The second stalk composed of the b- and δ-subunits connects F0 to F1 via an α-subunit in the Escherichia coli ATP synthase

The b- and delta-subunits of the Escherichia coli ATP synthase are critical for binding ECF1 to the F-0 part, and appear to constitute the stator necessary for holding the alpha(3)beta(3) hexamer as the c-epsilon-gamma domain rotates during catalysis. Previous studies have determined that the b- subunits are dimeric for a large part of their length, and interact with the F-1 part through the delta-subunit (Rodgers, A. J. W., Wilkens, S., Aggeler, R, Morris, M. B., Howitt, S. M., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 31058-31064). To further study b-subunit interactions, three mutants were constructed in which Ser-84, Ala-144, and Leu-156, respectively, were replaced by Cys. Treatment of purified ECF1F0 from all three mutants with CuCl2 induced disulfide formation resulting in b-subunit dimer cross-link products. In addition, the mutant bL156C formed a cross-link from a b-subunit to an alpha-subunit via alpha Cys90. Neither b-b nor b-alpha cross-linking had significant effect on ATPase activities in any of the mutants. Proton pumping activities were measured in inner membranes from the three mutants. Dimerization of the b-subunit did not effect proton pumping in mutants bS84C or bA144C. In the mutant bL156C, CuCI2 treatment reduced proton pumping markedly, probably because of uncoupling caused by the b-alpha cross-link formation. The results show that the alpha-subunit forms part of the binding site on ECF1 for the b(2)delta domain and that the b-subunit extends all the way from the membrane to the top of the F-1 structure. Some conformational flexibility in the connection between the second stalk and F-1 appears to be required for coupled catalysis.