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. 2011 May 20;286(20):18056-65.
doi: 10.1074/jbc.M110.186841. Epub 2011 Mar 10.

Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer

Kenneth R Pryde  1 , Judy Hirst
Affiliations

Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer

Kenneth R Pryde et al. J Biol Chem. .

Abstract

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD(+) and O(2) reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD(+) potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.

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Figures

FIGURE 1.
FIGURE 1.
Measurements of H2O2 production and NADH oxidation by complex I in SMPs. H2O2 production (measured by the accumulation of resorufin, y axis scale) by SMPs (35 μg of protein ml−1) was initiated by 30 μm NADH, and NADH oxidation was measured alongside (the intensity of the NADH traces has been multiplied by 0.2). A, in the presence of rotenone, H2O2 production and NADH oxidation were linear (black traces, 1). 1 mm NAD+ (red traces, 2) decreased both rates to the background level recorded in the presence of DPI (blue traces, 3). B, the black trace (1) is reproduced from panel A. The blue trace (2) was recorded during catalytic NADH oxidation (no inhibitor) with Δp = 0 (in the presence of gramicidin). The red trace (3) was recorded during NADH oxidation in the presence of Δp. The H2O2 production traces (2B and 3B) curve in response to NADH oxidation. See “Experimental Procedures” for conditions.
FIGURE 2.
FIGURE 2.
NADH-induced H2O2 production in the presence of various respiratory chain inhibitors, in the presence and absence of Δp and ΔpH. H2O2 production was induced by 30 μm NADH and measured using the HRP-Amplex Red system; Δp was imposed by ATP hydrolysis. ΔpH was increased using 20 mm KCl or decreased using 20 mm KCl and 2 nm nigericin (see “Results”). See “Experimental Procedures” for conditions.
FIGURE 3.
FIGURE 3.
NADH-induced H2O2 production by complex I in SMPs with and without Δp compared with that by isolated complex I. The three curves have been normalized independently. The data are plotted using ESet = −0.335 V − RT/2F·ln{[NADH]/[NAD+]} (using 30 μm NADH and variable NAD+) and fit by the Nernst equation for a 2-electron cofactor with 2 distinct potentials described by their average value, Eav, and their separation, ΔE. Blue, isolated complex I (Eav = −0.353, ΔE = 0.079 V) (5). Green, complex I in SMPs inhibited by rotenone (Δp = 0 V) (Eav = −0.363, ΔE = 0.079 V). Black, complex I in SMPs inhibited by rotenone with Δp ∼ 0.16 V (generated by ATP hydrolysis) (Eav = −0.366, ΔE = 0.060 V). The NADH-OH independent rates, measured in NADH only (see Fig. 2), have been subtracted from the SMP data sets. See “Experimental Procedures” for conditions.
FIGURE 4.
FIGURE 4.
Measurement of Δp in SMPs hydrolyzing ATP and evaluation of ΔpH. A, the rate of NADH:fumarate oxidoreduction, recorded during ATP hydrolysis (1 mm ATP+MgSO4) to generate Δp and with 400 μm KCN to inhibit respiration depends on the difference between the NAD+ and fumarate potentials, ΔE = −0.335 V + 0.020 − RT/2F·ln{([NADH][fumarate])/([NAD+][succinate])}, set using [NADH] = 0.1 mm, [NAD+] = 1 mm, [succinate] = 0.5 mm, and [fumarate] = 0.025–40 mm. The rate is 0 when −2ΔE = 4Δp. When −2ΔE is close to 4Δp, the data vary linearly with potential, but at high and low potential the rates are determined kinetically; the points with arrows are in principal at infinite potential ([NADH] = [fumarate] = 0 shown at 0.11 V, [NAD+] = [succinate] = 0 shown at 0.18 V). Similar titrations varying the succinate, NAD+, and NADH concentrations gave, overall, Δp = 0.162 ± 0.005 V. B, the fluorescence of ACMA (500 nm) is quenched upon the addition of 100 μm ATP+MgSO4 to SMPs (112 μg ml−1) in varying KCl concentrations. 2 nm nigericin (a K+/H+ exchanger) or 20 μg ml−1 gramicidin (a cation transporter) abolish the quenching effect. Note that the ACMA fluorescence is affected by ATP (the final value varies). See “Experimental Procedures” for conditions.
FIGURE 5.
FIGURE 5.
H2O2 production during RET in the presence of various respiratory chain inhibitors and in the presence and absence of Δp and ΔpH. H2O2 production rates were measured using the HRP-Amplex Red detection system during the linear phases of the reactions and normalized to the value from the standard RET condition (20 mm succinate, 1 mm ATP+MgSO4, KCN). Δp was collapsed with gramicidin, and ΔpH increased using 20 mm KCl or decreased using 20 mm KCl and 2 nm nigericin (see “Results”). See “Experimental Procedures” for conditions.
FIGURE 6.
FIGURE 6.
Comparison of NADH- and RET-induced H2O2 production by complex I in SMPs. The two curves have been normalized independently. Black, NADH-induced H2O2 production by complex I in SMPs inhibited by rotenone with Δp ∼ 0.16 V (from Fig. 3). Red, RET induced H2O2 production by complex I in SMPs inhibited by KCN. The RET data are plotted using ESet = −0.020 V −RT/2F. In{[succinate]/[fumarate]} + 2.Δp (10 mm succinate, variable fumarate concentrations) and fit by the Nernst equation for a 2-electron cofactor with two distinct potentials described by their average value, Eav = −0.305 V, and their separation, ΔE = −0.020 V. The rotenone independent rates (see Fig. 5) have been subtracted. The asterisk marks the 0.024 V increase in Δp (measured at E1/2) that is required to overlay the RET-induced data on the NADH-induced data. See “Experimental Procedures” for conditions.
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