Intergenic suppression of the γM23K uncoupling mutation in F0F1 ATP synthase by βGlu-381 substitutions: the role of the β380DELSEED386 segment in energy coupling

Abstract

We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, gammaM23K, caused increased energy of interaction between gamma- and beta-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] gammaM23K is believed to form an ionized hydrogen bond with betaGlu-381 in the conserved beta380DELSEED386 segment. In this report, we further test the role of gamma-beta-subunit interactions by introducing a series of substitutions for betaGlu-381 and gammaArg-242, the residue which forms a hydrogen bond with betaGlu-381 in the wild-type enzyme. betaE381A, D, and Q were able to restore efficient coupling when co-expressed with gammaM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by gammaM23K. betaE381K by itself caused inefficient coupling, but opposite from the effect of gammaM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the betaE381K mutation perturbs the gamma-beta-subunit interaction and the local conformation of the beta380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. betaE381A, L, K, and R, and gammaR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the beta380DELSEED386 segment and its interactions with the gamma-subunit are involved in the mechanism of coupling.

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

“…For example, some amino acid replacements in the interaction region between the βD$)!ELSEED$)' segment and the γ subunit have the opposite effect. Mutations such as βE381K or γR242E decrease ∆H ‡ and ∆S ‡ and generally cause destabilization of the complex [49]. Furthermore, analysis of a multitude of mutant enzymes that have no differences in catalytic or transport characteristics from those of the wild type have unaltered transition-state thermodynamic parameters (results not shown).…”

“…Several mutations in key positions that block the transmission of conformational information have been identified. Amino acid substitutions in the interfaces between the β and γ subunits [43,49], the ε and c subunits [53], the ε and a subunits [55] and the γ and c subunits [42] perturb proper transmission and result in inefficient coupling.…”

“…When Acridine Orange fluorescence was used to monitor the formation of a pH gradient at pH 7.5, no proton pumping was detected for the Cys, Leu and Asn mutant enzymes (results not shown). We observed previously that membranes made from strain DK8 had a significant rate of intrinsic proton leak that would short-circuit a low level of proton transport [49]. In efforts to circumvent this problem, we increased the sensitivity of the assay by using a pH 8.0 buffer and the dye ACMA to monitor the formation of the ∆pH ( Figure 3A).…”

“…We have previously used Arrhenius analyses of the catalytic transition state of steady-state ATP hydrolysis to assess the effects of amino acid replacements in the F " sector on the catalytic mechanism [42,43,45,49]. These analyses have been especially revealing with respect to the functional and physical interactions between the rotor γ subunit and the stator α and β subunits as well as an amino acid change at the γ-c subunit interface, γE208K, that affected coupling efficiency [49].…”

“…These analyses have been especially revealing with respect to the functional and physical interactions between the rotor γ subunit and the stator α and β subunits as well as an amino acid change at the γ-c subunit interface, γE208K, that affected coupling efficiency [49].…”