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

The biological roles of the three natural F1FO-ATPase inhibitors, epsilon, zeta, and IF1, on cell physiology remain controversial. The zeta subunit is a useful model for deletion studies since it mimics mitochondrial IF1, but in the F1FO-ATPase of Paracoccus denitrificans (PdF1FO), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (Pd Delta zeta) with a deleted zeta gene to determine its role in cell growth and bioenergetics. The results show that the lack of zeta in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous zeta gene. Removal of zeta increased the coupled PdF1FO-ATPase activity without affecting the PdF1FO-ATP synthase turnover, and the latter was not affected at all by zeta reconstitution in vitro. Therefore, zeta works as a unidirectional pawl-ratchet inhibitor of the PdF1FO-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.