Background: Complex II is not considered a significant contributor to mitochondrial ROS production. Results: Complex II generates ROS in both the forward reaction, from succinate, and the reverse reaction, from the reduced ubiquinone pool. Conclusion: Occupancy and reduction state of the flavin dictate its ROS producing behavior. Significance: Based on the maximum rates observed, complex II may be a contributor to physiological ROS production.

Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.

Background: At the redox potential of NADH/NAD ϩ , at least four mitochondrial sites produce superoxide/H 2 O 2 .

Background: Ten mitochondrial sites of superoxide/H 2 O 2 generation are known, but their contributions in vivo are undefined. Results: We assessed their rates ex vivo in conditions mimicking rest and exercise. Conclusion: Sites I Q and II F generated half the signal at rest. During exercise, rates were lower, and site I F dominated. Significance: Contributing sites ex vivo probably reflect those in vivo.

The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.

Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bellshaped relationship to the reduction state of cytochrome b 566 , suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q o ). Imposition of a membrane potential changed the relationships between superoxide production and b 566 reduction and between b 562 and b 566 redox states, suggesting that b 562 reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q o site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b 566 to depend on the b 562 redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b 566 was reduced, and reduction of b 562 stabilized the semiquinone when b 566 was oxidized. Thus, superoxide production at Q o depends on the reduction states of b 566 and b 562 and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.Mitochondria generate superoxide during oxidative metabolism. Damage caused by this and other reactive oxygen species (ROS) 2 has been implicated in the pathology of many diseases and the general phenomenon of aging (1-4). Isolated mitochondria generate superoxide from a number of sites, including the electron transport chain complexes I and III, glycerol 3-phosphate, 2-oxoglutarate, and pyruvate dehydrogenases and perhaps complex II and electron transfer flavoprotein-quinone oxidoreductase (5-7). The quinone (Q)-binding sites of NADH:Q oxidoreductase (complex I) and the cytochrome bc 1 complex (complex III) have the highest maximum rates of superoxide production (7,8). Superoxide from complex III is thought to arise from the reaction of oxygen with a semiquinone produced in the Q o site (the quinone binding site on the outer or cytosolic face of the protein) (9, 10).Complex III operates by a Q-cycle mechanism (11-13). Quinol is oxidized in the Q o site by a bifurcated electron transfer reaction that directs the two electrons down divergent paths. The first electron is passed down the high potential chain from the Rieske Fe-S center through cytochrome c 1 to cytochrome c and complex IV. The second electron is passed down the low potential chain from cytochrome b 566 through cytochrome b 562...

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 H2O2) at different sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site IF) 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 IIIQo) was calibrated to the reduction state of endogenous cytochrome b566. 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 IF and IIIQo. The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site IF was the dominant superoxide producer. Under non-phosphorylating conditions, overall rates were higher and sites IF, IIIQo and unidentified sites (perhaps the complex I site of quinone reduction, site IQ) all made substantial contributions to measured H2O2 production.

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD ؉ -reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH 2 oxidation via the Q cycle. Stepwise oxidation of the QH 2 /Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD ؉ redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I F ) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I Q ). Superoxide production by mitochondrial complex I (NADHubiquinone (Q)2 oxidoreductase) has been demonstrated using the isolated complex (1, 2), submitochondrial particles (3-7), and intact mitochondria isolated from a number of sources (8 -14). Isolated mammalian (bovine) complex I produces superoxide from the reduced flavin of the flavin mononucleotide (FMN) moiety (1, 2, 15). When compared over a range of intramitochondrial NADH/NAD ϩ ratios, superoxide production by complex I during forward electron transport in intact mitochondria is also maximal when the NAD pool is highly reduced (9,14,17).However, complex I can also produce superoxide at very high rates under conditions that drive the reduction of NAD ϩ by reverse electron transport. Superoxide production during reverse electron transport has been demonstrated with intact mitochondria (10,11,18) and well coupled submitochondrial particles (7,19). An important distinction is that the rate of superoxide production by isolated mitochondria during reverse electron transport can be severalfold higher than the maximum rate when the flavin is fully reduced by the addition of NADHgenerating substrates plus rotenone or other complex I Q site inhibitors (10,20). The high rate of superoxide production during reverse electron transport is inhibited by complex I Q site inhibitors and is highly sensitive to the pH gradient across the mitochondrial membrane (⌬pH), as well as to uncoupling and declining pro...