Replacement of the F0F1 ATP synthase γ subunit Met-23 with Lys (γM23K) perturbs coupling efficiency between transport and catalysis (Shin, K., Nakamoto, R. K., Maeda, M., and Futai, M. (1992) J. Biol. Chem. 267, 20835-20839). We demonstrate here that the γM23K mutation causes altered interactions between subunits. Binding of δ or ε subunits stabilizes the α3β3γ complex, which becomes destabilized by the mutation. Significantly, the inhibition of F1 ATP hydrolysis by the ε subunit is no longer relieved when the γM23K mutant F1 is bound to F0. Steady state Arrhenius analysis reveals that the γM23K enzyme has increased activation energies for the catalytic transition state. These results suggest that the mutation causes the formation of additional bonds within the enzyme that must be broken in order to achieve the transition state. Based on the x-ray crystallographic structure of Abrahams et al. (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the additional bond is likely due to γM23K forming an ionized hydrogen bond with one of the βGlu-381 residues. Two second site mutations, γQ269R and γR242C, suppress the effects of γM23K and decrease activation energies for the γM23K enzyme. We conclude that γM23K is an added function mutation that increases the energy of interaction between γ and β subunits. The additional interaction perturbs transmission of conformational information such that ε inhibition of ATPase activity is not relieved and coupling efficiency is lowered.
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