Abstract
The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator. In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b. Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c. In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c. The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane. The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity. ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme. The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions. The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide. These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.
Highlights
Through F0 is reversibly coupled to ATP synthesis or hydrolysis in catalytic sites on F1
The cross-linking of Cys at position 2 of subunit b with Cys at positions 74, 75, and 78 of subunit c supports the previously assumed topological arrangement of these subunits, in which the N-terminal end of subunits b and the C-terminal end of subunit c extend to the periplasmic surface of the membrane (Fig. 7)
Lotscher et al [69] had previously demonstrated crosslinking of the ␣-carboxyl group of the C-terminal residue of subunit c, i.e. Ala-79, with the amino group of phosphatidylethanolamine in a reaction catalyzed by the water-soluble carbodiimide, 1-ethyl-[3-(dimethylamino)propyl]-carbodiimide
Summary
Cross-linking and modeling experiments do indicate that helix-1 of subunit c should be packed on the inside of the ring and helix-2 on the outside [19, 21], and this predicted packing is suggested by the x-ray diffraction map [40]. The elongated, polar, and largely helical C-terminal domain is thought to be anchored to the lipid bilayer by a single Nterminal ␣-helix, which should extend to the periplasmic surface of the bacterial inner membrane [47, 48]. Electron micrographs indicate a second stalk at the periphery of the F1F0 molecule that is presumed to represent a dimer of b subunits extending from F0 to the top of F1 [55,56,57,58]. We report experiments, using subunit b-c cross-link formation, that support the idea of subunit c movement relative to a subunit b stator, as is predicted from rotary models for ATP synthase function
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