Movement of the helical domain of the ε subunit is required for the activation of thermophilic F1-ATPase

The inhibitory effect of epsilon subunit in F-1-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed: a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B,, and Capaldi, R, A. (1999) J, Biol, Chem. 274, 28351-28355), We introduced two cysteine residues at the interface of N-terminal beta -sandwich domain (S48C) and C-terminal cu-helical domain (N125C) of epsilon subunit, The alpha (3)beta (3)gamma epsilon complex containing the reduced form of this mutant epsilon subunit showed suppressed ATPase activity and gradual activation during the measurement. This activation pattern was similar to the complex with the wild type epsilon subunit, The conformation of the mutant epsilon subunit must be fixed and similar to the reported three-dimensional structure of the isolated epsilon subunit, when the intramolecular disulfide bridge was formed on this subunit by oxidation. This oxidized mutant epsilon subunit could form the alpha (3)beta (3)gamma epsilon complex but did not show any inhibitory effect. The complex was converted to the activated state, and the cross-link in the mutant epsilon subunit in the complex was efficiently formed in the presence of ATP-Mg, whereas no cross-link was observed without ATP-Mg, suggesting the conformation of the oxidized mutant epsilon subunit must be similar to that in the activated state complex. A non-hydrolyzable analog of ATP, 5'-adenylyl-beta,gamma -imidodiphosphate, could stimulate the formation of the cross-link on the epsilon subunit, Furthermore, the cross-link formation was stimulated by nucleotides even when this mutant epsilon subunit was assembled with a mutant alpha (3)beta (3)gamma complex lacking non-catalytic sites. These results indicate that binding of ATP to the catalytic sites induces a conformational change in the epsilon subunit and triggers transition of the complex from the suppressed state to the activated state.