How mitochondria produce reactive oxygen species

Abstract

The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O2(*-)) is the proximal mitochondrial ROS, and in the present review I outline the principles that govern O2(*-) production within the matrix of mammalian mitochondria. The flux of O2(*-) is related to the concentration of potential electron donors, the local concentration of O2 and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O2(*-) production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Deltap (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is a high NADH/NAD+ ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Deltap and NADH/NAD+ ratio, the extent of O2(*-) production is far lower. The generation of O2(*-) within the mitochondrial matrix depends critically on Deltap, the NADH/NAD+ and CoQH2/CoQ ratios and the local O2 concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O2(*-) generation by mitochondria in vivo from O2(*-)-production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to measure mitochondrial O2(*-) and H2O2 formation in vivo, as uncertainty about these values hampers studies on the role of mitochondrial ROS in pathological oxidative damage and redox signalling.

Figures

Figure 1. Overview of mitochondrial ROS production

ROS production by mitochondria can lead to oxidative damage to mitochondrial proteins, membranes and DNA, impairing the ability of mitochondria to synthesize ATP and to carry out their wide range of metabolic functions, including the tricarboxylic acid cycle, fatty acid oxidation, the urea cycle, amino acid metabolism, haem synthesis and FeS centre assembly that are central to the normal operation of most cells. Mitochondrial oxidative damage can also increase the tendency of mitochondria to release intermembrane space proteins such as cytochrome c (cyt c) to the cytosol by mitochondrial outer membrane permeabilization (MOMP) and thereby activate the cell's apoptotic machinery. In addition, mitochondrial ROS production leads to induction of the mitochondrial permeability transition pore (PTP), which renders the inner membrane permeable to small molecules in situations such as ischaemia/reperfusion injury. Consequently, it is unsurprising that mitochondrial oxidative damage contributes to a wide range of pathologies. In addition, mitochondrial ROS may act as a modulatable redox signal, reversibly affecting the activity of a range of functions in the mitochondria, cytosol and nucleus.

Figure 2. Measurement of H2O2 production by isolated mitochondria

The production of O2•− within the mitochondrial matrix, intermembrane space and outer membrane leads to the formation of H2O2 from SOD-catalysed dismutation. Some O2•− can react directly with nitric oxide (NO•) to form peroxynitrite (ONOO−). There are also sources outside mitochondria that produce H2O2 directly. The H2O2 efflux from mitochondria can be measured following reaction with a non-fluorescent substrate such as Amplex Red in conjunction with horseradish peroxidase (HRP) to form a fluorescent product, resorufin. Within mitochondria H2O2 is degraded by glutathione peroxidases (GPx) or peroxiredoxins (Prx) which depend on glutathione (GSH) and thioredoxin-2 (Trx) for their reduction respectively. Glutathione disulfide (GSSG) is reduced back to GSH by glutathione reductase (GR). Trx is reduced by thioredoxin reductase-2 (TrxR). Both enzymes receive reducing equivalents from the NADPH pool, which is kept reduced by the Δp-dependent transhydrogenase (TH), and by isocitrate dehydrogenase (ICDH). Note that many mitochondrial preparations, particularly those from the liver, contain large amounts of catalase contamination. The effects of such extramitochondrial H2O2 sinks are not indicated here as they are usually accounted for by appropriate H2O2 calibration curves in the presence of mitochondria. ox, oxidized; red, reduced. An animated version of this Figure can be seen at http://www.BiochemJ.org/bj/417/0001/bj4170001add.htm.

Figure 3. Modes of mitochondrial operation that lead to O2•− production

There are three modes of mitochondrial operation that are associated with O2•− production. In mode 1, the NADH pool is reduced, for example by damage to the respiratory chain, loss of cytochrome c during apoptosis or low ATP demand. This leads to a rate of O2•− formation at the FMN of complex I that is determined by the extent of FMN reduction which is in turn set by the NADH/NAD+ ratio. Other sites such as αKGDH may also contribute. In mode 2, there is no ATP production and there is a high Δp and a reduced CoQ pool which leads to RET through complex I, producing large amounts of O2•−. In mode 3, mitochondria are actively making ATP and consequently have a lower Δp than in mode 2 and a more oxidized NADH pool than in mode 1. Under these conditions, the flux of O2•− within mitochondria is far lower than in modes 1 and 2, and the O2•− sources are unclear.

Figure 4. Production of O2•− by complex I

The cartoon of complex I is a chimaera modelled on the hydrophobic arm of Yarrowia lipolytica obtained by electron microscopy [132] and the crystal structure of the hydrophilic arm from Thermus thermophilus [82]. The location of the FMN and the FeS centres in the water-soluble arm are indicated, along with the putative CoQ-binding site. In mode 1, there is extensive O2•− production from the FMN in response to a reduced NADH pool. In mode 2, a high Δp and a reduced CoQ pool lead to RET and a high flux of O2•− from the complex. The site of this O2•− production is uncertain, hence the question mark, but may be associated with the CoQ-binding site(s).

Figure 5. Possible mechanisms of mitochondrial redox signalling

The production of H2O2 from mitochondria is a potential redox signal. H2O2 generated by mitochondria can reversibly alter the activity of proteins with critical protein thiols by modifying them to intra- or inter-protein disulfides, or to mixed disulfides with GSH. These modifications can occur on mitochondrial, cytosolic or nuclear enzymes, carriers or transcription factors, transiently altering their activities. The change in activity can be reversed by reducing the modified protein thiol by endogenous thiol reductants such as GSH or thioredoxin. As the extent of H2O2 production from mitochondria will depend on factors such as Δp or the redox state of the NADH pool, it can act as a retrograde signal to the rest of the cell, reporting on mitochondrial status. This signal can then lead to the short-term modification of, for example, pathways supplying substrates to the mitochondria. Alternatively, longer-term modifications can occur through modifying redox-sensitive transcription factors that adjust the production of mitochondrial components. In addition, external signals may modify O2•− production by the respiratory chain by post-translational modification. Alteration of the activity of mitochondrial peroxidases could also modulate H2O2 efflux from mitochondria to the rest of the cell. It is also possible that secondary redox signals, such as lipid peroxidation products derived from H2O2, could act as secondary redox signals.