PMID:10882728

Citation	Jones, PC, Hermolin, J, Jiang, W and Fillingame, RH (2000) Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme. J. Biol. Chem. 275:31340-6
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.
Links	PubMed Online version:10.1074/jbc.M003687200
Keywords	Catalysis; Cross-Linking Reagents/pharmacology; Dimerization; Escherichia coli/enzymology; Hydrolysis; Intracellular Membranes/metabolism; Lipid Bilayers/chemistry; Microscopy, Electron; Models, Biological; Plasmids/metabolism; Protein Structure, Tertiary; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/metabolism; Protons; Spectrometry, Fluorescence; Time Factors
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