Probing conformations of the β subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), α3β3γ, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three β subunits have the “Closed” conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two β subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701–5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant β(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the ε subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the ε subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.