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Summary Using high-throughput screening we identified small molecules that suppress superoxide and/or H 2 O 2 production during reverse electron transport through mitochondrial respiratory complex I (site I Q ) without affecting oxidative phosphorylation (suppressors of site I Q electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site I Q is a normal contributor to mitochondrial superoxide-H 2 O 2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H 2 O 2 production by site I Q is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H 2 O 2 production by site I Q is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site I Q to cell physiology and pathology and have great potential as therapeutic leads.

The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.

H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ~40 pmol H2O2 · min−1 · mg protein−1, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ~200 pmol H2O2 · min−1 ~ mg protein−1 under conditions of compromised antioxidant defense and reduced ubiqui-none pool. Thus complex II and the ETF system both contribute to H2O2 production during fatty acid oxidation under appropriate conditions.

Mitochondria constitute an important topic of biomedical enquiry (one paper in every 154 indexed in PubMed since 1998 is retrieved by the keyword 'mitochondria') because of widespread recognition of their importance in cell physiology and pathology. Mitochondrial dysfunction is widely implicated in ageing and in the diseases of ageing, through dysfunction in adenosine triphosphate (ATP) synthesis, Ca(2+) homeostasis, central metabolic pathways or radical production. Nonetheless, the mechanisms and regulation of superoxide and hydrogen peroxide formation by mitochondria remain poorly described. Measurement of the capacities of different sites of superoxide and hydrogen peroxide production in isolated skeletal muscle mitochondria show that the maximum capacities of sites in complexes I, II and III and in several associated redox enzymes greatly exceed the native rates observed in the absence of respiratory chain inhibitors. In vitro, the native rates and the relative importance of different sites both depend on the substrate being oxidized, with sites IQ, IIF, GPDH, IF and IIIQo each being important with particular substrates. The techniques involved in measuring rates from each site should become applicable to cell cultures and in vivo in the future.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species (ROS), which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies but its role remains controversial. Using high-throughput screening we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.

Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H 2 O 2 ) at various sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site I F ) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site III Qo ) was calibrated to the reduction state of endogenous cytochrome b 566 . We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites I F and III Qo . The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site I F was the dominant superoxide producer. Under nonphosphorylating conditions, overall rates were higher, and sites I F and III Qo and unidentified sites (perhaps the complex I site of quinone reduction, site I Q ) all made substantial contributions to measured H 2 O 2 production.

This review describes the method of fluorescence correlation spectroscopy (FCS) and its applications. FCS is used for investigating processes associated with changes in the mobility of molecules and complexes and allows researchers to study aggregation of particles, binding of fluorescent molecules with supramolecular complexes, lipid vesicles, etc. The size of objects under study varies from a few angstroms for dye molecules to hundreds of nanometers for nanoparticles. The described applications of FCS comprise various fields from simple chemical systems of solution/micelle to sophisticated regulations on the level of living cells. Both the methodical bases and the theoretical principles of FCS are simple and available. The present review is concentrated preferentially on FCS applications for studies on artificial and natural membranes. At present, in contrast to the related approach of dynamic light scattering, FCS is poorly known in Russia, although it is widely employed in laboratories of other countries. The goal of this review is to promote the development of FCS in Russia so that this technique could occupy the position it deserves in modern Russian science.

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.