Abstract
F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.
Highlights
Bacterial ATP synthases are autoinhibited by subunit ⑀
An earlier study reported that deletion of 10 C-terminal residues from E. coli ⑀ allowed normal growth yield on succinate [23], indicating that in vivo ATP synthesis is more effective than with ⑀⌬5. These results suggest that residues between ⑀128 –133 are important for inhibition of ATP synthesis
Summary—Overall, our results are consistent with the idea that the ⑀CTD may be fine-tuned in different bacterial species to regulate ATP synthesis and hydrolysis functions according to the distinct metabolic/environmental demands of each species [2, 9]
Summary
Bacterial ATP synthases are autoinhibited by subunit ⑀. Results: Altering the regulatory interactions of ⑀ increases inhibition of ATP synthesis and reduces respiratory growth of E. coli. Conclusion: The ⑀ subunit can have distinct regulatory interactions during ATP synthesis versus hydrolysis. In the inhibited state of the Escherichia coli enzyme, the final segment of ⑀CTD is deeply buried but has few specific interactions with other subunits. This region of the ⑀CTD is variable or absent in other bacteria that exhibit strong ⑀-inhibition in vitro. The results suggest that the ⑀CTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP
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