The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

The peripheral stalk of F1F0 ATP synthase is essential for the binding of F-1 to F-0 and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b' subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463-5471). The spatial arrangement of the chimeric b and b' subunits, abbreviated Tb and Tb', has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between Tb-A83C and Tb'(A90C), indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, non-staggered parallel conformation may also exist.