Article

Production of Reactive Oxygen Species by Complex I (NADH:Ubiquinone Oxidoreductase) from Escherichia coli and Comparison to the Enzyme from Mitochondria †

Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom.
Biochemistry (Impact Factor: 3.02). 04/2008; 47(12):3964-71. DOI: 10.1021/bi702243b
Source: PubMed

ABSTRACT

The generation of reactive oxygen species by mitochondrial complex I (NADH:ubiquinone oxidoreductase) is considered a significant cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Defining its mechanism is important for the formulation of causative connections between complex I defects and pathological effects. Oxygen is probably reduced at two sites in complex I, one associated with NADH oxidation in the mitochondrial matrix and the other associated with ubiquinone reduction in the membrane. Here, we study complex I from Escherichia coli, exploiting similarities and differences in the bacterial and mitochondrial enzymes to extend our knowledge of O2 reduction at the active site for NADH oxidation. E. coli and bovine complex I reduce O2 at essentially the same rate, with the same potential dependence (set by the NAD (+)/NADH ratio), showing that the rate-determining step is conserved. The potential dependent rate of H2O2 production does not correlate to the potential of the distal [2Fe-2S] cluster N1a in E. coli complex I, excluding it as the point of O2 reduction. Therefore, our results confirm previous proposals that O2 reacts with the fully reduced flavin mononucleotide. Assays for superoxide production by E. coli complex I were prone to artifacts, but dihydroethidium reduction showed that, upon reducing O2, it produces approximately 20% superoxide and 80% H2O2. In contrast, bovine complex I produces 95% superoxide. The results are consistent with (but do not prove) a specific role for cluster N1a in determining the outcome of O2 reduction; possible reaction mechanisms are discussed.

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Available from: Martin Stephen King, Dec 19, 2013
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    • "Although this apparent bifurcation was taken to support the previous suggestion that N1a plays a role in minimizing/preventing reactive oxygen species production by the flavin [42], the same bifurcation does not occur in the mitochondrial enzyme because cluster N1a cannot be reduced by NADH [33] (see Figure 2). Even in the E. coli enzyme there is no data to support shuttling of electrons between the flavin and cluster N1a to prevent flavin radical formation [43] and furthermore, decreasing the reduction potential of the cluster in E. coli so that it cannot be reduced by NADH had no effect on reactive oxygen species production [13]. It is interesting to note that in fumarate reductase the fully reduced flavin produces either H 2 O 2 or superoxide depending on the oxidation state of a proximal [2Fe-2S] cluster [44]. "
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    ABSTRACT: Complex I (NADH:ubiquinone oxidoreductase) is critical for respiration in mammalian mitochondria. It oxidizes NADH produced by the Krebs' tricarboxylic acid cycle and β-oxidation of fatty acids, reduces ubiquinone, and transports protons to contribute to the proton-motive force across the inner membrane. Complex I is also a significant contributor to cellular oxidative stress. In complex I, NADH oxidation by a flavin mononucleotide, followed by intramolecular electron transfer along a chain of iron-sulfur clusters, delivers electrons and energy to bound ubiquinone. Either at cluster N2 (the terminal cluster in the chain) or upon the binding/reduction/dissociation of ubiquinone/ubiquinol, energy from the redox process is captured to initiate long-range energy transfer through the complex and drive proton translocation. This review focuses on current knowledge of how the redox reaction and proton transfer are coupled, with particular emphasis on the formation and role of semiquinone intermediates in both energy transduction and reactive oxygen species production. "This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt".
    Preview · Article · Dec 2015 · Biochimica et Biophysica Acta
    • "Some argue that the FMN is the sole source of ROS generation via the fully reduced FMN [6] [7]. And of the ROS produced, a vast majority is superoxide for the mammalian protein [8]. Others contend either the Fe–S cluster N2 or a semiquinone (SQ) bound to the Q reductase site is responsible for the majority of ROS generation [9] [10] [11]. "

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    • "During respiration, the NADH is oxidized to NAD+ and the NAD+/NADH ratio has been recognized as a key regulator in energy metabolism, aging, and immunological functions [19]. For example, decreases in NAD+ or in the NAD+/NADH are associated with increased production of superoxide by the mitochondria and subsequent alteration of the mitochondrial redox system [20–22]. "
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    ABSTRACT: An alcohol use disorder increases the risk of invasive and antimicrobial resistant community-acquired pneumonia and tuberculosis. Since the alveolar macrophage (AM) orchestrates the immune response in the alveolar space, understanding the underlying mechanisms by which alcohol suppresses AM phagocytosis is critical to improving clinical outcomes. In the alveolar space, chronic alcohol ingestion causes severe oxidative stress and depletes antioxidants which are critical for AM function. The mitochondrion is important in maintaining cellular redox balance and providing the ATP critical for phagocytosis. The focus of this study was to understand how alcohol triggers mitochondrial reactive oxygen species (ROS), stimulates cellular oxidative stress, and induces AM dysfunction. The current study also investigated the capacity of the mitochondrial targeted antioxidant, mitoTEMPOL (mitoT), in modulating mitochondrial oxidative stress, and AM dysfunction. Using in vitro ethanol exposure and AMs from ethanol-fed mice, ethanol promoted mitochondrial dysfunction including increased mitochondrial ROS, decreased mitochondrial membrane potential, and decreased ATP. Treatment with mitoT reversed these effects. Ethanol-induced decreases in phagocytosis and cell viability were also attenuated with mitoT. Therefore, antioxidants targeted to the mitochondria have the potential to ameliorate ethanol-induced mitochondrial oxidative stress and subsequent decreases in AM phagocytosis and cell viability.
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