Casey L. Quinlan

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

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. 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 H2O2 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 IIF). 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.

Sites of reactive oxygen species generation by mitochondria oxidizing different substrates

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.

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Background: At the redox potential of NADH/NAD+, at least four mitochondrial sites produce superoxide/H2O2. Results: We compare their capacities in situ in isolated mitochondria. Conclusion: Maximum capacities of complexes were 2-oxoglutarate dehydrogenase > pyruvate dehydrogenase > branched-chain 2-oxoacid dehydrogenase > complex I. Significance: H2O2 production from 2-oxoacid dehydrogenases can be considerable but may previously have been misattributed to complex I. Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.

Evidence for two sites of superoxide production by mitochondrial NADH-ubiquinone oxidoreductase (complex I).

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 QH2 oxidation via the Q cycle. Stepwise oxidation of the QH2/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 IF) 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 IQ).

Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

Background: Ten mitochondrial sites of superoxide/H2O2 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 IQ and IIF generated half the signal at rest. During exercise, rates were lower, and site IF dominated. Significance: Contributing sites ex vivo probably reflect those in vivo. 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.

The Mechanism of Superoxide Production by the Antimycin-inhibited Mitochondrial Q-cycle

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 bell-shaped relationship to the reduction state of cytochrome b566, 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 (Qo). Imposition of a membrane potential changed the relationships between superoxide production and b566 reduction and between b562 and b566 redox states, suggesting that b562 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 Qo 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 b566 to depend on the b562 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 b566 was reduced, and reduction of b562 stabilized the semiquinone when b566 was oxidized. Thus, superoxide production at Qo depends on the reduction states of b566 and b562 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.

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

Background: Oxidation of glycerol 3-phosphate generates superoxide/H2O2 from multiple sites within mitochondria. Results: Some of the superoxide/H2O2 originates specifically from mGPDH, but much can come from complex II; this demands a reassessment of prior investigations. Conclusion: The ubiquinone binding site in mGPDH produces superoxide to both sides of the inner membrane. Significance: mGPDH can generate superoxide at rates comparable with other major sites. 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 H2O2 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 H2O2 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 H2O2. Importantly, we demonstrate that a substantial portion of H2O2 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.

Native rates of superoxide production from multiple sites in isolated mitochondria measured using endogenous reporters.

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.

Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production – a correction using glutathione depletion

The production of H2O2 by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H2O2. However, matrix enzymes such as the glutathione peroxidases can consume H2O2 and compete with efflux of H2O2, causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1‐chloro‐2,4‐dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H2O2, consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H2O2 formation from matrix‐directed superoxide by up to two‐fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2–2.5 nmol H2O2·min−1·mg protein−1. Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H2O2 efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H2O2 efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB‐pretreated rate = control rate + [1.43 × (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.

Measurement of Proton Leak and Electron Leak in Isolated Mitochondria

Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton and electron leaks. In this chapter, methods are presented to measure proton and electron leak activities in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasised.