Generation of Reactive Oxygen Species by Mitochondrial Complex I: Implications in Neurodegeneration

Mitochondrial Complex I [NADH Coenzyme Q (CoQ) oxidoreductase] is the least understood of respiratory complexes. In this review we emphasize some novel findings on this enzyme that are of relevance to the pathogenesis of neurodegenerative diseases. Besides CoQ, also oxygen may be an electron acceptor from the enzyme, with generation of superoxide radical in the mitochondrial matrix. The site of superoxide generation is debated: we present evidence based on the rational use of several inhibitors that the one-electron donor to oxygen is an iron-sulphur cluster, presumably N2. On this assumption we present a novel mechanism of electron transfer to the acceptor, CoQ. Complex I is deeply involved in pathological changes, including neurodegeneration. Complex I changes are involved in common neurological diseases of the adult and old ages. Mitochondrial cytopathies due to mutations of either nuclear or mitochondrial DNA may represent a useful model of neurodegeneration. In this review we discuss Parkinson’s disease, where the pathogenic involvement of Complex I is better understood; the accumulated evidence on the mode of action of Complex I inhibitors and their effect on oxygen radical generation is discussed in terms of the aetiology and pathogenesis of the disease. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

The site of production of superoxide radical in mitochondrial Complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2

The mitochondrial respiratory chain is a powerful source of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. We have investigated the role of Complex I in superoxide radical production in bovine heart submitochondrial particles and found, by combined use of specific inhibitors of Complex I and by Coenzyme Q (CoQ) extraction from the particles, that the one-electron donor in the Complex to oxygen is a redox center located prior to the binding sites of three different types of CoQ antagonists, to be identified with a Fe-S cluster, most probably N2 on the basis of several known properties of this cluster. Short chain CoQ analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective in promoting superoxide formation.     

Mitochondrial production of oxygen radical species and the role of Coenzyme Q as an antioxidant

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), which is considered as the pathogenic agent of many diseases and of aging. We have investigated the role of complex I in superoxide radical production and found by the combined use of specific inhibitors of complex I that the one-electron donor to oxygen in the complex is a redox center located prior to the sites where three different types of Coenzyme Q (CoQ) competitors bind, to be identified with an Fe-S cluster, most probably N2, or possibly an ubisemiquinone intermediate insensitive to all the above inhibitors. Short-chain Coenzyme Q analogs enhance superoxide formation, presumably by mediating electron transfer from N2 to oxygen. The clinically used CoQ analog, idebenone, is particularly effective, raising doubts on its safety as a drug. Cells counteract oxidative stress by antioxidants. CoQ is the only lipophilic antioxidant to be biosynthesized. Exogenous CoQ, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases. The plasma membrane oxidoreductase and DT-diaphorase are two such systems, likewise, they are overexpressed under oxidative stress conditions.     

Mitochondrial Complex I: structural and functional aspects

This review examines two aspects of the structure and function of mitochondrial Complex I (NADH Coenzyme Q oxidoreductase) that have become matter of recent debate. The supramolecular organization of Complex I and its structural relation with the remainder of the respiratory chain are uncertain. Although the random diffusion model [C.R. Hackenbrock, B. Chazotte, S.S. Gupte, The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembranes 18 (1986) 331-368] has been widely accepted, recent evidence suggests the presence of supramolecular aggregates. In particular, evidence for a Complex I-Complex III supercomplex stems from both structural and kinetic studies. Electron transfer in the supercomplex may occur by electron channelling through bound Coenzyme Q in equilibrium with the pool in the membrane lipids. The amount and nature of the lipids modify the aggregation state and there is evidence that lipid peroxidation induces supercomplex disaggregation. Another important aspect in Complex I is its capacity to reduce oxygen with formation of superoxide anion. The site of escape of the single electron is debated and either FMN, iron-sulphur clusters, and ubisemiquinone have been suggested. The finding in our laboratory that two classes of hydrophobic inhibitors have opposite effects on superoxide production favours an iron-sulphur cluster (presumably N2) is the direct oxygen reductant. The implications in human pathology of better knowledge on these aspects of Complex I structure and function are briefly discussed.     

Studies On The Role Of Ubiquinone In The Control Of The Mitochondrial Respiratory Chain

This study examines the possible role of Coenzyme Q (CoQ. ubiquinone) in the control of mitochondrial electron transfer. The CoQ concentration in mitochondria from different tissues was investigated by HPLC. By analyzing the rates of electron transfer as a function of total CoQ concentration, it was calculated that, at physiological CoQ concentration NADH cytochrome c reductase activity is not saturated. Values for theoretical V_max could not be reached experimentally for NADH oxidation, because of the limited mis-cibility of CoQ₁₀ with the phospholipids. On the other hand, it was found that CoQ₃ could stimulate α-glycerophosphate cytochrome c reductase over three-fold. Electron transfer being a diffusion-coupled process. we have investigated the possibility of its being subjected to diffusion control. A reconstruction study of Complex I and Complex III in liposomes showed that NADH cytochrome c reductase was not affected by changing the average distance between complexes by varying the protein: lipid ratios. The results of a broad investigation on ubiquinol cytochrome c reductase in bovine heart submitochondrial particles indicated that the enzymic rate is not diffusion-controlled by ubiquinol. whereas the interaction of cytochrome c with the enzyme is clearly diffusion-limited     

Supercomplex organization of the mitochondrial respiratory chain and the role of the Coenzyme Q pool: pathophysiological implications

In this review we examine early and recent evidence for an aggregated organization of the mitochondrial respiratory chain. Blue Native Electrophoresis suggests that in several types of mitochondria Complexes I, III and IV are aggregated as fixed supramolecular units having stoichiometric proportions of each individual complex. Kinetic evidence by flux control analysis agrees with this view, however the presence of Complex IV in bovine mitochondria cannot be demonstrated, presumably due to high levels of free Complex. Since most Coenzyme Q appears to be largely free in the lipid bilayer of the inner membrane, binding of Coenzyme Q molecules to the Complex I-III aggregate is forced by its dissociation equilibrium; furthermore free Coenzyme Q is required for succinate-supported respiration and reverse electron transfer. The advantage of the supercomplex organization is in a more efficient electron transfer by channelling of the redox intermediates and in the requirement of a supramolecular structure for the correct assembly of the individual complexes. Preliminary evidence suggests that dilution of the membrane proteins with extra phospholipids and lipid peroxidation may disrupt the supercomplex organization. This finding has pathophysiological implications, in view of the role of oxidative stress in the pathogenesis of many diseases.     

Mitochondrial Complex I defects in aging

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed.The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase.A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats.In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases. (Mol Cell Biochem 174: 329–333, 1997)     

New developments on the functions of coenzyme Q in mitochondria

The notion of a mobile pool of coenzyme Q (CoQ) in the lipid bilayer has changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising complexes I and III; in this model, the electron transfer is thought to be mediated by tunneling or microdiffusion, with a clear kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound quinone, besides being required for electron transfer from other dehydrogenases to complex III. The mechanism of CoQ reduction by complex I is analyzed regarding recent developments on the crystallographic structure of the enzyme, also in relation to the capacity of complex I to generate superoxide. Although the mechanism of the Q-cycle is well established for complex III, involvement of CoQ in proton translocation by complex I is still debated. Some additional roles of CoQ are also examined, such as the antioxidant effect of its reduced form and the capacity to bind the permeability transition pore and the mitochondrial uncoupling proteins. Finally, a working hypothesis is advanced on the establishment of a vicious circle of oxidative stress and supercomplex disorganization in pathological states, as in neurodegeneration and cancer.     

Altered redox status of coenzyme Q9 reflects mitochondrial electron transport chain deficiencies in Caenorhabditis elegans

Mitochondrial disorders are often associated with primary or secondary CoQ10 decrease. In clinical practice, Coenzyme Q10 (CoQ10) levels are measured to diagnose deficiencies and to direct and monitor supplemental therapy. CoQ10 is reduced by complex I or II and oxidized by complex III in the mitochondrial respiratory chain. Therefore, the ratio between the reduced (ubiquinol) and oxidized (ubiquinone) CoQ10 may provide clinically significant information in patients with mitochondrial electron transport chain (ETC) defects. Here, we exploit mutants of Caenorhabditis elegans (C. elegans) with defined defects of the ETC to demonstrate an altered redox ratio in Coenzyme Q9 (CoQ9), the native quinone in these organisms. The percentage of reduced CoQ9 is decreased in complex I (gas-1) and complex II (mev-1) deficient animals, consistent with the diminished activity of these complexes that normally reduce CoQ9. As anticipated, reduced CoQ9 is increased in the complex III deficient mutant (isp-1), since the oxidase activity of the complex is severely defective. These data provide proof of principle of our hypothesis that an altered redox status of CoQ may be present in respiratory complex deficiencies. The assessment of CoQ10 redox status in patients with mitochondrial disorders may be a simple and useful tool to uncover and monitor specific respiratory complex defects.     

Functional role of mitochondrial respiratory supercomplexes

Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based upon specific interactions between individual respiratory complexes. These supercomplexes were found to be functionally relevant by flux control analysis and to confer a kinetic advantage to NAD-linked respiration (channelling). However, the Coenzyme Q pool is still required for FAD-linked oxidations and for the proper equilibrium with Coenzyme Q bound in the supercomplex. Channelling in the cytochrome c region probably also occurs but does not seem to confer a particular kinetic advantage. The supramolecular association of individual complexes strongly depends on membrane lipid amount and composition and is affected by lipid peroxidation; it also seems to be modulated by membrane potential and protein phosphorylation. Additional properties of supercomplexes are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or IV, and prevention of excessive generation of reactive oxygen species. The dynamic character of the supercomplexes allows their involvement in metabolic adaptations and in control of cellular signalling pathways. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

The role of Coenzyme Q in mitochondrial electron transport

In mitochondria, most Coenzyme Q is free in the lipid bilayer; the question as to whether tightly bound, non-exchangeable Coenzyme Q molecules exist in mitochondrial complexes is still an open question.We review the mechanism of inter-complex electron transfer mediated by ubiquinone and discuss the kinetic consequences of the supramolecular organization of the respiratory complexes (randomly dispersed vs. super-complexes) in terms of Coenzyme Q pool behavior vs. metabolic channeling, respectively, both in physiological and in some pathological conditions. As an example of intra-complex electron transfer, we discuss in particular Complex I, a topic that is still under active investigation.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

The participation of coenzyme Q in free radical production and antioxidation

Published experimental data pertaining to the participation of coenzyme Q as a site of free radical formation in the mitochondrial electron transfer chain and the conditions required for free radical production have been reviewed critically. The evidence suggests that a component from each of the mitochondrial NADH-coenzyme Q, succinate-coenzyme Q, and coenzyme QH₂-cytochrome c reductases (complexes I, II, and III, most likely a nonheme iron-sulfur protein of each complex, is involved in free radical formation. Although the semiquinone form of coenzyme Q may be formed during electron transport, its unpaired electron most likely serves to aid in the dismutation of superoxide radicals instead of participating in free radical formation. Results of studies with electron transfer chain inhibitors make the conclusion dubious that coenzyme Q is a major free radical generator under normal physiological conditions but may be involved in superoxide radical formation during ischemia and subsequent reperfusion. Experiments at various levels of organization including subcellular systems, intact animals, and human subjects in theclinical setting, support the view that coenzyme Q, mainly in its reduced state, may act as an antioxidant protecting a number of cellular membranes from free radical damage.     

Structural and functional organization of Complex I in the mitochondrial respiratory chain

Metabolic flux control analysis of NADH oxidation in bovine heart submitochondrial particles revealed high flux control coefficients for both Complex I and Complex III, suggesting that the two enzymes are functionally associated as a single enzyme, with channelling of the common substrate, Coenzyme Q. This is in contrast with the more accepted view of a mobile diffusable Coenzyme Q pool between these enzymes. Dilution with phospholipids of a mitochondrial fraction enriched in Complexes I and III, with consequent increased theoretical distance between complexes, determines adherence to pool behavior for Coenzyme Q, but only at dilution higher than 1:5 (protein:phospholipids), whereas, at lower phospholipid content, the turnover of NADH cytochrome c reductase is higher than expected by the pool equation.     

Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species

We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2′,7′-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q₂) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q₁ as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decyl-ubiquinone. This behaviour relates the prooxidant CoQ₁ activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH-DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron-sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Structural and functional organization of the mitochondrial respiratory chain: A dynamic super-assembly

The structural organization of the mitochondrial oxidative phosphorylation (OXPHOS) system has received large attention in the past and most investigations led to the conclusion that the respiratory enzymatic complexes are randomly dispersed in the lipid bilayer of the inner membrane and functionally connected by fast diffusion of smaller redox components, Coenzyme Q and cytochrome c. More recent investigations by native gel electrophoresis, however, have shown the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis has demonstrated that Complexes I and III in mammalian mitochondria and Complexes I, III, and IV in plant mitochondria kinetically behave as single units with control coefficients approaching unity for each single component, suggesting the existence of substrate channelling within the supercomplexes. The reasons why the presence of substrate channelling for Coenzyme Q and cytochrome c was overlooked in the past are analytically discussed. The review also discusses the forces and the conditions responsible for the formation of the supramolecular units. The function of the supercomplexes appears not to be restricted to kinetic advantages in electron transfer: we discuss evidence on their role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Finally, there is increasing evidence that disruption of the supercomplex organization leads to functional derangements responsible for pathological changes.     

Coenzyme Q10 Alleviated Behavioral Dysfunction and Bioenergetic Function in an Animal Model of Depression

Neurochemical Research - Coenzyme Q10 (CoQ10) is a natural compound, is involved in the mitochondrial electron transfer chain (ETC) and plays an important pattern in adenosine triphosphate (ATP)...     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.