F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of ε subunit in response to proton motive force and ADP/ATP balance

The epsilon subunit in F0F1-ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin down-state and an extended up-state, and the enzyme with the up-fixed epsilon cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. ( 2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560 - 6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of epsilon are fully extended to insert the C terminus into a deeper position in the central cavity of F-1 than was thought previously. (ii) Without a nucleotide, epsilon is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state epsilon can bind an ATP analogue, 2', 3'-O-( 2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state epsilon. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F0F1-ATPase/synthase would transform the epsilon subunit into the up-state conformation and change gear to the mode for ATP synthesis.