Inhibition of ATP Hydrolysis by Thermoalkaliphilic F1Fo-ATP Synthase Is Controlled by the C Terminus of the ε Subunit

ABSTRACT The F1Fo-ATP synthases of alkaliphilic bacteria exhibit latent ATPase activity, and for the thermoalkaliphile Bacillus sp. strain TA2.A1, this activity is intrinsic to the F1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F1 complexes from this thermoalkaliphile. Like the native F1Fo-ATP synthase, the recombinant TA2F1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the ε subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F1 mutant with either a truncated ε subunit [i.e., TA2F1(εΔC)] or substitution of basic residues in the second α-helix of ε with nonpolar alanines [i.e., TA2F1(ε6A)] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F1Fo-ATP synthase and TA2F1(εWT) complex with proteases revealed that the ε subunit was resistant to proteolytic digestion. In contrast, the ε subunit of TA2F1(ε6A) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that ε was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the ε subunit in the entire holoenzyme, we reconstituted recombinant TA2F1 complexes with F1-stripped native membranes of strain TA2.A1. The reconstituted TA2FoF1(εWT) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F1Fo-ATP synthase in native membranes. Reconstituted TA2FoF1(ε6A) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the ε subunit also inhibits ATPase activity of TA2FoF1.

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