ROS (reactive oxygen species) are considered to be a major cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Complex I (NADH:ubiquinone oxidoreductase) is one of the main contributors to superoxide production by mitochondria, and knowledge of its mechanism of O2 reduction is required for the formulation of causative connections between complex I defects and pathological effects. There is evidence for two distinct (but not mutually exclusive) sites of O2 reduction by complex I. Studies of the isolated enzyme largely support the participation of the reduced flavin mononucleotide in the active site for NADH oxidation, and this mechanism is supported in mitochondria by correlations between the NAD(P)+ potential and O2 reduction. In addition, studies of intact mitochondria or submitochondrial particles have suggested a mechanism involving the quinone-binding site, supported by observations during reverse electron transport and the use of ‘Q-site’ inhibitors. Here, we discuss extant data and models for O2 reduction by complex I. We compare results from the isolated enzyme with results from intact mitochondria, aiming to identify similarities and differences between them and progress towards combining them to form a single, unified picture.

ROS (reactive oxygen species) production by complex I in the cell

The electron transport chain in mitochondria is a significant source of cellular ROS. Historically, respiratory complexes I and III have been considered two major sources of superoxide and H2O2 in mitochondria, with their relative contributions, determined by the effects of appropriate inhibitors, being tissue-specific [13]. However, there is mounting evidence that complex I produces most of the ROS generated in intact mammalian mitochondria in vitro [46], suggesting that ROS generation by complex III is of limited physiological importance [7,8]. Although there are numerous complex cellular mechanisms in place to help minimize the harmful effects of ROS [9,10], intracellular ROS production is implicated in many mitochondrial pathologies. The free radical theory of aging proposes that it is a major determinant of life span [11,12] through the accumulation of damage to mitochondrial proteins, lipids and DNA [13,14].

It is difficult to draw direct causative connections between increased ROS production by a specific enzyme and a pathological effect. Even when a mitochondrial defect can be attributed to complex I, it is hard to determine whether the effects observed arise from a loss of complex I activity, an increase in its ROS production, or both. Robinson and co-workers have provided evidence that complex I deficiency is associated with the excessive production of hydroxyl radicals in a host of mitochondrial pathologies, including fatal infantile lactic acidosis, cardiomyopathy with cataracts, Leigh's disease, cataracts and developmental delay, and lactic acidaemia in the neonatal period followed by mild symptoms [1517]. Similarly, when superoxide production and complex I activity were compared quantitatively in cultured skin fibroblasts from 7 control subjects and 21 children with inherited isolated complex I deficiency, superoxide production was significantly increased in all but two of the deficient cell lines, and an inverse relationship between superoxide production and enzyme activity was observed [18]. Sherer et al. [19] investigated oxidative stress as a mechanism for rotenone toxicity in models relevant to Parkinson's disease, and suggested that complex I-mediated oxidative damage, rather than rotenone-induced ATP depletion, is responsible for the rotenone-induced degeneration of dopaminergic neurons. Furthermore, it has been proposed that the decreased activity of complex I in brain mitochondria from Parkinson's subjects results directly from oxidative damage to its subunits [20].

Two possible sites of oxygen reduction in complex I

The structure of the hydrophilic arm of complex I from Thermus thermophilus [21] provides the best current model for the structure of complex I from mitochondria (Figure 1). It shows that most of the cofactors in the enzyme are shielded from solvent, and therefore unlikely to react with molecular O2 at a significant rate. It is most likely that O2 accesses the ‘live’ parts of the enzyme at each end of the cofactor chain: either the flavin moiety or the quinone-binding site. There is evidence to support both possibilities, and of course they are not mutually exclusive.

Two possible sites of ROS production by complex I: the NADH and quinone-binding sites

Figure 1
Two possible sites of ROS production by complex I: the NADH and quinone-binding sites

The structure of the hydrophilic domain of complex I from T. thermophilus [21] is superimposed on the structure of complex I from E. coli determined by electron microscopy [39]. The iron–sulfur clusters are buried below the solvent-accessible surface. An enlarged view of the flavin site is shown in the inset, and the proposed quinone-binding site is indicated.

Figure 1
Two possible sites of ROS production by complex I: the NADH and quinone-binding sites

The structure of the hydrophilic domain of complex I from T. thermophilus [21] is superimposed on the structure of complex I from E. coli determined by electron microscopy [39]. The iron–sulfur clusters are buried below the solvent-accessible surface. An enlarged view of the flavin site is shown in the inset, and the proposed quinone-binding site is indicated.

O2 reduction by the reduced flavin

In isolated complex I, in the absence of a protonmotive force, there is substantial evidence that superoxide is produced only by the reduced flavin (see Figure 2) [22]. Superoxide and H2O2 production by isolated complex I have been detected in the presence of NADH only (Figure 2A), and all the electrons from NADH oxidation were conserved in O2 reduction. Bovine complex I reduces more than 90% of the O2 to superoxide, but Escherichia coli complex I, which behaves otherwise very similarly, appears to reduce a significant proportion of the O2 directly to H2O2 [23]. The difference may result from the different potentials of the distal [2Fe-2S] cluster. Superoxide (or H2O2) production is dependent on the ratio of NADH to NAD+, so at a constant NADH concentration it is inhibited by NAD+ (Figure 2A). These results can be explained by considering the population of the fully reduced flavin (Figures 2D and 2E): the nucleotides react with the flavin much faster than O2 does, so they establish a ‘pre-equilibrium’ and the experiment shown is equivalent to a redox titration [22].

Reduced flavin is the source of ROS production by isolated complex I

Figure 2
Reduced flavin is the source of ROS production by isolated complex I

(AC) Superoxide production detected using acetylated cytochrome c. (A) The addition of NADH to complex I initiates superoxide production; the subsequent addition of NAD+ causes a significant decrease in its rate (symbols indicate the correlation to D). (B) The subsequent addition of decylubiquinone (Q) causes a decrease in rate as the NADH is oxidized (grey trace). (C) Addition of Q in the presence of piericidin A (pier. A) or rotenone (rot.) does not change the rate of superoxide production. (D) The rate of ROS production (detected as H2O2 using Amplex Red) as a function of the NAD+/NADH potential, fitted to the potential-dependent population of the reduced flavin. (E) The half-height potential (from D) as a function of pH, compared with equivalent data for the flavin determined by EPR [40]. Adapted from [22] with permission. © 2006 the National Academy of Sciences.

Figure 2
Reduced flavin is the source of ROS production by isolated complex I

(AC) Superoxide production detected using acetylated cytochrome c. (A) The addition of NADH to complex I initiates superoxide production; the subsequent addition of NAD+ causes a significant decrease in its rate (symbols indicate the correlation to D). (B) The subsequent addition of decylubiquinone (Q) causes a decrease in rate as the NADH is oxidized (grey trace). (C) Addition of Q in the presence of piericidin A (pier. A) or rotenone (rot.) does not change the rate of superoxide production. (D) The rate of ROS production (detected as H2O2 using Amplex Red) as a function of the NAD+/NADH potential, fitted to the potential-dependent population of the reduced flavin. (E) The half-height potential (from D) as a function of pH, compared with equivalent data for the flavin determined by EPR [40]. Adapted from [22] with permission. © 2006 the National Academy of Sciences.

A number of studies in intact mitochondria have linked ROS production to the flavin in complex I. Diphenyleneiodonium reacts specifically with the flavin in complex I, and inhibits ROS production by rat heart mitochondria under conditions that favour succinate-supported reverse electron transport [4]. Kushnareva et al. [24] used β-hydroxybutyrate to poise the potential of the NAD+ pool in rat heart mitochondria, and in a ‘redox titration’ of ROS production against NAD+ potential they determined an E1/2 value of –0.392 V, close to values from isolated complex I and to the potential of the flavin (Figures 2D and 2E). In a detailed study using rat brain mitochondria, Starkov and Fiskum [25] elucidated the relationships between ROS production, NAD(P)H potential and membrane potential during forward electron transfer, and proposed that the ROS-producing site of complex I is in redox equilibrium with the pyridine nucleotides, so that its degree of reduction depends on the NAD+/NADH ratio. Interestingly, transduction of the alternative NADH dehydrogenase from Saccharomyces cerevisiae into rat dopaminergic and human neuroblastoma cell lines significantly decreases their ROS production in the presence of rotenone. It is unlikely that the dehydrogenase exerts a direct effect on complex I, rather it probably acts to decrease the NADH concentration and increase the NAD+ potential and thus suppresses ROS production indirectly, consistent with the flavin site model described [26].

Complex I inhibition and reverse electron transfer

Information about the site(s) of ROS production of complex I is provided by differences in the rates of ROS production (observed in mitochondria and submitochondrial particles) in the forward and reverse electron transfer directions, and in the presence and absence of inhibitors. Table 1 compares the results from a selection of representative studies on isolated mitochondria (making no attempt to account for variations in experimental conditions or procedures). It is clear that the trends are conserved between studies, but the absolute values are highly variable, perhaps because the studies used different tissues and sources of mitochondria. During forward electron transport, driven by NADH-linked substrates, complex I exhibits only minimal ROS production, but the addition of a ‘Q-site inhibitor’ (where Q is ubiquinone), such as rotenone, results in a significant increase in its rate. During reverse electron transport, driven by succinate, ROS production by complex I is increased significantly, and in this case, inhibited by the addition of Q-site inhibitors. Qualitatively, these observations are consistent with the site of ROS production being located ‘upstream’ of the inhibitor-binding site. During conditions for forward electron transport, rotenone prevents the electrons from exiting the enzyme and so they ‘back-up’ on to NADH, and they are available for ROS formation. During reverse electron transport, the enzyme's cofactors are reduced, unless rotenone prevents the electrons from moving upstream to the site of ROS production. Qualitatively, this behaviour is consistent with that expected from the flavin site (see above), with the degree of flavin reduction being set by the NAD+/NADH ratio [22,24,25]. Note that the relative potentials of the flavin and NAD+ mean that substantial levels of NAD+ reduction are required before the flavin is significantly reduced.

Table 1
Comparison of the results from a selection of recent studies of ROS production in intact mitochondria

Rates are expressed as pmol of H2O2/min per mg of protein.

 Rat heart [35Rat heart +Mn2+ [35Rat brain [36Rat brain [4Rat heart [24Rat brain [24Rat heart [30Rat glutathione-depleted heart [30Rat brain [37Human cortex [37Rat skeletal muscle [31,32Rat liver [38
Glutamate/pyruvate+malate 4±2 20±10 30±5 35±6 <20 58±49 40±20 30±30 ∼30 24±7 
Glu/pyruvate+malate+rotenone (A220±30 290±60 434±9 250±20 201±29 372±39 150±45 420±150 680±250 270±100 300±100 70±9 
Succinate (B630±120 1120±150 1388±59 390±30 294±20 264±14 1150±510 2430±970 800±270 130±80 2650±250 201±20 
Succinate+rotenone 60±10 130±30 174±18  53±50 53±50 97±880 ∼100   3±3 
Ratio of B to A 2.86 3.86 3.20 1.56 1.46 0.71 7.67 5.79 1.18 0.48 8.83 2.87 
 Rat heart [35Rat heart +Mn2+ [35Rat brain [36Rat brain [4Rat heart [24Rat brain [24Rat heart [30Rat glutathione-depleted heart [30Rat brain [37Human cortex [37Rat skeletal muscle [31,32Rat liver [38
Glutamate/pyruvate+malate 4±2 20±10 30±5 35±6 <20 58±49 40±20 30±30 ∼30 24±7 
Glu/pyruvate+malate+rotenone (A220±30 290±60 434±9 250±20 201±29 372±39 150±45 420±150 680±250 270±100 300±100 70±9 
Succinate (B630±120 1120±150 1388±59 390±30 294±20 264±14 1150±510 2430±970 800±270 130±80 2650±250 201±20 
Succinate+rotenone 60±10 130±30 174±18  53±50 53±50 97±880 ∼100   3±3 
Ratio of B to A 2.86 3.86 3.20 1.56 1.46 0.71 7.67 5.79 1.18 0.48 8.83 2.87 

In isolated complex I in the presence of NADH, Kussmaul and Hirst [22] found that the addition of decylubiquinone (an ubiquinone analogue) stops superoxide production, because catalysis rapidly converts the NADH into NAD+; rotenone and piericidin A did not affect the rate of superoxide production except to stop catalysis (see Figures 2B and 2C). Similarly, the addition of rotenone or piericidin A to mitochondrial membranes in the presence of stigmatellin (a complex III inhibitor) and a defined NADH concentration did not affect H2O2 production [22]. In similar experiments using purified Yarrowia lipolytica complex I, the effects of adding ubiquinone and an inhibitor were strongly dependent on the hydrophilicity of the quinone [27]. The addition of the hydrophilic ubiquinone-1 caused large increases in the rate of superoxide production, attributed to its reduction at a hydrophilic, non-physiological site on complex I. Similar behaviour was observed using submitochondrial particles, particularly from the hydrophilic ubiquinone analogue idebenone [28]. Addition of rotenone to Y. lipolytica complex I in the presence of the physiological Q9 only had little effect [27]. Vinogradov and Grivennikova [29] reported that superoxide production by complex I in submitochondrial particles in 1 mM NADH was inhibited by NAD+, but showed only a small increase on the addition of rotenone (0.6−0.7 nmol·min−1·mg−1). Furthermore, NADH-driven superoxide production by Y. lipolytica complex I incorporated into proteoliposomes [27], and by complex I in submitochondrial particles [29], did not respond significantly to the presence of an uncoupler. These observations are all consistent with superoxide production by the reduced flavin alone, governed by the NAD+ potential.

Are ROS produced at the Q-binding site in complex I also?

In the presence of NADH-linked substrates and a Q-site inhibitor, the NAD+ pool is expected to be close to completely reduced, so superoxide production by the flavin of complex I should be maximal [25]. If this is true, then superoxide production by the flavin during reverse electron transport cannot exceed this rate. In Table 1, a number of studies show a greater than 2-fold rate increase during reverse electron transport, with the value of Lambert and Brand [31,32] being particularly high. Although NAD+ potentials in the different conditions have not been established experimentally alongside rates of superoxide production, the high (up to 10-fold) increases observed would require significant differences in NAD+ potential, suggesting that alternative explanations should be explored.

First, the reason why such different ratios are observed in different studies is not completely clear. To the best of our knowledge, the highest rates have not been observed in submitochondrial particles, or from an isolated complex I, so effects from other enzymes in the mitochondrial matrix, or differences between the H2O2 produced inside the mitochondrion and detected outside it (possibly affected by the NADP+ and glutathione potentials [30]), cannot be excluded. However, high rates of ROS production during reverse electron transfer are only observed at high membrane potentials (and are very sensitive to small decreases in it) [25,31], so variations in how well coupled the mitochondria are may affect the results. Lambert and Brand [32] have also suggested that the presence of phosphate in many studies dissipates ΔpH, and so precludes observation of the highest rates of superoxide formation. Further observations that are not explained by the flavin model have been described in [31,32]. Nigericin (which collapses ΔpH) collapses the elevated rates of superoxide production observed during reverse electron transport to a level consistent with that produced by forward electron transport in the presence of an inhibitor; during inhibited forward electron transport, the production of ΔpH by ATP hydrolysis causes superoxide production to increase. Different Q-site inhibitors (rotenone, piericidin A and myxothiazol) promote this effect to different extents, and may result in different levels of superoxide formation in the forward direction also. However, although the apparent effects of ΔpH are striking, ΔpH-dependent superoxide production is not independent of the matrix pH.

Here, we consider that evidence for superoxide production by the reduced flavin, determined by the NAD+ potential, is convincing, and that the flavin mechanism is able to explain most of the results described. However, it may not adequately explain the increased ROS production during reverse electron transfer in the presence of ΔpH, so that a second site of ROS production in complex I should be considered. Lambert and Brand [31] proposed that superoxide is formed by a semiquinone radical, and EPR studies have described several QH (ubisemiquinone) radicals formed under different conditions [33,34]. However, the ‘fast-relaxing’ semiquinone that is formed in the presence of a protonmotive force during both forward and reverse electron transport is abolished by inhibitors such as rotenone and piericidin A (as well as by uncouplers), so its presence does not correlate with the rate of superoxide production. Cluster N2, immediately upstream of the Q-site, has also been proposed to be the locus of ROS production [28]. It is hoped that future research using submitochondrial particles and isolated complex I, building on knowledge of the conditions under which the putative second site of superoxide production is most active, will help to define further the locations and mechanisms of O2 reduction.

Integration of Structures, Spectroscopies and Mechanisms: Second Joint German/British Bioenergetics Conference, a Biochemical Society Focused Meeting held at University of Edinburgh, U.K., 2–4 April 2008. Organized by Ulrich Brandt (Frankfurt, Germany), Steve Chapman (Edinburgh, U.K.), Peter Heathcoate (Queen Mary, University of London, U.K.), John Ingledew (St Andrews, U.K.), Mike Jones (Bristol, U.K.), Bernd Ludwig (Frankfurt, Germany), Fraser MacMillan (University of East Anglia, Norwich, U.K.), Hartmut Michel (Max-Planck-Institute for Biophysics, Frankfurt am Main, Germany), Peter Rich (University College London, U.K.) and John Walker (MRC Dunn Human Nutrition Unit, Cambridge, U.K.). Edited by Ulrich Brandt and Peter Rich.

Abbreviations

     
  • Q

    ubiquinone

  •  
  • ROS

    reactive oxygen species

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