Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme.
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.
Jones
P C
PC
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Hermolin
J
J
Jiang
W
W
Fillingame
R H
RH
D002384:Catalysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008854:Microscopy, Electron; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011522:Protons; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D003432:Cross-Linking Reagents; D008051:Lipid Bilayers; D011522:Protons; D006180:Proton-Translocating ATPases