THE 2ND STALK COMPOSED OF THE B-SUBUNITS AND DELTA-SUBUNITS CONNECTS F-0 TO F-1 VIA AN ALPHA-SUBUNIT IN THE ESCHERICHIA-COLI ATP SYNTHASE

The b- and delta-subunits of the Escherichia coli ATP synthase are cri tical for binding ECF1 to the F-0 part, and appear to constitute the s tator necessary for holding the alpha(3)beta(3) hexamer as the c-epsil on-gamma domain rotates during catalysis. Previous studies have determ ined 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 Ca paldi, R. A. (1997) J. Biol. Chem. 272, 31058-31064). To further study b-subunit interactions, three mutants were constructed in which Ser-8 4, Ala-144, and Leu-156, respectively, were replaced by Cys. Treatment of purified ECF1F0 from all three mutants with CuCl2 induced disulfid e formation resulting in b-subunit dimer cross-link products. In addit ion, the mutant bL156C formed a cross-link from a b-subunit to an alph a-subunit via alpha Cys90. Neither b-b nor b-alpha cross-linking had s ignificant effect on ATPase activities in any of the mutants. Proton p umping activities were measured in inner membranes from the three muta nts. Dimerization of the b-subunit did not effect proton pumping in mu tants bS84C or bA144C. In the mutant bL156C, CuCI2 treatment reduced p roton pumping markedly, probably because of uncoupling caused by the b -alpha cross-link formation. The results show that the alpha-subunit f orms part of the binding site on ECF1 for the b(2)delta domain and tha t the b-subunit extends all the way from the membrane to the top of th e F-1 structure. Some conformational flexibility in the connection bet ween the second stalk and F-1 appears to be required for coupled catal ysis.