Inhibition of thermophilic F1-ATPase by the ε subunit takes different path from the ADP-Mg inhibition

Abstract

The F₁-ATPase, the soluble part of FoF₁-ATP synthase, is a rotary molecular motor consisting of α₃β₃γδε. The γ and ε subunits rotate relative to the α₃β₃δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F₁-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F₁-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α₃β₃γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

Keywords: ADP inhibited form; ATP synthase; regulation.

Figure 1.

Time-course of the rotation of beads attached to TF₁(−ε), TF₁(ε^WT) and TF₁(ε^R126A). Typical examples of time-course of bead rotation at 200 μM ATP with (A) TF₁(−ε), (B) TF₁(ε^WT) and (C) TF₁(ε^R126A) are shown. Data with similar bead sizes are shown.

Figure 2.

Distribution of inactive state durations. The durations of the inactive state were measured and their distributions are shown. Samples used are indicated on the top of each column. The ATP concentrations were 20 μM, 200 μM, and 2 mM, respectively from the top to the bottom rows. Each distribution was fitted with the sum of two exponential functions: y = N₁ exp (−t/τ_i1) + N₂ exp (−t/τ_i2). The best-fit values of τ_i1, τ_i2, N₁, and N₂ are shown on each panel. Solid lines show the best-fit curves. τ_i2 and N₂ for TF₁(ε^WT) with 20 μM ATP could not be obtained due to no convergence.

Figure 3.

Distribution of active state durations. The durations of the active state were measured and their distributions are shown. Samples used are indicated on the top of each column. The ATP concentrations were 20 μM, 200 μM, and 2 mM, respectively from the top to the bottom rows. Each distribution was fitted with a single exponential function: y = N exp (−t/τ_a). The best-fit values of τ_a and N are shown on each panel. Solid lines show the best-fit curves. Insets in TF(ε^R126A) with 20 and 200 μM are enlargements along the x-axis.

Figure 4.

Schematic model of the relationship between ε inhibition and ADP-Mg inhibition. F₁, F₁*ε and F₁*ADP-Mg represent F₁ in active state, ε inhibited state and ADP-Mg inhibited state, respectively. The time constant for each path at 200 μM ATP is shown. The time constant for recovery from ε inhibition was taken from τ_i1 for TF₁(ε^R126A). Values shown in parentheses, which represent time constants for the ADP-Mg inhibition, are from τ_a and τ_i1 for TF₁(−ε). The time constant for the ε inhibition is calculated as 1/(1/τ_a(TF₁(ε^R126A)) − 1/τ_a(TF₁(−ε))) assuming that τ_a(TF₁(ε^R126A)) contained contributions both from the ε inhibition and from the ADP-Mg inhibition (τ_a(TF₁(−ε))).

Figure 5.

Comparison of relative ATPase activity and occupancy of the active state. The occupancy of the active state was the sum of the durations in the active state divided by the same sum plus the sum for the inactive state. The obtained occupancy for TF₁(ε^R126A) was divided by that for TF₁(−ε) and plotted against the ATP concentration (closed circles). ATPase activity of TF₁(ε^R126A) was divided by that of TF₁(−ε) and plotted against the ATP concentration (open squares).

Figure 6.

Activation of TF₁(ΔNC) by the addition of ε^R126A. The ATPase activity of TF₁(ΔNC) was measured by the decrease of absorbance at 340 nm using the NADH-coupled ATP-regenerating system at 50 μM ATP. The reaction was initiated by the addition of TF₁(ΔNC) lacking ε at the time indicated by the left arrowhead. ε^WT, ε^R126A (360 nM), or buffer (−ε), was added at the time indicated by the right arrowhead. Vertical and horizontal bars denote 0.01 absorbance and 200 s, respectively.