*2.5. ATP Synthase*

*M. tb* possesses a F1F0 ATP synthase consisting of two functional domains, a membrane-embedded F0 unit and an external F1 domain, which is linked by central and periphery stalks on which F0 rotates [78]. This rotation is powered by a flow of protons down the electrochemical gradient of the PMF, and drives a cycle of conformational changes in the catalytic F1 domain, resulting in successive ADP binding and entrapment, ADP phosphorylation to form ATP and ATP release [79]. Conventionally, the ATP synthase is also capable of ATPase activity when intracellular ATP levels are high and the PMF is low, hydrolysing ATP while pumping protons across the cytoplasm to re-energise the PMF [80]. In mycobacteria however, the ATP synthase has been characteristically observed to have suppressed ATP hydrolysis activity, which has been postulated to be an adaptation to conserving ATP under low oxygen tensions [39,78,81,82].

In *M. tb*, the F1F0 ATP synthase is encoded by the atpBEFHAGDC operon and is essential for the viability of replicating and non-growing *M. tb* [31–33,83], highlighting its critical role in ATP production and in maintaining a respiratory electron flow in all metabolic states [84]. Even though intracellular ATP levels in non-replicating *M. tb* are significantly reduced compared to replicating bacilli, basal levels of ATP are still maintained in non-replicating states [19,21]. The essentiality of the ATP synthase in these conditions is underlined by the fact that its pharmacological inhibition by BDQ, a specific ATP synthase inhibitor, kills hypoxic, non-replicating *M. tb* [83].

Interestingly, the mycobacterial ATP synthase is deprived of efficient ATP hydrolysis activity [82]. As a step towards uncovering the molecular basis of the extreme latency of ATP hydrolysis, a crystal structure of the *M. smeg* catalytic F1 domain revealed similarities with the *Caldalkalibacillus thermarum* F1-ATPase, which also hydrolyses ATP poorly [85]. This is likely due to an arrest in the catalytic rotary cycle of the F1 component, resulting in the inability to release products of ATP hydrolysis. The ε-subunit of the F1 module of *M. tb*, whose structure has been recently solved by nuclear magnetic resonance (NMR), has been implicated in the regulation of ATP hydrolysis. The removal of its C-terminal resulted in an increased ATP hydrolysis rate and decreased ATP synthesis [86]. Furthermore, Saw et al., demonstrated that this site is amenable to chemical inhibition in *M. smeg* by epigallocatechin gallate, the most abundant catechin in green tea [87]. Other subunits involved in the regulation of ATP hydrolysis include the γand α- subunits of the F1 module [88,89]. Altogether, these advances in the understanding of the F1F0 ATP synthase aid in identifying new, specific inhibitors that either block *de novo* ATP synthesis and/or activate ATP hydrolysis, with the aim of depleting the residual pool of ATP in *M. tb*.
