Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme.

TitleInsights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme.
Publication TypeJournal Article
Year of Publication2000
AuthorsJones, PC, Hermolin, J, Jiang, W, Fillingame, RH
JournalJ Biol Chem
Date Published2000 Oct 06
KeywordsCatalysis, Cross-Linking Reagents, Dimerization, Escherichia coli, Hydrolysis, Intracellular Membranes, Lipid Bilayers, Microscopy, Electron, Models, Biological, Plasmids, Protein Structure, Tertiary, Proton-Translocating ATPases, Protons, Spectrometry, Fluorescence, Time Factors

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.

Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.M003687200
PubMed ID10882728
Grant ListGM23105 / GM / NIGMS NIH HHS / United States