Proton-Translocating NADH-Ubiquinone Oxidoreductase: Interaction with Artificial Electron Acceptors, Inhibitors, and Potential Medicines.

Proton-translocating NADH-ubiquinone oxidoreductase (complex I) catalyzes the oxidation of NADH by ubiquinone accompanied by the transmembrane transfer of four protons, thus contributing to the formation of a proton motive force () across the coupling membranes of mitochondria and bacteria, which drives ATP synthesis in oxidative phosphorylation. In recent years, great progress has been achieved in resolving complex I structure by means of X-ray crystallography and high-resolution cryo-electron microscopy, which has led to the formulation of detailed hypotheses concerning the molecular mechanism of coupling of the redox reaction to vectorial proton translocation. To test and probe proposed mechanisms, a comprehensive study of complex I using other methods including molecular dynamics and a variety of biochemical studies such as kinetic and inhibitory analysis is required.

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

F·F-ATP synthases/ATPases (F·F) 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·F 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·F in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis.

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 K 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.

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

Proton-translocating F×F-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-directed inhibitor, blocks the synthesis and hydrolysis of ATP with a significant difference in the affinity [Zharova, T. V.

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.

Correlation of synovial caspase-3 concentration and the photodynamic effectiveness in osteoarthritis treatment.

Background: The present study focuses on investigation of Intra-articular PDT mechanisms for OA treatment. Also, a search for determination of the most effective dose of chlorin e6 (Ce6) for anti-inflammatory PDT of OA was carried out.

Methods: The study was carried out on laboratory animals (11 Chinchilla rabbits, 1 year, 2.

Functional heterogeneity of F·FH-ATPase/synthase in coupled Paracoccus denitrificans plasma membranes.

F·FH-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.

Gonarthritis photodynamic therapy with chlorin e6 derivatives.

Background: The new methods of osteoarthritis treatment are in constant demand due to the complexity of the early diagnosis and therapy. Specific features of Сhlorin e6 derivative (Ce6) accumulation in knee joint tissues and the efficiency of photodynamic therapy (PDT) of gonarthritis were studied.

Methods: The experimental research was conducted on the model of posttraumatic gonarthritis on rabbits.

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.

Oxidative phosphorylation and respiratory control phenomenon in Paracoccus denitrificans plasma membrane.

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.

Paracoccus denitrificans proton-translocating ATPase: kinetics of oxidative phosphorylation.

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.

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.

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.

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.

Proton-translocating ATP-synthase of Paracoccus denitrificans: ATP-hydrolytic activity.

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.

Kinetic mechanism of mitochondrial NADH:ubiquinone oxidoreductase interaction with nucleotide substrates of the transhydrogenase reaction.

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.

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.

Catalytic properties of mitochondrial NADH-ubiquinone reductase (Complex I).

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.

Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites.

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.

A competitive inhibition of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) by ADP-ribose.

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.

Novel oxaloacetate effect on mitochondrial Ca2+ movement.

Mitochondrial Ca2+ movement was investigated in the presence of oxaloacetate, which is widely known as a 'Ca(2+)-releasing' agent [1978, Proc. Natl. Acad.

[Oxaloacetate-dependent calcium transport in rat liver mitochondria].

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.

[Status of liver mitochondria and rat tissue creatine kinase during administration of antivitamin K].

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.

[Metabolic function of isolated liver mitochondria during various conditions of vitamin D and K supply and administration of pelentane].

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.

[Functional changes in the pituitary-adrenal system during the action of electroacupuncture on patients with spinal osteochondrosis].

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.