F(0).F(1)-ATP synthase in tightly coupled inside-out vesicles derived from Paracoccus denitrificans catalyzes rapid respiration-supported ATP synthesis, whereas their ATPase activity is very low. In the present study, the conditions required to reveal the Deltamu(H+)-generating ATP hydrolase activity of the bacterial enzyme have been elucidated. Energization of the membranes by respiration results in strong activation of the venturicidin-sensitive ATP hydrolysis, which is coupled with generation of Deltamũ(H+). Partial uncoupling stimulates the proton-translocating ATP hydrolysis, whereas complete uncoupling results in inhibition of the ATPase activity. The presence of inorganic phosphate is indispensable for the steady-state turnover of the Deltamũ(H+)-activated ATPase. The collapse of Deltamũ(H+) brings about rapid deactivation of the enzyme, which has been subjected to pre-energization. The rate and extent of the deactivation depend on protein concentration, i.e. the more vesicles are present in the assay mixture, the higher the rate and extent of the deactivation is seen. Sulfite and the ADP-trapping system protect ATPase against the Deltamũ(H+) collapse-induced deactivation, whereas phosphate delays the rate of deactivation. A low concentration of ADP (<1 microm) increases the rate of deactivation. Taken together, the results suggest that latent proton-translocating ATPase in P. denitrificans is kinetically equivalent to the previously characterized ADP(Mg2+)-inhibited, azide-trapped bovine heart mitochondrial F(0).F(1)-ATPase (Galkin, M. A., and Vinogradov, A. D. (1999) FEBS Lett. 448, 123-126). A Deltamũ(H+)-sensitive mechanism operates in P. denitrificans that prevents physiologically wasteful consumption of ATP by F(0).F(1)-ATPase (synthase) complex when the latter is unable to maintain certain value of Deltamũ(H+).