F1·Fo ATP Synthase/ATPase: Contemporary View on Unidirectional Catalysis.

F1·Fo-ATP synthases/ATPases (F1·Fo) are molecular machines that couple either ATP synthesis from ADP and phosphate or ATP hydrolysis to the consumption or production of a transmembrane electrochemical gradient of protons. Currently, in view of the spread of drug-resistant disease-causing strains, there is an increasing interest in F1·Fo as new targets for antimicrobial drugs, in particular, anti-tuberculosis drugs, and inhibitors of these membrane proteins are being considered in this capacity. However, the specific drug search is hampered by the complex mechanism of regulation of F1·Fo in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis. In this review, we consider the current state of the problem of "unidirectional" F1·Fo catalysis found in a wide range of bacterial F1·Fo and enzymes from other organisms, the understanding of which will be useful for developing a strategy for the search for new drugs that selectively disrupt the energy production of bacterial cells.

Proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans plasma membranes catalyzes FMN-independent reverse electron transfer to hexaammineruthenium (III).

NADH-OH, the specific inhibitor of NADH-binding site of the mammalian complex I, is shown to completely block FMN-dependent reactions of P. denitrificans enzyme in plasma membrane vesicles: NADH oxidation (in a competitive manner with Ki of 1 nM) as well as reduction of pyridine nucleotides, ferricyanide and oxygen in the reverse electron transfer. In contrast to these activities, the reverse electron transfer to hexaammineruthenium (III) catalyzed by plasma membrane vesicles is insensitive to NADH-OH. To explain these results, we hypothesize the existence of a non-FMN redox group of P. denitrificans complex I that is capable of reducing hexaammineruthenium (III), which is corroborated by the complex kinetics of NADH: hexaammineruthenium (III)-reductase activity, catalyzed by this enzyme. A new assay procedure for measuring succinate-driven reverse electron transfer catalyzed by P. denitrificans complex I to hexaammineruthenium (III) is proposed.

Interaction of Venturicidin and Fo·F1-ATPase/ATP Synthase of Tightly Coupled Subbacterial Particles of Paracoccus denitrificans in Energized Membranes.

Proton-translocating Fo×F1-ATPase/synthase that catalyzes synthesis and hydrolysis of ATP is commonly considered to be a reversibly functioning complex. We have previously shown that venturicidin, a specific Fo-directed inhibitor, blocks the synthesis and hydrolysis of ATP with a significant difference in the affinity [Zharova, T. V. and Vinogradov, A. D. (2017) Biochim. Biophys. Acta, 1858, 939-944]. In this paper, we have studied in detail inhibition of Fo×F1-ATPase/synthase by venturicidin in tightly coupled membranes of Paracoccus denitrificans under conditions of membrane potential generation. ATP hydrolysis was followed by the ATP-dependent succinate-supported NAD+ reduction (potential-dependent reverse electron transfer) catalyzed by the respiratory chain complex I. It has been demonstrated that membrane energization did not affect the affinity of Fo×F1-ATPase/synthase for venturicidin. The dependence of the residual ATP synthase activity on the concentration of venturicidin approximated a linear function, whereas the dependence of ATP hydrolysis was sigmoidal: at low inhibitor concentrations venturicidin strongly inhibited ATP synthesis without decrease in the rate of ATP hydrolysis. A model is proposed suggesting that ATP synthesis and ATP hydrolysis are catalyzed by two different forms of Fo×F1.

Inhibition of respiratory complex I by 6-ketocholestanol: Relevance to recoupling action in mitochondria.

6-Ketocholestanol (kCh) is known as a mitochondrial recoupler, i.e. it abolishes uncoupling of mitochondria by such potent agents as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3,5-di(tert-butyl)-4-hydroxybenzylidenemalononitril (SF6847) [Starkov et al., 1997]. Here, we report data on the kCh-induced inhibition of both NADH-oxidase and NADH-ubiquinone oxidoreductase activities of the respiratory complex I in bovine heart submitochondrial particles (SMP). Based on the absence of such inhibition with hexaammineruthenium (III) (HAR) as the complex I electron acceptor, the kCh effect could be associated with the ubiquinone-binding centre of this respiratory enzyme. In isolated rat liver mitochondria (RLM), kCh inhibited oxygen consumption with the glutamate/malate, substrates of NAD-linked dehydrogenases, while no inhibition of RLM respiration was observed with succinate, in agreement with the absence of the kCh effect on the succinate oxidase activity in SMP. Three kCh analogs (cholesterol, 6α-hydroxycholesterol, and 5α,6α-epoxycholesterol) exhibited no effect on the NADH oxidase activities in both SMP and RLM. Importantly, the kCh analogs were ineffective in the recoupling of RLM treated with CCCP or SF6847. Therefore, interaction of kCh with the complex I may be involved in the kCh-mediated mitochondrial recoupling.

Functional heterogeneity of Fo·F1H+-ATPase/synthase in coupled Paracoccus denitrificans plasma membranes.

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.

ATPase/synthase activity of Paracoccus denitrificans Fo·F1 as related to the respiratory control phenomenon.

The time course of ATP synthesis, oxygen consumption, and change in the membrane potential in Paracoccus denitrificans inside-out plasma membrane vesicles was traced. ATP synthesis initiated by the addition of a limited amount of either ADP or inorganic phosphate proceeded up to very low residual concentrations of the limiting substrate. Accumulated ATP did not decrease the rate of its synthesis initiated by the addition of ADP. The amount of residual ADP determined at State 4 respiration was independent of ten-fold variation of Pi or the presence of ATP. The pH-dependence of Km for Pi could not be fitted to a simple phosphoric acid dissociation curve. Partial inhibition of respiration resulted in a decrease in the rate of ATP synthesis without affecting the ATP/ADP reached at State 4. At pH8.0, hydrolysis of ATP accumulated at State 4 was induced by a low concentration of an uncoupler, whereas complete uncoupling results in rapid inactivation of ATPase. At pH7.0, no reversal of the ATP synthase reaction by the uncoupler was seen. The data show that ATP/ADP×Pi ratio maintained at State 4 is not in equilibrium with respiratory-generated driving force. Possible mechanisms of kinetic control and unidirectional operation of the Fo·F1-ATP synthase are discussed.

Energy-linked binding of Pi is required for continuous steady-state proton-translocating ATP hydrolysis catalyzed by F0.F1 ATP synthase.

The presence of medium Pi (half-maximal concentration of 20 microM at pH 8.0) was found to be required for the prevention of the rapid decline in the rate of proton-motive force (pmf)-induced ATP hydrolysis by Fo.F1 ATP synthase in coupled vesicles derived from Paracoccus denitrificans. The initial rate of the reaction was independent of Pi. The apparent affinity of Pi for its "ATPase-protecting" site was strongly decreased with partial uncoupling of the vesicles. Pi did not reactivate ATPase when added after complete time-dependent deactivation during the enzyme turnover. Arsenate and sulfate, which was shown to compete with Pi when Fo.F1 catalyzed oxidative phosphorylation, substituted for Pi as the protectors of ATPase against the turnover-dependent deactivation. Under conditions where the enzyme turnover was not permitted (no ATP was present), Pi was not required for the pmf-induced activation of ATPase, whereas the presence of medium Pi (or sulfate) delayed the spontaneous deactivation of the enzyme which was induced by the membrane de-energization. The data are interpreted to suggest that coupled and uncoupled ATP hydrolysis catalyzed by Fo.F1 ATP synthases proceeds via different intermediates. Pi dissociates after ADP if the coupling membrane is energized (no E.ADP intermediate exists). Pi dissociates before ADP during uncoupled ATP hydrolysis, leaving the E.ADP intermediate which is transformed into the inactive ADP(Mg2+)-inhibited form of the enzyme (latent ATPase).

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo.F1-ATP synthase.

Fo.F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP.Mg2+ -induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319-12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 x 10(-6) M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo.F1-ATP synthase.

Energy-dependent transformation of F0.F1-ATPase in Paracoccus denitrificans plasma membranes.

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 Deltamu(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 Deltamu(H+)-activated ATPase. The collapse of Deltamu(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 Deltamu(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 Deltamu(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 Deltamu(H+).

In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.

The channel-forming antibiotic alamethicin was used to permeabilize mitochondrial membranes for the low molecular mass hydrophilic substrates NADH and ATP. Alamethicin-treated mitochondria show high rotenone-sensitive NADH oxidase, NADH-quinone reductase, and oligomycin-sensitive and carboxyatractylate-insensitive ATPase activities. Alamethicin does not affect Complex I and ATPase activities in inside-out submitochondrial particles. Permeabilized mitochondria quantitatively retain their aconitase and iso-citrate dehydrogenase activities. Electron microscopy of alamethicin-treated mitochondria reveals no disruption of their outer and inner membranes. From the results obtained it is recommended, that alamethicin be used for the in situ catalytic assay of intramitochondrially located enzymes.