Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

F O F 1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli , counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F O F 1 . Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits ( b -α, b -β, b -δ, and b-a ) were screened and combined with each other to discriminate between the two b subunits ( i.e. b I and b II ). The results show the b dimer to be located at a non-catalytic α/β cleft, with b I close to subunit α, whereas b II is proximal to subunit β. Furthermore, b I can be linked to subunit δ as well as to subunit a . Among the subcomplexes formed were a-b I -α, b II -β, α- b I - b II -β, and a-b I -δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b I is functionally related to the single b subunit present in mitochondrial ATP synthase. Background: The peripheral stator stalk of Escherichia coli ATP synthase contains two b subunits. Results: Using disulfide bond formation, one b subunit was cross-linked to a , α, and δ and the other to β. Conclusion: The b subunits adopt distinct positions within the stator to generate stability. Significance: The different positions imply different roles in counteracting the torque generated by the rotor.