Large conformational changes of the ɛ subunit in the bacterial F ₁ F ₀ ATP synthase provide a ratchet action to regulate this rotary motor enzyme

Abstract

The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the ␥ and subunits in the F1 part and c subunit ring in the F0 part, rotates relative to a stator composed of ␣3␤3␦ab2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the subunit plays a key role by acting as a switch of this motor. Two different arrangements of the subunit have been visualized recently. The first has been observed in beef heart mitochondrial F1-ATPase where the C-terminal portion is arranged as a two-␣-helix hairpin structure that extends away from the ␣3␤3 region, and toward the position of the c subunit ring in the intact F 1F0. The second arrangement was observed in a structure determination of a complex of the ␥ and subunits of the Escherichia coli F1-ATPase. In this, the two Cterminal helices are apart and extend along the ␥ to interact with the ␣ and ␤ 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 1F0, confirming that both conformations of the subunit exist in the enzyme complex. With the C-terminal domain of toward the F0, 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 subunit in the F1F0 complex and argue for a ratchet function of this subunit.

Rotor/Stator Interactions of the ϵ Subunit in Escherichia coli ATP Synthase and Implications for Enzyme Regulation

“…With an 80% yield in the cross-linking reaction, it was found that ATP hydrolysis was inhibited to a greater extent than ATP synthesis (75% versus 25%). A similar differential inhibition of hydrolysis and synthesis activities was reported earlier (35) with ⑀ trapped in the open conformation (Fig. 1C).…”

“…This possibility was suggested by the open conformation of ⑀ (Fig. 1C) observed in an ⑀/␥-fragment complex (45) and by a cross-linking experiment, which showed that ⑀ can adopt a similar conformation in EcF 0 F 1 (35). However, the latter study did not eliminate the possibility that the N-terminal domain of ⑀ might have to detach from the c-ring before the C-terminal domain of ⑀ can interact with the ␣ 3 ␤ 3 hexamer (25).…”

“…The closed conformation of ⑀ can also be fixed in EcF 0 F 1 by disulfide cross-linking of substituted cysteines without loss of ATP synthesis activity (34,35). However, for some bacterial and chloroplast synthases, the C-terminal domain of ⑀ appears to modulate the ATPase activity in a manner that suggests a possible regulatory role (34, 36 -38).…”

“…1C), would interact with the bottom of the ␣ 3 ␤ 3 hexamer. A disulfide cross-link (␥L99C-⑀S118C) was used to confirm that the Cterminal domain of ⑀ can adopt this open conformation in EcF 0 F 1 (35). However, this structure is also unable to account for ␤-⑀ cross-links involving ⑀S108C, because the ␤-carbons of ⑀Ser-108 and the nearest ␤Asp-380 are still far too distant (ϳ25 Å; Fig.…”

ATP Synthase with Its γ Subunit Reduced to the N-terminal Helix Can Still Catalyze ATP Synthesis

“…Fixing ⑀ in either up conformation by cross-linking to ␥ has been shown to impair ATP hydrolysis but not synthesis. Freezing ⑀ in the down position inhibited neither reaction (29,30).…”