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TheE subunit of bacterial F[sub0]F[sub1]-ATP synthase (F[sub0]F[sub1]), 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 F[sub0]F[sub1] (EF[sub0]F[sub1]) mutant in which the &3x20AC; subunit lacked the C-terminal domain (F[sub0]F[sub1][supϵΔC]), and ATP synthesis driven by acid-base transition (ΔpH) and the K[sup+]-valinomycin diffusion potential (ΔΨ) was compared in detail with that of the wild-type enzyme (F[sub0]F[sub1][supϵWT]). The turnover numbers (kcat) of F[sub0]F[sub1][supϵWT] were severalfold lower than those of F[sub0]F[sub1][supϵΔC]. F[sub0]F[sub1][supϵWT] showed higher Michaelis constants (K,,). The dependence of the activities of F[sub0]F[sub1][supϵWT] and F[sub0]F[sub1][supϵΔC] 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 F[sub0]F[sub1][supϵWT] also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of F[sub0]F[sub1][supϵΔC]. These results suggest that the C-terminal domain of the ϵ subunit of EF[sub0]F[sub1] slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the γ subunit. [ABSTRACT FROM AUTHOR]

It has been proposed that C-terminal two α-helices of the ∈ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the γ subunit and C terminus of the β subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic/3 subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the β subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 °C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 °C reached to 0.67 mM, a consistent value to assume that the ∈ subunit acts as a sensor of ATP concentration in the cell. [ABSTRACT FROM AUTHOR]

F1-ATPase (F1) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α3β3 stator ring. The 3 catalytic sites of F1 reside on the interface of the α and β subunits of the α3β3 ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F1 remains controversial.Westudied the role of the arginine finger by analyzing the rotation of a mutant F1 with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F1 showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F1 carrying a single copy of the β mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATPbinding rate. [ABSTRACT FROM AUTHOR]

The ∈ subunit in F[sub 0]F[sub 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., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560-6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of ∈ are fully extended to insert the C terminus into a deeper position in the central cavity of F[sub 1] than was thought previously. (ii) Without a nucleotide, ∈ is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state ∈ can bind an ATP analogue, 2',3'O-(2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state ∈. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F[sub 0]F[sub 1]-ATPase/synthase would transform the ∈ subunit into the up-state conformation and change gear to the mode for ATP synthesis. [ABSTRACT FROM AUTHOR]

V 1 -ATPase (V 1 ), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis-derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V 1 from the pathogen Enterococcus hirae (EhV 1 ). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 10 7 m -1 s -1 At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, -80 and -40° backward steps were observed. The time constants of the pauses before -80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV 1 , including substeps largely different from those of F 1 -ATPases., (© 2019 Iida et al.)

F(1)-ATPase is a rotary molecular motor in which the gamma-subunit rotates against the alpha(3)beta(3) cylinder. The unitary gamma-rotation is a 120 degrees step comprising 80 and 40 degrees substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on gamma-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F(1)-ATPase from thermophilic Bacillus PS3 was found below 9 degrees C as an intervening pause before the 80 degrees substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F(1)(beta E190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 degrees C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F(1), carrying a single copy of beta E190D, was observed at 18 degrees C. The hybrid F(1) showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated beta E190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of beta before or after ATP binding.

The epsilon 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 alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon 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 epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5'-Adenyl-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.