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

The transmembrane sector of the F0F1 rotary ATP synthase is proposed to org anize with an oligomeric ring of c subunits, which function as a rotor, int eracting with two b subunits at the periphery of the ring, the b subunits f unctioning 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 o f N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of su bunit c, In each case, a maximum of 50% of the b subunit could be cross-lin ked 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 Nand 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 mode l for ATP synthase function, but unexpectedly, cross-linking did not lead t o inhibition of ATPase activity. ATP hydrolysis and proton pumping are ther efore 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 init ially occupied non-neighboring positions. The movement or exchange of subun its at the position adjacent to subunit b was blocked by dicyclohexylcarbod iimide. These experiments provide a biochemical verification that the oligo meric c-ring can move with respect to the b-stator and provide further supp ort for a rotary catalytic mechanism in the ATP synthase.