Reprint of: Ubisemiquinone Is the Electron Donor for Superoxide Formation by Complex III of Heart Mitochondria.

Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

Suggested involvement of ketone bodies in the pathogenesis of the metabolic syndrome.

Untreated brain mitochondria are strong producers of H2O2. High peroxide production (in the presence of glutamate and pyruvate) is strictly succinate-dependent. Importantly, it is inhibited by the ketone body acetoacetate (AcAc) starting at 10 µM (maximal effect at 0.5mM). Butyrate derives from the fermentation of prebiotics, is present physiologically in the colon and is a strong producer of AcAc: indeed butyrate induces in the colon the transcription of mitochondrial 3-hydroxy-3-methyl glutarylCoA (HMGCoA) synthase, a key enzyme in ketone body synthesis. Obesity and insulin resistance were shown to be dependent on increased permeability of the colon epithelium to bacterial lipopolysaccharide (LPS); the process is evident particularly upon ingestion of lipids (a peroxidative event, inhibited by vitamin E) and is likely sensitive to AcAc. The oxidation of butyrate and the production of AcAc in the colon appear to be inhibited by high luminal sulphides and high NH3, a situation that presumably facilitates LPS permeation (on the contrary beta-hydroxy-butyrate oxidation is not inhibited). It is proposed that these damaging events may be opposed by the delivery of ketone bodies directly to the colon.

The control of mitochondrial succinate-dependent H2O2 production.

In brain mitochondria succinate activates H(2)O(2) release, concentration dependently (starting at 15 µM), and in the presence of NAD dependent substrates (glutamate, pyruvate, ß-hydroxybutyrate). We report that TCA cycle metabolites (citrate, isocitrate, α-ketoglutarate, fumarate, malate) individually and quickly inhibit H(2)O(2) release. When they are present together at physiological concentration (0.2, 0.01, 0.15, 0.12, 0.2 mM respectively) they decrease H(2)O(2) production by over 60% at 0.1-0.2 mM succinate. The degree of inhibition depends on the concentration of each metabolite. Acetoacetate is a strong inhibitor of H(2)O(2) release, starting at 10 µM and acting quickly. It potentiates the inhibition induced by TCA cycle metabolites. The action of acetoacetate is partially removed by ß-hydroxybutyrate. Removal is minimal at 0.1 mM acetoacetate, and is higher at 0.5 mM acetoacetate. We conclude that several inhibitors of H(2)O(2) release act jointly and concentration dependently to rapidly set the required level of H(2)O(2) generation at each succinate concentration.

Different effects of SNP and GSNO on mitochondrial O2 .- /H 2O2 production.

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H(2)O(2) release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ((.)NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its (.)NO release) induces an inhibition of the succinate dependent H(2)O(2) production consistent with a (.)NO dependent covalent modification. However maximal inhibition of the succinate dependent H(2)O(2) release is obtained in the presence of low GSNO (20-100 µM), but not with SNP. This inhibition appears independent of (.)NO release since µM GSNO does not affect mitochondrial respiration, or the H(2)O(2) detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O (2) (.-) since GSNO chemically competes with NBT and cytochrome C in O (2) (.-) detection.

Succinate is the controller of O2-/H2O2 release at mitochondrial complex I : negative modulation by malate, positive by cyanide.

Mitochondrial production of H(2)O(2) is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 microM, maximal H(2)O(2) production at 600 microM succinate). Malate counteracts rapidly the succinate induced increased H(2)O(2) release and moves the succinate dependent H(2)O(2) production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN(-)) was used to mimic NO and CO. In the presence of G/P and succinate (300 microM), CN(-) progressively increased the H(2)O(2) release rate, starting at 1.5 microM. The succinate dependent H(2)O(2) production curve was moved to the left by 30 microM CN(-). The V(max) was little modified. We conclude that succinate is the controller of mitochondrial H(2)O(2) production, modulated by malate and CN(-). We propose that succinate promotes an interaction between Complex II and Complex I, which activates O(2)(-) production.

Clorgyline and other propargylamine derivatives as inhibitors of succinate-dependent H(2)O(2) release at NADH:UBIQUINONE oxidoreductase (Complex I) in brain mitochondria.

Complex I is the main O(2)(-) producer of the mitochondrial respiratory chain. O(2)(-) release is low with NAD-linked substrates and increases strongly during succinate oxidation, which increases the QH(2)/Q ratio and is rotenone sensitive. We show that the succinate dependent O(2)(-) production (measured as H(2)O(2) release) is inhibited by propargylamine containing compounds (clorgyline, CGP 3466B, rasagiline and TVP-1012). The inhibition does not affect membrane potential and is unaffected by DeltapH modifications. Mitochondrial respiration is similarly unaffected. The propargylamines inhibition of O(2)(-)/H(2)O(2) production is monitored also in the presence of the Parkinson's disease toxin dopaminochrome which stimulates O(2)(-) release. Propargylamine-containing compounds are the first pharmacological inhibitors described for O(2)(-) release at Complex I.

Long chain fatty acyl-CoA modulation of H(2)O (2) release at mitochondrial complex I.

Complex I is responsible for most of the mitochondrial H(2)O(2) release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H(2)O(2) 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 H(2)O(2) 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 DeltapH or Deltap 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.

Succinate modulation of H2O2 release at NADH:ubiquinone oxidoreductase (Complex I) in brain mitochondria.

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.

Dopamine-derived dopaminochrome promotes H(2)O(2) release at mitochondrial complex I: stimulation by rotenone, control by Ca(2+), and relevance to Parkinson disease.

Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1-0.2 mum)- and respiration-dependent generation of H(2)O(2) at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5-30 nm). The rotenone effect required that contaminating Ca(2+) (8-10 mum) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O(2), generating O(2)(.) and then H(2)O(2). Mitochondrial removal of H(2)O(2) monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca(2+) levels. Thus, the H(2)O(2) formed accumulated in the medium if contaminating Ca(2+) was present; in the absence of Ca(2+), H(2)O(2) was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular O(2)(.) release via activation of NADPH oxidase in the microglia. In turn, O(2)(.) oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.

Respiration-dependent removal of exogenous H2O2 in brain mitochondria: inhibition by Ca2+.

In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect approximately 40%, IC50, 12 microm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60-70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 microm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased DeltaPsi, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks.