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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 K i 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

F 1 ·F o -ATP synthases/ATPases (F 1 ·F o ) 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 F 1 ·F o 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 F 1 ·F o 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" F 1 ·F o catalysis found in a wide range of bacterial F 1 ·F o 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.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Proton-translocating F o ×F 1 -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 F o -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 F o ×F 1 -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 F o ×F 1 -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 F o ×F 1 .

F o ·F 1 H + -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(Mg 2+ )-induced deactivation nor the energy-dependent enzyme activation. V max , not apparent Km ATP 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 F o ·F 1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of F o ·F 1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Changes in respiratory activity, transmembrane electric potential, and ATP synthesis as induced by additions of limited amounts of ADP and P(i) to tightly coupled inverted (inside-out) Paracoccus denitrificans plasma membrane vesicles were traced. The pattern of the changes was qualitatively the same as those observed for coupled mitochondria during the classical State 4-State 3-State 4 transition. Bacterial vesicles devoid of energy-dependent permeability barriers for the substrates of oxidation and phosphorylation were used as a simple experimental model to investigate two possible mechanisms of respiratory control: (i) in State 4 phosphoryl transfer potential (ATP/ADP × P(i)) is equilibrated with proton-motive force by reversibly operating F(1)·F(o)-ATPase (thermodynamic control); (ii) in State 4 apparent "equilibrium" is reached by unidirectional operation of proton motive force-activated F(1)·F(o)-ATP synthase. The data support the kinetic mechanism of the respiratory control phenomenon.

The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate (P(i)). Apparent K(m) values for ADP and P(i) were 7-11 and 60-120 µM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg²(+)). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly: K(m) value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent K(m) values for ADP and P(i) (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006) Biochemistry, 45, 14552-14558).

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).

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.

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+).

Tightly coupled membranes of Paracoccus denitrificans catalyze oxidative phosphorylation but are incapable of ATP hydrolysis. The conditions for observation and registration of the venturicidin-sensitive ATPase activity of subbacterial particles derived from this organism are described. The ATP hydrolytic activity does not appear after prolonged incubation in the presence of pyruvate kinase and phosphoenol pyruvate (to remove ADP), EDTA (to remove Mg2+) and/or inorganic phosphate, whereas the activity dramatically increases after energization of the membranes. ATP hydrolysis by -activated ATPase is coupled with electric potential formation. Inorganic phosphate prevents and azide promotes a decline of the enzyme activity during ATP hydrolysis. The addition of uncouplers results in rapid and complete inactivation of ATPase. The -dependent ATPase activity increases upon dilution of the membranes. The results are discussed as evidence for the presence of distinct ATP-synthase and ATP-hydrolase states of F(o)F(1) complex in the coupling membranes (Vinogradov, A. D. (1999) Biochemistry (Moscow), 64, 1219-1229). The proposal is made that part of the free energy released from oxidoreduction in the respiratory chain is used to maintain active conformation of the energy-transducing proteins.

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., (Copyright 2003 Elsevier Science (USA))

The effects of Tinopals (cationic benzoxazoles) AMS-GX and 5BM-GX on NADH-oxidase, NADH:ferricyanide reductase, and NADH --> APAD+ transhydrogenase reactions and energy-linked NAD+ reduction by succinate, catalyzed by NADH:ubiquinone oxidoreductase (Complex I) in submitochondrial particles (SMP), were investigated. AMS-GX competes with NADH in NADH-oxidase and NADH:ferricyanide reductase reactions (K(i) = 1 micro M). 5BM-GX inhibits those reactions with mixed type with respect to NADH (K(i) = 5 micro M) mechanism. Neither compound affects reverse electron transfer from succinate to NAD+. The type of the Tinopals' effect on the NADH --> APAD+ transhydrogenase reaction, occurring with formation of a ternary complex, suggests the ordered binding of nucleotides by the enzyme during the reaction: AMS-GX and 5BM-GX inhibit this reaction uncompetitively just with respect to one of the substrates (APAD+ and NADH, correspondingly). The competition between 5BM-GX and APAD+ confirms that NADH is the first substrate bound by the enzyme. Direct and reverse electron transfer reactions demonstrate different specificity for NADH and NAD+ analogs: the nicotinamide part of the molecule is significant for reduced nucleotide binding. The data confirm the model suggesting that during NADH --> APAD+ reaction, occurring with ternary complex formation, reduced nucleotide interacts with the center participating in NADH oxidation, whereas oxidized nucleotide reacts with the center binding NAD+ in the reverse electron transfer reaction.

The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD+) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH (Ki = 40 microM) than with APAD+ (Ki = 150 microM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.

Qualitative and quantitative characteristics of the reactions catalyzed by the most complex and least understood proton translocating unit of the mammalian respiratory chain (NADH-ubiquinone oxidoreductase, Complex I) are described for enzyme preparations differing in degree of resolution--from intact mitochondria to homogeneous small enzyme fragments. Special attention is given to the problems and pitfalls of reliable interpretation of the kinetic analysis of the enzyme activities. Detailed analysis of the problems concerning the slow active/inactive reversible enzyme transition is provided.

Considerable quantitative variations in the competitive inhibition of NADH oxidase activity of bovine heart submitochondrial particles (SMP) by different samples of NAD- were observed. ADP-ribose (ADPR) was identified as the inhibitory contaminating substance responsible for variations in the inhibition observed. ADPR competitively inhibits NADH oxidation with Ki values (25 degrees C, pH 8.0) of 26 microM, 30 microM, and 180 microM for SMP, purified Complex I and three-subunit NADH dehydrogenase (FP), respectively. ADPR decreases NADH-induced flavin reduction and prolongs the cyclic bleaching of FP during aerobic oxidation of NADH. Ki for inhibition of the rotenone-sensitive NADH oxidase in SMP by ADPR does not depend on delta mu H+. The initial rate of the energy-dependent NAD+ reduction by succinate is insensitive to ADPR. The inhibitor increases the steady-state level of NAD+ reduction reached during aerobic succinate-supported reverse electron transfer catalyzed by tightly coupled SMP. The results obtained are consistent with the proposal on different nucleotide-binding sites operating in the direct and reverse reactions catalyzed by the mitochondrial NADH-ubiquinone reductase.

Mitochondrial Ca2+ movement was investigated in the presence of oxaloacetate, which is widely known as a 'Ca(2+)-releasing' agent [1978, Proc. Natl. Acad. Sci. USA 75, 1690-1694]. It is demonstrated that rat liver mitochondrial are capable of net Ca2+ accumulation from the oxaloacetate supplemented assay mixture. Both the membrane energization and the cation uniport at the expense of oxaloacetate are shown to be specifically blocked by either arsenite or ammonium chloride. With respiratory inhibitors present, ADP is shown to be a prerequisite for a high Ca2+ capacity, which can be alternatively enlarged with a concomitant loss of the arsenite effect by an addition of an NADP(+)-specific reductant (isocitrate). Arsenite-sensitive production of NADPH is observed, thus suggesting coupling between pyridine nucleotide transhydrogenation and the cation uniport in mitochondria. The role of such a coupling mechanism in the uniporter-mediated Ca2+ fluxes in mitochondria is discussed.

The effect of oxaloacetate on Ca2+ transport in isolated rat liver mitochondria has been studied. Under aerobic conditions in the presence of oxaloacetate mitochondria accumulate Ca2+ in a ruthenium red- and uncoupler-sensitive way. Oxaloacetate catalyzes also the slow (5 nM Ca2+/min/mg protein) uptake of limited amounts of calcium by the mitochondria in the presence of respiratory chain and ATPase inhibitors. Under these conditions ADP, pyruvate, succinate and isocitrate increase both the rate of oxaloacetate-dependent Ca2+ transport and the amount of the accumulated cation. In all cases studied (with the exception of isocitrate) the oxaloacetate-dependent Ca2+ uptake was blocked by low concentrations of arsenite. Oxaloacetate added to mitochondria in the presence of respiratory chain and ATPase inhibitors increases the [NAD+]. [NADPH]/[NADH].[NADP+] ratio and stimulates the transmembrane potential generation in the mitochondria. Ammonium chloride decreases the rates of the oxaloacetate-dependent Ca2+ uptake. The data obtained suggest that the oxaloacetate-dependent Ca2+ uptake by the mitochondria first demonstrated in this study is mediated by energy-dependent mitochondria transhydrogenase. These results are discussed in connection with oxaloacetate-induced Ca2+ release from mitochondria.

It has been shown that 16-18 days administration of antivitamin K (pelentan) leads to two-fold increase of prothrombin time in adult rats but does not influence the soluble brain, renal, heart, muscle and serum creatine kinase activity. No effect on metabolic function of isolated liver mitochondria has been found in contrast to the vitamin K deficient rats. Mitochondria were characterized by high value of respiration control; substance oxidation rates and internal mitochondrial Ca2+ content do not differ in the level from those of control animals. From the obtained results and data published in literature a conclusion can be drawn about only particular similarity (prothrombin time) between the antivitamin K administration and alimentary vitamin K deficit.

Alimentary deficiency of vitamin K caused a decrease in the rate of respiration in presence of ADP and in the rate of oxidative phosphorylation in the presence of succinate. Administration of the antivitamin K pelentane, excess of vikasol and deficiency of vitamin D did not affect these parameters. As distinct from controls and rats treated with pelentane, transport of calcium was decreased in presence of all the substrates studied in mitochondria isolated from liver tissue of animals deprived of vitamins K and D as well as of animals treated with vikasol excess. At the same time, accumulation of calcium led to time-dependent inhibition of respiratory chain if NAD-dependent substrates were used. Possible reasons of dissimilarity observed are discussed; the phenomenon found may occur due to exhaustion of the mitochondrial pyridine nucleotides pool. The data obtained suggest that antivitamins K altered only some parameters of body status (prothrombin time) similarly to the alterations observed in alimentary deficiency of vitamin K.

A session of low-frequency electroacupuncture (EAP) of the nerve points of the cervicobrachial or lumbosacral regions was performed in 61 patients with vertebral osteochondrosis. An EAP therapeutic effect, analgetic one in particular, was found to depend on cortisol and aldosterone blood levels: reduced levels of the hormones related to EAP procedure appeared associated with a positive therapeutic response.