Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

Abstract

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.

Figure 1

Comparison of the arrangement of the ɛ subunit (mitochondrial δ subunit) between (A) bovine mitochondria and (B) E. coli F₁-ATPase. The bottom part of the subunit γ is shown in blue, the subunit ɛ (mitochondrial δ) in green, and c subunit ring in light gray. The positions of Cys residues mutated in this study are shown with space-filling spheres in red. The bottom parts of an α and a β subunit of α₃β₃ are shown in white. The residues are numbered from the E. coli sequence. The two models were created based on the coordinates of the bovine heart MF₁-ATPase (1E79), and E. coli γɛ subunits (1FS0) and the unrefined c_α model (1Q01) in the Protein Data Bank.

Figure 2

Formation of the ɛ–cc′ and γ–ɛ cross-links via disulfide bonds in the EF₁F₀ mutants. Inner membranes from the wild-type and the two EF₁F₀ mutants were exposed to 100 μM CuCl₂ to induce the cross-linking. As a control, 1 mM DTT was added instead of CuCl₂. The samples were loaded on the SDS/PAGE (15%). The dissociation buffer for SDS/PAGE contained 40 mM N-ethylmaleimide but no reducing agent. The cross-linked products were identified with anti-γ, ɛ, and c subunit immunoblotting, respectively (the data from the anti-γ, and c subunit antibodies are not shown.).

Figure 3

Effect of cross-linking on the ATP hydrolysis and ATP-driven proton translocation. (A) Effect of the cross-linking on ATPase activity and inhibitor sensitivity. The inner membranes from wild-type and mutants were treated with CuCl₂ or DTT as described in Fig. 2. The ATPase activity was measured in the presence of an ATP regenerating system. Samples were also incubated for 60 min at 23°C with 40 μM of DCCD, the specific inhibitor, before ATPase activity was measured. The inhibition was calculated as percentage of the activity without DCCD treatment. Inner membrane reacted with DTT, filled; CuCl₂, diagonal stripes. (B) Effect of the cross-linking on ATP-driven proton translocation. The proton pumping ability of the inner membranes from wild-type and mutants was determined by monitoring the decrease of the fluorescence intensity of ACMA. Before the assay, the inner membranes were treated with DTT or CuCl₂ as described in Fig. 2. At the time indicated by arrow heads, 0.5 mM NADH, 10 mM KCN, 2 mM ATP, and 3.6 μM nigericin were added respectively. Inner membrane treated with DTT (solid line), with CuCl₂ (dotted line). Vertical bar, 30% of relative fluorescence; horizontal bar, 100 s.

Figure 4

Effect of cross-linking on ATP synthesis. The inner membranes from wild-type and mutants were exposed to 2 mM NADH at 37°C to generate a proton gradient. The data show the amount of ATP produced by 1 mg of inner membrane protein. Solid line, DTT; dashed line, CuCl₂-treated membranes as described in Fig. 2. Before the assay, the samples were reacted with (open circle) or without (filled square) 40 μM DCCD for 60 min at 23°C.