Murphy 2009 Biochem J

Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1-13.

» PMID:19061483 Open Access

Murphy MP (2009) Biochem J

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 Δp (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 Δp and NADH/NAD⁺ ratio, the extent of O₂^•− production is far lower. The generation of O₂^•− within the mitochondrial matrix depends critically on Δp, 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 signaling.

Selected quotes

• In contrast with the difficulties of assessing O₂^•− directly, intramitochondrial O₂^•− flux can be readily measured in isolated mitochondria following its dismutation to H₂O₂ by MnSOD and subsequent diffusion from the mitochondria [11,54,55].

• not all H₂O₂ produced within the mitochondrial matrix will survive to efflux from the mitochondria, owing to matrix peroxidases that consume H₂O₂ [1,61–63]. These include peroxiredoxins 3 and 5 [64], catalase [65,66] and glutathione peroxidases 1 and 4 [67], with the peroxiredoxins probably of the greatest significance [64].

• mitochondrial ROS production is also reported to increase under conditions of very low [O₂], which is paradoxical and seems to contradict the dependence of mitochondrial O₂^•− production on [O₂] given in eqn (2). These hypoxic effects are seen in cultured cells when ambient O₂ is decreased from 21 % O₂ to 1–3 % O₂ [120,121]. This corresponds to an equilibrium [O₂] of 10–20 μM, although the local [O₂] around mitochondria will be lower.

• the question remains of how lowering [O₂] can increase H₂O₂ efflux from mitochondria. .. the question remains of how lowering [O₂] can increase H₂O₂ efflux from mitochondria. When isolated mitochondria were maintained at low [O₂], ROS production decreased as the [O₂] was lowered from approx. 5 μM O₂ to anoxia [29].

• Clearly, more work is required to unravel the mechanism of increased mitochondrial ROS production during hypoxia, but it remains an intriguing puzzle, and our understanding of mitochondrial ROS production will be incomplete until there is a satisfactory explanation for this phenomenon.

• Since it was first published by Chance and colleagues [4,12], this value of 1–2 % of respiration going to O₂^•− has propagated through the literature and has been used erroneously to estimate mitochondrial O₂^•− production in vivo, even though the original authors made it clear that it only applied to particular experimental conditions [12].

• when glutamate/malate are used as substrates, H₂O₂ production accounts for approx. 0.12 % of respiration [27].

• Secondly, measurements on isolated mitochondria are generally made using air-saturated medium containing ∼ 200 μM O₂. As mitochondrial O₂^•− production is probably proportional to [O₂] and the physiological [O₂] around mitochondria is approx. 10–50 μM, O₂^•− production may be 5–10-fold lower than for isolated mitochondria in the same state.

• The third and most important factor limiting extrapolation of in vitro O₂^•− production to the situation in vivo is that mitochondria in vivo are likely to be making ATP and will thus be operating in mode 3 with a lowered Δp and relatively oxidized NADH and CoQ pools. Consequently, their rates of H₂O₂ efflux are negligible compared with modes 1 or 2.

Cited by

• Komlodi et al (2022) Hydrogen peroxide production, mitochondrial membrane potential and the coenzyme Q redox state measured at tissue normoxia and experimental hyperoxia in heart mitochondria. MitoFit Preprints 2021 (in prep)
  
• Komlódi T, Schmitt S, Zdrazilova L, Donnelly C, Zischka H, Gnaiger E. Oxygen dependence of hydrogen peroxide production in isolated mitochondria and permeabilized cells. MitoFit Preprints (in prep).

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MitoFit 2021 Tissue normoxia, MitoFit 2021 AmR