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


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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    • "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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    • "Impact of complex I lo..., Mitochondrion (2013), Complex I is acknowledged as a main contributor to superoxide production by mitochondria, where O 2 reacts with reduced flavin mononucleotide (Esterhazy et al., 2008; Kussmaul and Hirst, 2006). ROS (reactive oxygen species) are considered to be a major cause of cellular oxidative stress. "
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    • "The dependence of the rate of ROS production on the ambient redox potential calculated from the ratio [NADH]/ [NAD + ] is shown in Fig. 2. The experimental curve, which is well reproducible and similar for wild type and the NuoCD R274A and NuoM E144A variants, is less steep and positively shifted (Fig. 2A) from a Nernstian n = 2 curve with Em = −350 mV, characteristic for FMN of E. coli Complex I (Euro et al., 2008b). These data are in very good agreement with that obtained on E. coli Complex I by Esterhazy et al. (2008). In the mitochondrial enzyme the rate of O2 @BULLET− formation is half-maximal at −359 mV (Kussmaul and Hirst, 2006). "
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