The Biological Role of the ζ Subunit as Unidirectional Inhibitor of the F1FO-ATPase of Paracoccus denitrificans

Abstract

The biological roles of the three natural FF-ATPase inhibitors, ε, ζ, and IF, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF, but in the FF-ATPase of Paracoccus denitrificans (PdFF), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdFF-ATPase activity without affecting the PdFF-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdFF-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

The 3 × 120° rotary mechanism of Paracoccus denitrificans F 1 -ATPase is different from that of the bacterial and mitochondrial F 1 -ATPases

“…Δζ preparation when LDAO is included in the assay (37). Additionally, the reconstitution of the ζ-subunit led to a complete inhibition of PdF 1 γCC ATP hydrolytic activity, in agreement with previous studies (26, 32) (SI Appendix, Fig.…”

“…purified from P. denitrificans cells (0.14 units per milligram of protein) but similar to the activity of pure F 1 obtained from a P. denitrificans ζ-knockout strain (PdΔζ; 0.87 units permilligram of protein) (37). Furthermore, the addition of 0.1% LDAO resulted in an 11-fold increase in activation, showing a specific activity of 56.7 ± 3.6 s −1 (± SE) ( Fig.…”

“…Recently, two P. denitrificans mutants lacking the ζ-subunit gene were studied. One ζ-knockout causes a specific growth defect associated with the activation of the ATP hydrolytic activity of PdF 1 F o (37). However, the other knockout caused only a moderate increase in PdF 1 F o ATP hydrolysis, which is insufficient to activate the membrane ATPase (74).…”

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions

“…[ATP] was reported as µmol (mg prot) −1 (Palikaras and Tavernarakis 2016;Mendoza-Hoffmann et al 2018).…”

Paracoccus denitrificans Oxidative Phosphorylation: Retentions, Gains, Losses, and Lessons En Route to Mitochondria

“…Recently there have been two reports of studies in which a gene knock out of the zeta subunit has been made. Varghese et al could find no effect on growth or in vitro ATPase activity whereas other work reported impaired growth of cells. Clearly the role of the zeta subunit, also found in other alpha‐proteobacteria, requires more attention.…”