3glu381 substitutions implicate a role in energy coupling for the β380delseed386 loop of the fof1 atp synthase

The Escherichia coli F0F1 ATP synthase links the transport of protons across a membrane and the catalysis of ATP synthesis or hydrolysis. We previously reported that changing a single amino acid within the 7 subunit, 7M23K, caused a profound perturbation in energy coupling by increasing the energy of interaction between the γ and β subunits (Al-Shawi, M. K., Ketchum, C. J., and Nakamoto, R. K. (1997) J. Biol. Chem. 272, 2300-2306). The crystal structure of bovine mitochondrial F1 predicts that 7M23K is in a position to influence function based on its proximity to the conserved 33DELSEED386 loop and likely forms an ionized hydrogen bond with 3Glu381. The structure also suggests that βGlu381 directly interacts with 7Arg242 in the wild-type enzyme. We tested the involvement of 3Glu381 and γArg242 in the coupling mechanism by introducing a series of substitutions at both positions. Of these mutations βE381A, D and Q restored ATP-dependent H+ pumping to the γM23K mutant enzyme, thus confirming a functional interaction between these residues. Significantly, 3E381K alone uncoupled transport and catalysis. 3E381A, L, K and R, and 7R242L and E mutations disturbed enzyme assembly and stability to varying degrees. These results provide the first functional evidence that the 33aDELSEED3S6 loop and its interactions with the 7 subunit are involved in the coupling mechanism.