Disruption of mitochondrial redox circuitry in oxidative stress

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Abstract

The present review and commentary considers oxidative stress as a disruption of mitochondrial redox circuitry rather than an imbalance of oxidants and antioxidants. Mitochondria contain two types of redox circuits, high-flux pathways that are central to mechanisms for ATP production and low-flux pathways that utilize sulfur switches of proteins for metabolic regulation and cell signaling. The superoxide anion radical (hereafter termed “superoxide”, O2radical dot), a well known free radical product of the high-flux mitochondrial electron transfer chain, provides a link between the high-flux and low-flux pathways. Disruption of electron flow and increased superoxide production occurs due to inhibition of electron transfer in the high-flux pathway, and this creates aberrant “short-circuit” pathways between otherwise non-interacting components. A hypothesis is presented that superoxide is not merely a byproduct of electron transfer but rather is generated by the mitochondrial respiratory apparatus to serve as a positive signal to coordinate energy metabolism. Electron mediators such as free Fe3+ and redox-cycling agents, or potentially free radical scavenging agents, could therefore cause oxidative stress by disrupting this normal superoxide signal. Methods to map the regulatory redox circuitry involving sulfur switches (e.g., redox-western blotting of thioredoxin-2, redox proteomics) are briefly presented. Use of these approaches to identify sites of disruption in the mitochondrial redox circuitry can be expected to generate new strategies to prevent toxicity and, in particular, promote efforts to re-establish proper electron flow as a means to counteract pathologic effects of oxidative stress.

Introduction

Accumulation of data on redox signaling pathways and lack of major benefits in intervention trials with free radical scavengers [1], [2], [3], [4], [5], [6], [7], [8], [9] has prompted efforts to refine the definition of oxidative stress. The definition of oxidative stress as “a disturbance in the prooxidant–antioxidant balance in favor of the former”[10], implies that a disturbance due to prooxidant conditions can be corrected by addition of appropriate antioxidants. However, redox mechanisms function in cell signaling, and cells are very sensitive to loss of these regulatory and control systems [11]. Consequently, an alternate definition for oxidative stress is “a disruption of redox signaling and control”[12]. These two concepts have recently been incorporated into a new definition as “an imbalance between oxidants and antioxidants in favor of the oxidants, leading to a disruption of redox signaling and control and/or molecular damage”[13]. However, this definition suggests that disruption of signaling and control is caused by an imbalance of prooxidants and antioxidants; from the perspective of oxidative stress in the mitochondria, this may be inverted in cause and effect.

In the present review and commentary, I consider an alternative definition that may provide a better description of oxidative stress in mitochondria: “a disruption of electron transfer reactions leading to an oxidant/antioxidant imbalance and oxidative damage to macromolecules”. This provides the basis to view the complex issues of oxidative stress in mitochondria in terms of disruption of normal redox biology, with reactive oxygen species (ROS) such as the free radical superoxide anion (O2radical dot) and hydrogen peroxide being intermediates connecting two types of redox circuits, high-flux circuits that function in energy metabolism and low-flux circuits that function in control of metabolism and cell signaling. This focuses attention on the disruption of electron flow from the high-flux metabolic pathways to low-flux signaling and control pathways as the fundamentally important process characterizing oxidative stress in mitochondria.

The first section summarizes the foundation for this view of oxidative stress derived from historical concepts of cellular respiration. These historical concepts lead to a first principle that mitochondria evolved as redox organelles containing electron transfer machinery which have very high rates of transfer compared to other oxidation–reduction processes in cells. The next section addresses a second principle, that sulfur switches are components of low-flux redox circuits which function in cell signaling and metabolic control. This section is based upon accumulating knowledge concerning thiol/disulfides in redox signaling in the cell cytoplasm and nuclei. In mitochondria, interference with high-flux electron transfer causing disruption of low-flux signaling and control mechanisms provides a simple and logical way to define oxidative stress. The following section addresses the principle that ROS are products of the high-flux metabolic pathways connecting these with the low-flux redox signaling and control pathways. This section includes the speculative hypothesis that O2radical dot is generated by the electron transport chain as a signaling molecule, providing a signal that the electron transport pathway is competent for generation of the protonmotive force needed for ATP synthesis. The last section addresses practical aspects of this new “definition” of oxidative stress, namely that oxidation of specific proteins can serve as reporters of disruption of redox pathways and thereby improve mechanistic details of oxidative stress [14], [15]. Use of these approaches to identify and eliminate dietary and environmental agents which disrupt mitochondrial redox circuitry, such as those implicated in Parkinson's disease [16], and develop therapeutic interventions to re-establish proper electron flow could provide novel strategies to protect against oxidative stress.

Section snippets

Historical precedents to the concept that mitochondrial oxidative stress represents a disruption of mitochondrial redox circuits

In the 1920s, Warburg found a complex iron-containing substance in tissue which functioned in oxidative processes and was inhibited by cyanide or sulfide. He referred to this substance as the “Atmungsferment”, meaning respiratory enzyme, a kinetically active component for reduction of molecular oxygen. About the same time, Keilin used spectral changes of cytochromes in response to oxidants and reductants to elucidate the major components of electron transfer in the respiratory chain [17]. He

First principle: the mitochondrial electron transfer chain contains high-flux redox circuits that are effectively insulated from other electron transfer pathways

Although discussions of mitochondrial oxidative stress often begin with consideration of ROS being produced as an unavoidable byproduct of respiration, I feel that a more appropriate starting principle concerns the nature of the mitochondrial electron transport chain. This chain is quantitatively the most significant redox system in aerobic organisms, accounting for perhaps 98–99% of all O2 consumption. The pathway can be viewed as a high-flux electron transfer pathway because the rates of

Second principle: low-flux redox circuits involving sulfur switches function in cell signaling and control

Early evidence for redox signaling mechanisms was obtained from studies showing that low levels of H2O2 stimulated cell proliferation [32] and that high concentrations of thiols such as N-acetylcysteine (NAC) also alter signaling [11]. Studies to examine whether H2O2 effects are mediated indirectly via a general redox signal or whether H2O2 targets specific signaling pathways were performed using cells expressing NADPH oxidase-1 (Nox1), an enzyme which produces H2O2 and induces cell growth,

Third principle: superoxide anion produced by the high-flux pathways connects the high-flux circuits with the low-flux circuits

As illustrated in Fig. 2, inhibitors or electron mediators stimulate electron flow from high-flux electron transfer pathways to O2 to generate O2radical dot. With a steady-state redox potential for the NADH/NAD+ couple in the mitochondrial matrix about −300 mV [28], the energy available for 1-electron transfer from the NAD+/NADH couple is ample to reduce O2 to produce O2radical dot. If the reaction were near equilibrium in cells, then with O2 at a concentration of 10 μM and the E0 for O2/O2radical dot of −330 mV, the

Implications resulting from the definition of mitochondrial oxidative stress as a disruption of redox circuitry

Although much of what is known about mitochondrial oxidative stress is unaffected, a shift in definition of oxidative stress would have important implications concerning analytic methods for measurement, mechanisms of injury and strategies for intervention. In the following brief discussion, most of the implications apply regardless of whether O2radical dot is a toxic byproduct of respiration or a purposefully generated signaling molecule. Important differences, particularly concerning interventional

Summary and conclusion

The present review considers oxidative stress in mitochondria as a disruption of redox circuits rather than as an imbalance of oxidants and antioxidants. Two types of redox circuits are conceptually distinguished, those with metabolic functions and those with functions in metabolic control and cell signaling. The former circuits have high electron transfer rates relative to the latter. Oxidative stress occurs when electron transfer through these high-flux circuits is disrupted by conditions

Acknowledgements

The research cited in this review that was performed in laboratory of the author was supported by NIH grant ES09047 and the Emory General Clinical Research Center grant M01 RR00039. The author greatly appreciates the thoughtful comments of an unidentified reviewer, as well as those of my colleagues, Y. Go, J. Johnson and P. Halvey.

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