In Escherichia coli F 1 F 0 -ATP synthase, the two b subunits form the second stalk spanning the distance between the membrane F 0 sector and the bulk of F 1. Current models predict that the stator should be relatively rigid and engaged in contact with F 1 at fixed points. To test this hypothesis, we constructed a series of deletion mutations in the uncF(b) gene to remove segments from the middle of the second stalk of the subunit. Mutants with deletions of 7 amino acids were essentially normal, and those with deletions of up to 11 amino acids retained considerable activity. Membranes prepared from these strains had readily detectable levels of F 1 -ATPase activity and proton pumping activity. Removal of 12 or more amino acids resulted in loss of oxidative phosphorylation. Levels of membrane-associated F 1 -ATPase dropped precipitously for the longer deletions, and immunoblot analysis indicated that reductions in activity correlated with reduced levels of b subunit in the membranes. Assuming the likely ␣-helical conformation for this area of the b subunit, the 11-amino acid deletion would result in shortening the subunit by approximately 16 Å. Since these deletions did not prevent the b subunit from participating in productive interactions with F 1 , we suggest that the b subunit is not a rigid rodlike structure, but has an inherent flexibility compatible with a dynamic role in coupling.The proton-translocating adenosine triphosphate (F 1 F 0 -ATP) synthases are responsible for the majority of ATP synthesis in most organisms (1-3). Enzymes of the F 1 F 0 -ATP synthase family are located in the mitochondria, chloroplast, and bacterial membranes. The energy of the electrochemical gradient across these membranes is used to drive ATP production. In the Escherichia coli enzyme, the F 1 sector consists of five dissimilar subunits (␣ 3 ,  3 , ␥, ␦, ⑀) and houses the catalytic sites for ATP synthesis or ATP hydrolysis, and the F 0 sector consists of three integral membrane subunits (a, b 2 , c 9 -12 ) and carries out proton conduction.Electron microscopy of E. coli F 1 F 0 -ATP synthase revealed that the F 1 and F 0 sectors are joined by two narrow stalks 40 -45 Å in length (4). Rotation of a central stalk complex comprising the ␥ and ⑀ subunits, the "rotor," is essential to the catalytic mechanism of F 1 F 0 -ATP synthase (5-7). A thinner peripheral second stalk consisting of the ␦ and two b subunits is thought to form the "stator," which holds the catalytic complex stationary while the central stalk rotates. The stator concept was strongly supported by the observation that the ␦ subunit can be fixed to a specific ␣ subunit without loss of activity (8). The amino-terminal hydrophobic domain of the b subunit forms a single transmembrane-spanning domain while the bulk of the protein extends toward the F 1 sector. More than a decade ago, proteolysis experiments showed that the cytoplasmic domain of the b subunit is required for binding F 1 to F 0 (9 -11). The b subunit is thought to interact with one of the  subunits (1...
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