoF1 lacking the two α-helices at the C terminus of the ϵ subunit
      FoF1ϵWT
      wild-type FoF1
      C12E8
      octaethylene glycol mono-n-dodecyl ether
      PAB
      4-aminobenzamidine dihydrochloride
      TNP-ATP
      2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate.
      is an enzyme that is responsible for ATP synthesis during oxidative phosphorylation and photosynthesis (
      • Boyer P.D.
      ,
      • Senior A.E.
      • Nadanaciva S.
      • Weber J.
      ,
      • Capaldi R.A.
      • Aggeler R.
      ). FoF1 is a complex of two rotary motors F1 and Fo, and the ATP synthesis/hydrolysis reaction that is reversibly catalyzed by F1 is coupled with proton transport across membrane-embedded Fo (
      • Noji H.
      • Yasuda R.
      • Yoshida M.
      • Kinosita Jr., K.
      ,
      • Yoshida M.
      • Muneyuki E.
      • Hisabori T.
      ,
      • Kinosita Jr., K.
      • Adachi K.
      • Itoh H.
      ). The subunit composition of bacterial F1 and Fo is α3β3γδϵ and ab2c10–15, respectively, and the γϵc10–15 complex rotates against the α3β3δab2 complex in FoF1.
      Among these subunits, ϵ is known to be an endogenous inhibitor of the ATP hydrolysis reaction catalyzed by F1 and FoF1 (
      • Sternweis P.C.
      • Smith J.B.
      ,
      • Capaldi R.A.
      • Schulenberg B.
      ,
      • Vik S.B.
      ,
      • Feniouk B.A.
      • Suzuki T.
      • Yoshida M.
      ). The inhibition of ATP hydrolysis by the ϵ subunit of Escherichia coli F1 (EF1) and FoF1 (EFoF1) has been extensively studied. Addition of ϵ to ϵ-depleted EF1 showed noncompetitive inhibition of ATP hydrolysis (
      • Sternweis P.C.
      • Smith J.B.
      ). The affinity of MgATP and MgADP to the high affinity site of the three catalytic β subunits of EF1 was decreased by ϵ (
      • Weber J.
      • Dunn S.D.
      • Senior A.E.
      ). It has been reported that the ϵ subunit of EF1 had no effect on the equilibrium between ATP and ADP·Pi but inhibited product release under unisite catalysis conditions (
      • Dunn S.D.
      • Zadorozny V.D.
      • Tozer R.G.
      • Orr L.E.
      ). Inhibition of EFoF1-mediated ATP hydrolysis by ϵ was also demonstrated in experiments involving partial digestion by a protease (
      • Mendel-Hartvig J.
      • Capaldi R.A.
      ). The inhibitory effect of the ϵ subunit of thermophilic Bacillus PS3 F1 (TF1) and FoF1 (TFoF1) has also been studied extensively. Slow binding and hydrolysis of TNP-ATP, a fluorescent ATP analog, under unisite catalysis conditions have been reported (
      • Kato Y.
      • Matsui T.
      • Tanaka N.
      • Muneyuki E.
      • Hisabori T.
      • Yoshida M.
      ). However, in contrast to EF1, inhibition by the ϵ subunit of TF1 was relieved slowly and apparently disappeared at high ATP concentrations ([ATP]). This is not due to the dissociation of ϵ from the F1 complex, because disappearance of inhibition at high [ATP] was also reported in TFoF1 in which ϵ is indispensable for stable complex formation (
      • Kato-Yamada Y.
      • Bald D.
      • Koike M.
      • Motohashi K.
      • Hisabori T.
      • Yoshida M.
      ). However, in contrast to ATP hydrolysis, there has been no detailed analysis of the effect of ϵ on ATP synthesis.
      The ϵ subunit has a molecular mass of 14 kDa and a two-domain structure consisting of an N-terminal 10-stranded β-sandwich and two C-terminal α-helices. Of these two domains, the C-terminal domain is responsible for inhibiting ATP hydrolysis, and the ϵ subunit in which this domain is absent does not have any inhibitory effect (
      • Kato-Yamada Y.
      • Bald D.
      • Koike M.
      • Motohashi K.
      • Hisabori T.
      • Yoshida M.
      ,
      • Xiong H.
      • Zhang D.
      • Vik S.B.
      ). Structural studies on isolated ϵ and its complex with the truncated γ subunit have shown that the C-terminal domain of ϵ adopts two different conformations, the “hairpin-folded” and “extended” states (Fig. 1) (
      • Wilkens S.
      • Dahlquist F.W.
      • McIntosh L.P.
      • Donaldson L.W.
      • Capaldi R.A.
      ,
      • Uhlin U.
      • Cox G.B.
      • Guss J.M.
      ,
      • Wilkens S.
      • Capaldi R.A.
      ,
      • Rodgers A.J.
      • Wilce M.C.
      ). These conformations are also found in the crystal structure of the bovine mitochondrial homolog of F1 and in the low resolution crystal structure of EF1 (
      • Gibbons C.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ,
      • Hausrath A.C.
      • Capaldi R.A.
      • Matthews B.W.
      ). Chemical modification of the ϵ subunit of EFoF1 indicated that the C-terminal domain is intrinsically flexible (
      • Ganti S.
      • Vik S.B.
      ). Cross-linking and fluorescence resonance energy transfer experiments supported the existence of multiple conformations of the ϵ subunit of EFoF1 and TFoF1 (
      • Schulenberg B.
      • Capaldi R.A.
      ,
      • Kato-Yamada Y.
      • Yoshida M.
      • Hisabori T.
      ,
      • Tsunoda S.P.
      • Rodgers A.J.
      • Aggeler R.
      • Wilce M.C.
      • Yoshida M.
      • Capaldi R.A.
      ,
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ,
      • Bulygin V.V.
      • Duncan T.M.
      • Cross R.L.
      ,
      • Iino R.
      • Murakami T.
      • Iizuka S.
      • Kato-Yamada Y.
      • Suzuki T.
      • Yoshida M.
      ,
      • Zimmermann B.
      • Diez M.
      • Zarrabi N.
      • Gräber P.
      • Börsch M.
      ).
      Another kind of “extended” state was proposed in TFoF1 (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ); however, for simplicity, we did not distinguish between the two extended forms in this manuscript.
      4Another kind of “extended” state was proposed in TFoF1 (
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ); however, for simplicity, we did not distinguish between the two extended forms in this manuscript.
      These studies have shown that the extended state inhibits ATP hydrolysis, whereas the hairpin-folded state does not.
      Figure thumbnail gr1
      FIGURE 1Crystal structures of the ϵ subunit (blue) of EF1 in the extended state (right, Protein Data Bank code 1JNV) (
      • Hausrath A.C.
      • Capaldi R.A.
      • Matthews B.W.
      ) and the δ subunit (equivalent to bacterial ϵ) of F1 from bovine mitochondria in the hairpin-folded state (left, Protein Data Bank code 1E79) (
      • Gibbons C.
      • Montgomery M.G.
      • Leslie A.G.
      • Walker J.E.
      ). The C-terminal α-helices of the ϵ subunit (enclosed by black lines) of EFoF1 were truncated by introducing a stop codon at the position of Asp-91.
      In contrast to ATP hydrolysis, the correlation between the conformation of ϵ and its effect on ATP synthesis has not been fully understood. In both EFoF1 and TFoF1, when ϵ was fixed in an extended state that inhibits ATP hydrolysis, no change was observed in the ATP synthesis activity driven by the proton motive force (Δμ) generated by the respiratory chain in the inverted membrane (
      • Tsunoda S.P.
      • Rodgers A.J.
      • Aggeler R.
      • Wilce M.C.
      • Yoshida M.
      • Capaldi R.A.
      ,
      • Suzuki T.
      • Murakami T.
      • Iino R.
      • Suzuki J.
      • Ono S.
      • Shirakihara Y.
      • Yoshida M.
      ). Based on these results, it has been proposed that ϵ functions as a ratchet that inhibits only ATP hydrolysis. However, Masaike et al. (
      • Masaike T.
      • Suzuki T.
      • Tsunoda S.P.
      • Konno H.
      • Yoshida M.
      ) reported that truncation of the C-terminal domain of ϵ increased the ATP synthesis activity of TFoF1, suggesting that the C-terminal domain of ϵ suppresses the ATP synthesis activity.
      In this study, we generated an EFoF1 mutant with a truncated ϵ subunit that did not contain the C-terminal α-helices (FoF1ϵΔC). This mutant was purified and reconstituted into a liposome, and the ATP synthesis rate was measured by the acid-base transition (ΔpH) and K+-valinomycin diffusion potential (ΔΨ) methods. The rate of ATP hydrolysis by solubilized EFoF1 was also measured. The activities of FoF1ϵΔC under various conditions were investigated and compared in detail with those of the wild-type enzyme (FoF1ϵWT). The results indicated that the ATP synthesis and hydrolysis activities of FoF1ϵΔC were much higher than those of FoF1ϵWT. The inhibitory mechanism of the C-terminal α-helices of the ϵ subunit of EFoF1 was discussed.

      Acknowledgments

      We thank Drs. Nobuhito Sone, Masatoshi Toei, Masahiro Nakano, and Ken Yokoyama for technical advice and members of the Noji laboratory for helpful discussions.

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