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. 2006 May 16;103(20):7607-12.
doi: 10.1073/pnas.0510977103. Epub 2006 May 8.

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Lothar Kussmaul  1 , Judy Hirst
Affiliations

The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria

Lothar Kussmaul et al. Proc Natl Acad Sci U S A. .

Abstract

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Superoxide production by isolated complex I in the presence and absence of catalytic substrates and products (A) and inhibitors (B). Superoxide production was monitored via Cyt c reduction [black traces, 0.02 absorption units (AU) scale marker, 550–541 nm]. NADH oxidation was monitored directly (gray traces, 0.2 AU scale marker, 340–541 nm). Complex I and phospholipids (and inhibitors when added) were present at the start of each measurement. For the inhibitor assays (B), the results were identical if the order of addition was Q − inhibitor − NADH, showing that prior occupancy of the binding site does not affect the results. Conditions were as follows: pH 7.5, 32°C, 30 μM NADH, 300 μM NAD+, 50 μM Cyt c, 100 μM Q (decylubiquinone), 100 μM QH2 (decylubiquinol), 0.4 mg ml−1 bovine heart polar phospholipids, 0.5 μM piericidin A, 2.5 μM rotenone, and 300 μM capsaicin.
Fig. 2.
Fig. 2.
The generation, interconversion, and detection of superoxide and hydrogen peroxide in complex I assays. CI, complex I; HRP, horseradish peroxidase; CAT, catalase; SOD, superoxide dismutase.
Fig. 3.
Fig. 3.
Competition assay showing that most of the electrons are conserved in superoxide formation, but that a small fraction of them form hydrogen peroxide directly. In the absence of Cyt c, all of the electrons form H2O2. In 150 μM Cyt c superoxide detection is stoichiometric (it outcompetes dismutation). However, ≈10% of the H2O2 is not susceptible to Cyt c. Amplex Red oxidation was measured at 557–620 nm (557 nm is an isosbestic point for Cyt c reduction). The curve was calculated by using constant O2•− production and simple bimolecular reactions and is illustrative only. CAT, catalase. Conditions were as follows: pH 7.5 and 32°C.
Fig. 4.
Fig. 4.
The rate of superoxide production by isolated complex I as a function of complex I, O2, and NADH concentration. The rate depends linearly on complex I (A) and O2 concentrations (B) (% saturated in air), 23°C. (C) Superoxide production in the presence of 300 nM NADH (average and standard deviations of three experiments). In the presence of the NADH regenerating system, [NADH] is maintained, and the assay is linear; in its absence, NADH is rapidly oxidized, and the rate drops to zero (control). (D) Dependence of O2•− production on [NADH], maintained by the regenerating system: KM(app) = 0.05 μM, Vmax = 49.1 nmol O2•− min−1 mg−1 (Inset, data recorded at high [NADH]). General conditions were as follows: pH 7.5 and 32°C.
Fig. 5.
Fig. 5.
The rate and potential of superoxide production by isolated complex I. (A) The rate of H2O2 generation measured by using Amplex Red plotted against the NADH:NAD+ potential (♦). NADH was repurified anaerobically, and the lowest potential points were checked by using the NADH-regenerating system. The two curves are from Eq. 2 and the potentials from EPR, shifted by + 18 mV. The sigmoidal curve describes the fully reduced flavin, and the peak-shaped curve refers to the flavin radical. (B) pH dependence of E1/2 measured by using Cyt c (♦) and calculated from the pH dependence of the individual flavin potentials (pKS = 8.1, pKR = 6.8, solid line; dashed line from ref. 30). Assay conditions were as follows: pH 7.5, 32°C, 50 μM Cyt c, 10 μM Amplex Red, and 2 units ml−1 HRP.
Fig. 6.
Fig. 6.
EPR spectra from complex I at high and low potential. The signals from clusters N2, N3, and N4 are present at 9 K in equal intensities at both potentials. Cluster N5 is observed in equal intensity but only at lower temperature (data not shown). Only the signal from cluster N1b is increased at −0.4 V, and there is no evidence for the signal from cluster N1a [gz = 2.00 (28)]. The difference signal at 9 K has not been assigned. Conditions were as follows: Complex I (pH 7.5 (0°C), 10 mg ml−1) was reduced with 1 mM NADH (≈−0.4 V) or with 1 mM NADH, 10 mM NAD+ (≈−0.3 V). The spectra were normalized by using the signal from cluster N2 (27). Microwave frequency, 9.39 GHz; microwave power, 1 mW; modulation amplitude, 5 G; time constant, 81.92 ms; conversion time, 20.48 ms.
Fig. 7.
Fig. 7.
The preequilibrium of species that determines the rate of formation of superoxide by the reduced flavin. KdNADH and KdNAD+ are dissociation constants, and KINADH and KINAD+ are inhibition constants. The catalytically productive states interconvert by hydride transfer (KH−). Species enclosed by braces are enzyme bound, and the boxed species is that which produces O2•−. For simplicity, the flavin radical and the FeS clusters are not considered.
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