Energy Coupling, Turnover, and Stability of the F0F1 ATP Synthase Are Dependent on the Energy of Interaction between γ and β Subunits

Abstract

Replacement of the F 0 F 1 ATP synthase ␥ subunit Met-23 with Lys (␥M23K) 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 ␥M23K mutation causes altered interactions between subunits. Binding of ␦ or ⑀ subunits stabilizes the ␣ 3 ␤ 3 ␥ complex, which becomes destabilized by the mutation. Significantly, the inhibition of F 1 ATP hydrolysis by the ⑀ subunit is no longer relieved when the ␥M23K mutant F 1 is bound to F 0 . Steady state Arrhenius analysis reveals that the ␥M23K 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 ␥M23K forming an ionized hydrogen bond with one of the ␤Glu-381 residues. Two second site mutations, ␥Q269R and ␥R242C, suppress the effects of ␥M23K and decrease activation energies for the ␥M23K enzyme. We conclude that ␥M23K is an added function mutation that increases the energy of interaction between ␥ and ␤ subunits. The additional interaction perturbs transmission of conformational information such that ⑀ inhibition of ATPase activity is not relieved and coupling efficiency is lowered.The F 0 F 1 ATP synthase links two disparate functions: transport of protons across a membrane and catalysis of ATP synthesis or hydrolysis (for reviews see Refs. 1-5). The fully cooperative mechanism of ATP hydrolysis requires a minimum of three different subunits in a complex containing ␣ 3 ␤ 3 ␥ 1 . The transport mechanism is most likely assembled from F 0 sector subunits. In the Escherichia coli complex, transport requires three different membrane-spanning subunits, a 1 b 2 c ϳ10 (6). In addition, two more soluble subunits, ␦ and ⑀, are needed to reconstitute catalytic and transport sectors so that they are coupled to carry out ATP-driven proton pumping or ⌬ Hϩ -driven ATP synthesis (7-10).Catalysis and transport mechanisms most likely communicate indirectly through a series of conformational and electrostatic interactions. Conformational changes relevant to the catalytic state of the enzyme or the presence of a ⌬ H ϩ have been detected by several methods including altered cross-linking patterns, protease susceptibility, environmentally sensitive fluorescent probes, accessibility of epitopes, x-ray diffraction, cryoelectron microscopy, and spectroscopic analyses (reviewed in Refs. 11 and 12). High resolution structural information based on crystals of the bovine mitochondrial F 1 has also provided a great deal of information about possible subunit interactions that may be involved in linking transport and catalysis (13).Mutagenic analysis has also yielded important information about the coupl...

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

“…We also point out that effects on the thermodynamic parameters of catalysis are easily missed if the level of wild-type and mutant activity happen to coincide near the assay temperature. For example, the ATPase activity of γM23K F " is the same as that of wild-type F " at 21 mC; however, the ∆H ‡ of the γM23K enzyme is 22 kJ\mol greater [43].…”

“…For a given reaction series that uses the same transition state there should be a quantitative relationship between enthalpy and entropy of activation, such as an isokinetic relationship [54]. We previously demonstrated that the transitionstate thermodynamic parameters from ATP synthases with a variety of mutations or from different sources could be fitted to an isokinetic line [43,45] such as that shown in Figure 5. In isokinetic relationships there is a linear free-energy relationship between two rate constants (k " and k # ) measured at two temperatures (T # T " ), which can be given by the equation : log k # l ajb log k " where a and b are constants [54].…”

“…Determination of the F " content in membrane preparations was performed by a quantitative immunoblot assay described previously [43]. Purified F " was used as a reference standard.…”

“…ATP hydrolysis rates were determined at 30 mC in a buffer containing 50 mM Hepes\KOH, 10 mM ATP, 5 mM MgSO % and 1 µM carbonyl cyanide mchlorophenylhydrazone (CCCP), pH 7.5, with 5 mM phosphoenolpyruvate, 50 µg\ml pyruvate kinase and 0.1-0.3 mg\ml membrane protein [45]. For the Arrhenius analysis, the pH was adjusted to 7.5 at the appropriate temperature [43]. ATP synthesis rates were determined at 30 mC as described previously [45].…”

“…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].…”