Zur Regulation der NAD-abhängigen Hydrogenase-Aktivität

Zusammenfassung Ruhende Zellen von Hydrogenomonas H 16 enthalten je Gramm Trockengewicht 0,7 mg ATP und 0,9 mg NAD. Bei der Fixierung von Kohlendioxyd und der Synthese des Speicherstoffs Poly--hydroxybuttersäure sinkt die intracelluläre ATP-Konzentration um 30% und das Redoxverhältnis NADH₂/NAD von 1,4 auf 0,46. Die NAD-abhängige Hydrogenase enthält NAD als Coenzym relativ fest gebunden. In Gegenwart von Wasserstoff wird dieses zu NADH₂ reduziert und das Enzym in eine aktive, reaktionsfähige Form umgewandelt. Die Geschwindigkeit der NAD-Reduktion ist infolge einer allosterischen Hemmung der NAD-abhängigen Hydrogenase durch ihr Reaktionsprodukt NADH₂ von dem Redoxverhältnis NADH₂/NAD abhängig. Hierdurch erhält das Enzym eine regulatorische Funktion für den von Folgereaktionen abhängigen Wasserstofftransport.

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Summary Resting cells of Hydrogenomonas strain H 16 contain 0.7 mg ATP and 0.9 mg NAD/g dry weight. During the fixation of carbon dioxide and the synthesis of the storage product poly--hydroxybutyric acid, the intracellular concentration of ATP decreases by 30% and the redox-ratio (NADH₂/NAD) decreases from 1.4 to 0.46.In the NAD-dependent hydrogenase the coenzyme NAD is bound to the enzyme. In the presence of hydrogen NAD is reduced to NADH₂ and the enzyme is converted to a reactive state. The velocity of NAD-reduction is related to the concentration of NADH₂ and the redox-ratio NADH₂/NAD. This allosteric inhibition of the NAD-dependent hydrogenase by its reaction product NADH₂ is responsible for the control of hydrogen transport exerted by consecutive hydrogen requiring reactions.

     

Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics

The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-P_i exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-P_i exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-P_i exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD⁺ reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling.     

NADH2- and succinate-dependent O2 uptake in chromatophores from Chromatium vinosum

The O₂ uptake linked to NADH₂ and succinate oxidation was observedin chromatophores from photosynthetically grown Chromatium vinosum. The maximal rate was 60–120 nmoles of O₂ uptake per minper {diaeresis}mole of bacterio-chlorophyll. The rate of O₂uptake linked to NADH₂ oxidation was higher in the neutral-to-acidicpH range than in the alkaline range, whereas that linked tosuccinate oxidation was higher in the alkaline range. The O₂ uptake linked to NADH₂ oxidation was inhibited by rotenone,amytal, antimycin A, KCN and NaN₃, while that linked to succinateoxidation was inhibited by antimycin A, KCN and NaN₃. Malate,citrate, pyruvate, acetate, -ketoglutarate, NADPH₂ and thiosulfatedid not serve as substrates for the O₂ uptake of isolated chromatophores. The rates of the O₂ uptake linked to both NADH₂ and succinateoxidation were not stimulated by adding uncouplers or underphosphorylating conditions. Little or no ATP was synthesizedin the dark, coupled to either NADH₂ or succinate oxidation,in spite of a high activity of photophosphorylation in Chromatiumchromatophores. (Received February 26, 1980; )     

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:

1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
2. The membrane preparation also catalyzed the reduction of fumarate with H₂S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H₂S and fumarate and between NADH and fumarate.
4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H₂S, or NADH, and the oxidation of DMNH₂ by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H₂S and NADH oxidation by DMN were not affected by the inhibitor.

It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.     

The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH₂ of high purity, the maximal specific rates were similar to those obtained with NAD and NADH₂, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH₂, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH₂ do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH₂. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH₂ than NAD and NADH₂. 5. Phosphate behaves as a competitive inhibitor towards NADP.     

New method of detection of certain bacterial oxidoreductases and transaminases by the Indicator reaction on agar

A method for detection of the following enzymes is described: glutamate oxalacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, 2. 6. 1. 1), lactate dehydrogenase (L-lactate: NAD oxidoreductase, 1. 1. 1. 27) and malate dehydrogenase (L-malate: NAD oxido-reductase, 1. 1. 1. 37), diffused from bacterial suspensions into agar, by means of a “sandwich” agar detector layer containing the specific substrates. The principle of the reaction: reduced nicotinamide-dinucleotide ? nicotinamide-adenine-dinucleotide (NADH₂?NAD) was used, in which fluorescence of NADH₂ (in u. v. light) disappears proportionately to the activity of the test enzyme in its diffusion zone.     

Two regimens of electrogenic cyclic redox chain operation in chromatophores of non-sulfur purple bacteria A study using antimycin A

Antimycin A causes a biphasic suppression of the light-induced membrane potential generation in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores incubated anaerobically. The first phase is observed at low antibiotic concentrations and is apparently due to its action as a cyclic electron transfer inhibitor. The second phase is manifested at concentrations which are greater than 1–2 μM and is due to uncoupling that may be connected with an antibiotic-induced dissipation of the electrochemical H⁺ gradient across the chromatophore membrane. The inhibitory effect of anti-mycin added at low concentrations under aerobic conditions is removed by succinate to a large extent. It is expected that the electrogenic cyclic redox chain in the bacterial chromatophores incubated under conditions of continuous illumination may function at two regimes: (1) as a complete chain involving all the redox components, and (2) as a shortened chain involving only the P-870 photoreaction center, ubiquinone and cytochrome c₂.     

Role of Copper Ions in the Regulation of L-Glutamate Biosynthesis

Cell-free extracts of Brevibacterium thiogenitalis culture grown in the presence of copper catalyzed the oxidation of NADH₂ and succinate through an electron transport chain which contained menaquinones and cytochromes a, b and c. On the other hand, extracts of cells grown in the absence of copper lacked cytochromes a and c, and contained cytochrome d.

These findings, as well as the results obtained in inhibition experiments, suggest that in copper-deficient cells the major part of NADH₂ was oxidized via a bypass in which the electrons were transferred directly from flavoprotein or cytochrome b to molecular oxygen.

Electron transport from these substrates to molecular oxygen resulted in ATP synthesis. The average P/O ratios in extracts of the copper-sufficient cells were 0.33 for generated NADH₂, 0.20 for added NADH₂, and 0.34 for succinate, and those in extracts of the copper-deficient cells were 0.15, 0.13 and 0.21, respectively. In addition, a linear relationship was found between the yield of L-glutamate from acetate and the P/Ο ratios with both NADH₂ and succinate as substrates.

From these results, it is reasonable to consider that the poor yield of L-glutamate from acetate in copper-deficient cells was due to a reduction in energy supply, which was caused by the low efficiency of oxidative phosphorylation.     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

The photoassimilation of succinate to hexose by Rhodospirillum rubrum

1. A pathway for the synthesis of hexose from succinate by Rhodospirillum rubrum is proposed. 2. With 2,3-¹⁴C₂-labelled succinate and fumarate as substrates in experiments with chromatophores and a soluble enzyme fraction of R. rubrum it was found that the products of succinate metabolism by the extracts were the same as in whole cells. It was also found that the light-dependent oxidation of succinate was catalysed by the chromatophores, but that all the other enzymes involved were in the soluble fraction. 3. By using specific assays the presence of all the enzymes required for the proposed pathway was demonstrated in the extracts and their specific activities were measured. 4. The overall rate of succinate assimilation was measured manometrically. The activities of the enzymes assayed were sufficient to account for the overall rate of assimilation. It is concluded that the proposed pathway represents the major mechanism for synthesis of hexose from succinate in R. rubrum. 5. The formation of alanine and aspartate was observed in experiments with isotopically labelled substrates, and possible synthetic pathways for these compounds are discussed.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

Analytical application of the stereospecific hydrogen exchange reaction between (R)-carnitine and water, catalyzed by (R)-carnitine dehydrogenase and alpha-lipoamide dehydrogenase (diaphorase)

A stereospecific hydrogen exchange between tritiated water and the hydrogen at C₃ of (R)-carnitine takes place under the coupled catalyses of (R)-carnitine dehydrogenase from Pseudomonas aeruginosa and α-lipoamide dehydrogenase (diaphorase) from pig heart. This exchange reaction can be used to synthesize (R)-(3-³H) -carnitine. The amount of tritium released from the C₃ position of (R)-(3-³H) -carnitine into water is decreased proportionally by the addition of non labelled (R)-carnitine, making possible a new sensitive assay for (R)-carnitine.     

The site of inhibition of dibromothymoquinone in mitochondrial respiration

Dibromothymoquinone (2,5-dibromo, 6-isopropyl, 3-methyl benzoquinone, DBMIB) is a quinone analogue recently introduced as a specific inhibitor of chloroplast photosynthesis at the level of plastoquinone. In beef heart mitochondria DBMIB inhibits the oxidation of both succinate and NAD linked substrates; the apparent K_I is 6 μM for βhydroxybutyrate oxidation and 61μM for succinate oxidation respectively. In sonic fragments NADH oxidation is also inhibited; however, the rotenone block of respiration can be partially bypassed by the autooxidation of reduced DBMIB. Under the same conditions succinoxidase of ETP is inhibited, as in intact mitochondria; autoxidation of DBMIB reduced by succinate can however be obtained in presence of detergents. Hexahydrocoenzyme Q₄ reverses the DBMIB inhibition of succinate in sonic fragments. The site of inhibition by DBMIB is the oxygen side of CoQ, since DBMIB can function as electron acceptor in the NADH-CoQ assay for site I energization in submitochondrial particles, studied by measuring the quenching of atebrin fluorescence.     

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P_i) and ATP are present. No reduction occurs in the absence of P_i, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Energy-linked reactions in photosynthetic bacteria: Pi in equilibrium with HOH oxygen exchange catalyzed by the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum

The inorganic phosphate-water oxygen exchange reaction has been studied in chromatophores of Rhodospirillum rubrum. Under appropriate conditions, chromatophores catalyzed this exchange at a rate of up to 150 μatom oxygen/h/mg bacteriochlorophyll. The reaction is largely inhibited by inhibitors of the membrane-bound inorganic pyrophosphatase, fluoride and methylene diphosphonate, and is not inhibited by oligomycin. These results indicate that the P_i ? HOH oxygen exchange is almost entirely due to the pyrophosphatase. In the presence of ADP, the exchange reaction was stimulated by about 40% and this portion of the exchange was sensitive to oligomycin, but not to fluoride or methylene diphosphonate. Thus this portion of the exchange can be attributed to the ATP synthese complex. The rates of the oxygen exchange reaction and other chromatophore-catyalyzed reactions are compared.     

Photo-induced electron transport and water state in Rhodospirillum rubrum chromatophores

It is shown that in bacterial chromatophores the pronounced changes in the free water content with a proton spin-spin relaxation time (T₂) of 10^?3—10^?2 s does not influence the efficiency of electron transfer from the photosynthetic reaction centre to the membrane pool of secondary acceptors. An abrupt inhibition of this process occurs only after the loss of the water with faster proton spin-spin relaxation time (T₂ of 10^?4 s). The process is reversible. The water fraction in question is obviously bound to the chromatophore proteins and forms the primary hydration layer.     

Long chain fatty acyl-CoA modulation of H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I

Complex I is responsible for most of the mitochondrial H₂O₂ release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H₂O₂ production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125–129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H₂O₂ release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on ΔpH or Δp and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.