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 signalling.

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How mitochondria produce reactive oxygen species.

Hydroperoxide metabolism in mammalian organs.

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2−•). Even though O2−• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments. We also discuss various physiological and pathological scenarios resulting from an increased steady state concentration of mitochondrial oxidants.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-7793.2003.00335.x

Mitochondrial formation of reactive oxygen species.

https://www.cell.com/cell/pdf/S0092-8674(05)00109-1.pdf

Mitochondria, Oxidants, and Aging

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H 2 O 2 indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H 2 O 2 generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H 2 O 2 . Succinate, the most effective substrate, yields H 2 O 2 at a rate of 0.5nmol/min per mg of protein in state 4. H 2 O 2 generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H 2 O 2 modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H 2 O 2 at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H 2 O 2 rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H 2 O 2 (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H 2 O 2 /mol of NADP + during the oxidation of NADPH. H 2 O 2 is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H 2 O 2 /mol of NAD + ). 8. Estimation of the rates of production of H 2 O 2 under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H 2 O 2 /min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H 2 O 2 on cellular metabolism.

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The cellular production of hydrogen peroxide

The role of H2O2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors

Ethanol oxidation by rat liver catalase (the ‘peroxidatic’ reaction) was studied quantitatively with respect to the rate of H2O2 generation, catalase haem concentration, ethanol concentration and the steady-state concentration of the catalase–H2O2 intermediate (Compound I). At a low ratio of H2O2-generation rate to catalase haem concentration, the rate of ethanol oxidation was independent of the catalase haem concentration. The magnitude of the inhibition of ethanol oxidation by cyanide was not paralleled by the formation of the catalase–cyanide complex and was altered greatly by varying either the ethanol concentration or the ratio of the rate of H2O2 generation to catalase haem concentration. The ethanol concentration producing a half-maximal activity was also dependent on the ratio of the H2O2-generation rate to catalase haem concentration. These phenomena are explained by changes in the proportion of the ‘catalatic’ and ‘peroxidatic’ reactions in the overall H2O2-decomposition reaction. There was a correlation between the proportion of the ‘peroxidatic’ reaction in the overall catalase reaction and the steady-state concentration of the catalase–H2O2 intermediate. Regardless of the concentration of ethanol and the rate of H2O2 generation, a half-saturation of the steady state of the catalase–H2O2 intermediate indicated that about 45% of the H2O2 was being utilized by the ethanol-oxidation reaction. The results reported show that the experimental results in the study on the ‘microsomal ethanol-oxidation system’ may be reinterpreted and the catalase ‘peroxidatic’ reaction provides a quantitative explanation for the activity hitherto attributed to the ‘microsomal ethanol-oxidation system’.

/pdf/the-characteristics-of-the-peroxidatic-reaction-of-catalase-12i198ztea.pdf

The characteristics of the "peroxidatic" reaction of catalase in ethanol oxidation.

Optical measurements of intracellular oxygen concentration of rat heart in vitro

The importance of measuring intracellular oxygen concentrations in tissues has, over the years, emerged as a basic parameter in the physiology and biochemistry of living tissues. The credibility of oxyhemoglobin determinations, even as refined A/V differences, is taxed especially in cases where inhomogeneous tissues with variable oxygen demands and oxygen supply are served. The formation of lactic acid in the venous blood is often used as a criterion of anoxia, but it also lacks credibility where inhomogeneous circulatory pathways are served and in addition, is questionable from the standpoint of whether the appearance of excess lactate is an unequivocal criterion of oxygen insufficiency. To indicate my empathy with polarographic techniques as they have been developed at the Johnson Foundation, I wish to recall the pioneering works of Bronk(1) Brink (2), Davies and Remond (3) that stand as landmarks in the exploration of tissue oxygen tension by microelectrode methods. I served my apprenticeship with them.

Nozomu Oshino

Papers

The cellular production of hydrogen peroxide

The role of H2O2 generation in perfused rat liver and the reaction of catalase compound I and hydrogen donors

The characteristics of the "peroxidatic" reaction of catalase in ethanol oxidation.

Optical measurements of intracellular oxygen concentration of rat heart in vitro

Basic principles of tissue oxygen determination from mitochondrial signals.