Deactivation of mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I): Extrinsically affecting factors.

Mitochondrial NADH:ubiquinone oxidoreductase (proton translocating respiratory complex I) serves several essential functions in cell metabolism: it maintains the intramitochondrial NADH/NAD+ ratio, contributes to generation of the proton-motive force, and participates in physiological and/or pathophysiological production of so-called reactive oxygen species. A characteristic feature of complex I is a slow, compared with its catalytic turnover, transformation to its inactive (deactivated) state, a phenomenon operationally called A/D transition. Here we report data on several extrinsic factors affecting deactivation as observed in coupled or uncoupled bovine heart submitochondrial particles. The time course of the strongly temperature-dependent deactivation deviates from first-order kinetics, and this deviation is abolished in the presence of an SH-group-specific reagent. The residual fraction of activity attained upon extensive deactivation shows the same kinetics of NADH oxidation as the fully active enzyme does. The rate of complex I deactivation is only slightly pH dependent within the range of 7.0-8.5 and significantly increases at higher pH. ATP∙(Mg) decreases the rate of complex I deactivation in coupled SMP, and this effect is abolished if the proton-motive force generating ATPase activity of Fo∙F1 is precluded. Taken together, the data show that an equilibrium between the A and D forms of complex I exists. Possible mechanistic aspects of the deactivation process are discussed.

FMN site-independent energy-linked reverse electron transfer in mitochondrial respiratory complex I.

A simple assay procedure for measuring ATP-dependent reverse electron transfer from ubiquinol to hexaammineruthenium (III) (HAR) catalyzed by mitochondrial respiratory complex I is introduced. The specific activity of the enzyme in this reaction and its sensitivity to the standard inhibitors and uncoupling are the same as with other well-known electron acceptors, NAD+ and ferricyanide. In contrast to the reactions with these acceptors, the energy-dependent HAR reduction is not inhibited by NADH-OH, the specific inhibitor of NADH-binding site. These results suggest that a catalytically competent electron connection exists between HAR and a redox component of complex I that is different from flavin mononucleotide bound at the substrate-binding site.

Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay.

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125-750 µM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria.

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.

Respiratory complex II: ROS production and the kinetics of ubiquinone reduction.

Bovine heart mitochondrial respiratory complex II generates ROS, mostly as superoxide, at the rate of about 20% of that detected during simultaneous operation of complex I and II when oxidation of ubiquinol is prevented by myxothiazol. ROS generating activity at different fumarate/succinate concentrations ratio implies that an enzyme component with a midpoint potential 40mV more positive than that of fumarate/succinate couple is the donor for one-electron reduction of oxygen. This suggests that the iron-sulfur cluster(s) is(are) involved in superoxide formation. Complex II-mediated ROS production exhibits a maximum at low (about 50µM) succinate concentration and gradually declines to zero activity upon further increase of the substrate. This phenomenology is explained and kinetically modeled to suggest a ping-pong mechanism of ROS generating activity where only dicarboxylate free reduced enzyme is oxidized by oxygen. The succinate:quinone reductase activity catalyzed by purified succinate:ubiquinone reductase also exhibits a ping-pong mechanism where only dicarboxylate free enzyme is oxidized by added quinone. Together these data suggest long distance interaction between the succinate (fumarate) binding and ubiquinone (ubiquinol) reactive sites.

Oxidation of NADH and ROS production by respiratory complex I.

Kinetic characteristics of the proton-pumping NADH:quinone reductases (respiratory complexes I) are reviewed. Unsolved problems of the redox-linked proton translocation activities are outlined. The parameters of complex I-mediated superoxide/hydrogen peroxide generation are summarized, and the physiological significance of mitochondrial ROS production is discussed. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

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.

Partitioning of superoxide and hydrogen peroxide production by mitochondrial respiratory complex I.

Membrane-bound respiratory complex I in inside-out submitochondrial particles (SMP) catalyzes both superoxide and hydrogen peroxide formation in NADH- and/or succinate-supported reactions. At optimal NADH concentration (50µM), the complex I-mediated process results in a formation of two superoxide anions and H(2)O(2) as the reaction products in approximately 0.7 ratio. Almost the same ratio is found for purified complex I (0.6) and for the aerobic succinate-supported reverse electron transfer reaction. Superoxide production is depressed at high, more physiologically relevant NADH concentrations, whereas hydrogen peroxide formation is insensitive to the elevated level of NADH. The rates of H(2)O(2) formation at variable NAD(+)/NADH ratios satisfactorily fit the Nernst equation for a single reactive two-electron donor component equilibrated with ambient midpoint redox potential of -347mV (0.13 NAD(+)/NADH ratio, pH 8.0). Half-maximal superoxide production rate proceeds at significantly higher NAD(+)/NADH ratio (0.33). Guanidine strongly stimulates NADH-supported hydrogen peroxide and superoxide production at any NADH concentration and activates NADH:ferricyanide and inhibits NADH:hexaammineruthenium (III) reductase activities while showing no effects on NADH oxidase of SMP. In the low range of NADH concentration, superoxide production rate shows a simple hyperbolic dependence on NADH with apparent K(m)(NADH) of 0.5µM, whereas sigmoidal dependence of hydrogen peroxide production is seen with half-maximal rate at 25µM NADH. We interpret the data as to suggest that at least two sites participate in complex I-mediated ROS generation: FMNH(-) that produces hydrogen peroxide, and an iron-sulfur center (likely N-2) that produces superoxide anion.

Mitochondrial hydrogen peroxide production as determined by the pyridine nucleotide pool and its redox state.

The rates of NADH-supported superoxide/hydrogen peroxide production by membrane-bound bovine heart respiratory complex I, soluble pig heart dihydrolipoamide dehydrogenase (DLDH), and by accompanying operation of these enzymes in rat heart mitochondrial matrix were measured as a function of the pool of pyridine nucleotides and its redox state. Each of the activities showed nontrivial dependence on nucleotide pool concentration. The NAD(+)/NADH ratios required for their half maximal capacities were determined. About half of the total NADH-supported H(2)O(2) production by permeabilized mitochondria in the absence of stimulating ammonium could be accounted for by DLDH activity. The significance of the mitochondrial NADH-dependent hydrogen peroxide production under physiologically relevant conditions is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I).

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production.

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287-1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H(2)O(2) production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.

What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4-State 3 transition) decreases the succinate- and glutamate plus malate-supported H2O2 production 8- and 1.3-times, respectively. NH4+ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H2O2 production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H2O2 diffusion by alamethicin treatment are only partially (approximately 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (approximately 6-fold) stimulated by ammonium chloride. NAD+ inhibits both Complex I-mediated and ammonium-stimulated H2O2 production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli.

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc(1) (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.

Ammonium-dependent hydrogen peroxide production by mitochondria.

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.

NADH/NAD+ interaction with NADH: ubiquinone oxidoreductase (complex I).

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I).

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K(d ( approximately 10(-8)M at pH 10.0).

Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I.

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly 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).

Topography and chemical reactivity of the active-inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane-matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.

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.