Review
Bioenergetics and the formation of mitochondrial reactive oxygen species

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The contribution of mitochondria to the manifestation of disease is ascribed largely to the production of reactive oxygen species (ROS), which are obligatory by-products of aerobiosis. Studies using isolated mitochondria have revealed multiple potential sites and circumstances of ROS production but the relevance of these to in situ conditions is limited. In this article, we focus on bioenergetic factors that promote ROS generation at physiologically relevant sites in mitochondria. Emphasis is given to ROS generation by complex I – the first component of the respiratory chain – and to how the NADH:NAD+ ratio regulates ROS formation. Complex I is a physiologically and pathologically relevant ROS-forming site that is important not only in normal mitochondrial energy production but also in the pathogenesis of Parkinson's disease, which is the second most common neurodegenerative disease.

Introduction

Stryer states that danger lurks in the reduction of O2 in mitochondria and that the strategy for its safe reduction is clear: the catalyst (the respiratory chain) must not release partly reduced intermediates [1]. Such an intermediate is the superoxide anion (O2radical dot), which is also classified as a reactive oxygen species (ROS). By definition, ROS also include the hydroxyl radical radical dotOH and the non-radical species hydrogen peroxide (H2O2) [2]. ROS have physiological roles in signal transduction [3], although they have received attention as potential damagers in a plethora of diseases affecting virtually all parts of the human body [3] and, most importantly, two vital organs – the heart [4] and the brain [5]. In addition, these molecules probably contribute to aging in people who are disease free [6].

Mitochondria are considered to be a major source of ROS, the formation of which is inevitable during normal oxidative metabolism, although it can increase greatly in pathological conditions. Oxidative stress caused by the effects of ROS in cells has been implicated in the pathogenesis of many diseases, probably most convincingly in neurodegenerative diseases. For example, Parkinson's disease (PD), Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis express distinct pathological and symptomatic features but there is overwhelming evidence that mitochondrial defects and oxidative stress have a role in the pathogenic processes of all these diseases [7]. It is unclear whether the accumulation of ROS is a primary event or a consequence of other cellular dysfunctions under these conditions. Understanding the mechanisms by which ROS are generated in mitochondria and the conditions that favor or influence this process could help to find effective means with which to protect cells against the injurious effect of excessive ROS formation.

In this article, we review mitochondrial ROS generation, focusing on bioenergetic aspects and on the role of complex I (an important ROS-forming site; see Glossary) in excessive ROS generation. Complex I deficiency in the brain has been linked to the pathogenesis of PD [8], emphasizing the crucial role of ROS generation by this complex in neurodegeneration and the impact of the regulation of ROS formation by key bioenergetic parameters.

Section snippets

ROS formation in mitochondria

ROS generation within mitochondria is closely associated with the primary function of these organelles, namely oxidative metabolism and ATP synthesis. The machinery of mitochondrial metabolism ultimately leads to ATP synthesis by the F1Fo-ATPase in the inner membrane of mitochondria (Figure 1). This process involves oxidation of metabolites in the tricarboxylic acid cycle, resulting in the formation of NADH and FADH2. These reducing equivalents supply electrons derived from the metabolism of

Dependence of mitochondrial ROS production on the NADH:NAD+ ratio

Hansford et al. [18] first demonstrated that H2O2 generation is associated with a high degree of pyridine nucleotide reduction. It is important to emphasize that succinate, which donates electrons to complex II via FADH2, was used in this study (Figure 1). With succinate as a substrate, most electrons run through complexes III and IV to oxygen, generating sufficient pmf to drive the flow of a proportion of electrons in reverse via complex I – an energetically uphill process termed ‘reverse

Dependence of mitochondrial ROS production on the pmf

The redox state of pyridine nucleotides, and other key bioenergetic parameters in mitochondria are controlled by the pmf. Because changes in ΔΨm usually reflect changes in the pmf, most studies address this parameter when studying ROS generation. Succinate-supported ROS production requires high ΔΨm [12]; a small decrease in ΔΨm due to uncoupling paralleled a substantial decrease in H2O2 formation in experiments on isolated heart and brain mitochondria 17, 32. The strong dependence of ROS

Pharmacological considerations

There is overwhelming evidence of the involvement of oxidative stress in tissue pathology. However, it is important to note that mitochondria not only initiate oxidative stress due to excess production of ROS but also enhance the overall tissue injury by becoming targets of their own damaging products [40]. The latter phenomenon is pertinent to at least two intramitochondrial sites of ROS generation: complex I and α-KGDH. ROS can inhibit complex I activity dose dependently [41], setting the

Concluding remarks

An important conclusion can be drawn from the relationship between mitochondrial ROS formation and energy production: mitochondria that perform their major physiological function, ATP synthesis, impose less oxidative danger on cells than do resting mitochondria, despite the multiple sites within these organelles that have the means (high redox potential), the motive (sequential electron flow in the midst of molecular oxygen) and the opportunity (thermodynamic favor) to create ROS.

Research in

Acknowledgements

We are most grateful to Gary Fiskum and Anatoly A. Starkov for critically reading the manuscript. The work by our group referred to in the text was supported by grants from Országos Tudományos Kutatási Alapprogram, Magyar Tudományos Akadémia, Nemzeti Kutatási és Technológiai Hivatal and Egeszsegügyi Tudományos Tanács (to V.A-V.).

Glossary

Antimycin
an inhibitor of complex III produced by Streptomyces bacteria.
Complex I
NADH–ubiquinone oxidoreductase. Complex I is an integral inner-membrane multiprotein complex that is exposed to both matrix and intermembrane space. It oxidizes NADH using coenzyme Q as an electron acceptor in a reversible reaction that is coupled with a proton-pump-generating transmembrane potential.
Complex II
succinate–ubiquinone oxidoreductase. Complex II (succinate dehydrogenase) is a flavoprotein that is located

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