Understanding differential aspects of microdiffusion (channeling) in the Coenzyme Q and Cytochrome c regions of the mitochondrial respiratory system.

Over the past decades, models of the organization of mitochondrial respiratory system have been controversial. The goal of this perspective is to assess this "conflict of models" by focusing on specific kinetic evidence in the two distinct segments of Coenzyme Q- and Cytochrome c-mediated electron transfer. Respiratory supercomplexes provide kinetic advantage by allowing a restricted diffusion of Coenzyme Q and Cytochrome c, and short-range interaction with their partner enzymes. In particular, electron transfer from NADH is compartmentalized by channeling of Coenzyme Q within supercomplexes, whereas succinate oxidation proceeds separately using the free Coenzyme Q pool. Previous evidence favoring Coenzyme Q random diffusion in the NADH-dependent electron transfer is due to downstream flux interference and misinterpretation of results. Indeed, electron transfer by complexes III and IV via Cytochrome c is less strictly dependent on substrate channeling in mammalian mitochondria. We briefly describe these differences and their physiological implications.

Shared and Distinctive Ultrastructural Abnormalities Expressed by Megakaryocytes in Bone Marrow and Spleen From Patients With Myelofibrosis.

Numerous studies have documented ultrastructural abnormalities in malignant megakaryocytes from bone marrow (BM) of myelofibrosis patients but the morphology of these cells in spleen, an important extramedullary site in this disease, was not investigated as yet. By transmission-electron microscopy, we compared the ultrastructural features of megakaryocytes from BM and spleen of myelofibrosis patients and healthy controls. The number of megakaryocytes was markedly increased in both BM and spleen. However, while most of BM megakaryocytes are immature, those from spleen appear mature with well-developed demarcation membrane systems (DMS) and platelet territories and are surrounded by platelets. In BM megakaryocytes, paucity of DMS is associated with plasma (thick with protrusions) and nuclear (dilated with large pores) membrane abnormalities and presence of numerous glycosomes, suggesting a skewed metabolism toward insoluble polyglucosan accumulation. By contrast, the membranes of the megakaryocytes from the spleen were normal but these cells show mitochondria with reduced crests, suggesting deficient aerobic energy-metabolism. These distinctive morphological features suggest that malignant megakaryocytes from BM and spleen express distinctive metabolic impairments that may play different roles in the pathogenesis of myelofibrosis.

Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their ex vivo Expansion Highly Dependent on Plasma Lipoproteins.

Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly in vivo. The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs. To test this hypothesis, we first compared the expression profiling of erythroblasts generated with or without dexamethasone. This analysis revealed differences in expression of 55 genes, 6 of which encoding proteins involved in lipid metabolism. These were represented by genes encoding the mitochondrial proteins 3-Hydroxymethyl-3-Methylglutaryl-CoA lyase, upregulated, and 3-Oxoacid CoA-Transferase1 and glycerol-3-phosphate acyltransferase1, both downregulated, and the proteins ATP-binding cassette transporter1 and Hydroxysteroid-17-Beta-Dehydrogenase7, upregulated, and cAMP-dependent protein kinase catalytic subunit beta, downregulated. This profiling predicts that dexamethasone, possibly by interfering with mitochondrial functions, impairs the intrinsic lipid metabolism making the synthesis of the plasma membrane of erythroid cells depend on lipid-uptake from external sources. Optical and electron microscopy analyses confirmed that the mitochondria of erythroblasts generated with dexamethasone are abnormal and that their plasma membranes present pebbles associated with membrane ruptures releasing exosomes and micro-vesicles. These results indicate that the lipid supplements of media currently available are not adequate for cRBCs. To identify better lipid supplements, we determined the number of erythroblasts generated in synthetic media supplemented with either currently used liposomes or with lipoproteins purified from human plasma [the total lipoprotein fraction (TL) or its high (HDL), low (LDL) and very low (VLDL) density lipoprotein components]. Both LDL and VLDL generated numbers of erythroid cells 3-2-fold greater than that observed in controls. These greater numbers were associated with 2-3-fold greater amplification of erythroid cells due both to increased proliferation and to resistance to stress-induced death. In conclusion, dexamethasone impairs lipid metabolism making ex vivo expansion of erythroid cells highly dependent on lipid absorbed from external sources and the use of LDL and VLDL as lipid supplements improves the generation of cRBCs.

The Combination of Physical Exercise with Muscle-Directed Antioxidants to Counteract Sarcopenia: A Biomedical Rationale for Pleiotropic Treatment with Creatine and Coenzyme Q10.

Sarcopenia represents an increasing public health risk due to the rapid aging of the world's population. It is characterized by both low muscle mass and function and is associated with mobility disorders, increased risk of falls and fractures, loss of independence, disabilities, and increased risk of death. Despite the urgency of the problem, the development of treatments for sarcopenia has lagged. Increased reactive oxygen species (ROS) production and decreased antioxidant (AO) defences seem to be important factors contributing to muscle impairment. Studies have been conducted to verify whether physical exercise and/or AOs could prevent and/or delay sarcopenia through a normalization of the etiologically relevant ROS imbalance. Despite the strong rationale, the results obtained were contradictory, particularly with regard to the effects of the tested AOs. A possible explanation might be that not all the agents included in the general heading of "AOs" could fulfill the requisites to counteract the complex series of events causing/accelerating sarcopenia: the combination of the muscle-directed antioxidants creatine and coenzyme Q10 with physical exercise as a biomedical rationale for pleiotropic prevention and/or treatment of sarcopenia is discussed.

Complex I function in mitochondrial supercomplexes.

This review discusses the functional properties of mitochondrial Complex I originating from its presence in an assembled form as a supercomplex comprising Complex III and Complex IV in stoichiometric ratios. In particular several lines of evidence are presented favouring the concept that electron transfer from Complex I to Complex III is operated by channelling of electrons through Coenzyme Q molecules bound to the supercomplex, in contrast with the hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I. This article is part of a Special Issue entitled Respiratory Complex I, edited by Volker Zickermann and Ulrich Brandt.

The Interplay Between Respiratory Supercomplexes and ROS in Aging.

SIGNIFICANCE: The molecular mechanism of aging is still vigorously debated, although a general consensus exists that mitochondria are significantly involved in this process. However, the previously postulated role of mitochondrial-derived reactive oxygen species (ROS) as the damaging agents inducing functional loss in aging has fallen out of favor in the recent past. In this review, we critically examine the role of ROS in aging in the light of recent advances on the relationship between mitochondrial structure and function. RECENT ADVANCES: The functional mitochondrial respiratory chain is now recognized as a reflection of the dynamic association of respiratory complexes in the form of supercomplexes (SCs). Besides providing kinetic advantage (channeling), SCs control ROS generation by the respiratory chain, thus providing a means to regulate ROS levels in the cell. Depending on their concentration, these ROS are either physiological signals essential for the life of the cell or toxic species that damage cell structure and functions. CRITICAL ISSUES: We propose that under physiological conditions the dynamic nature of SCs reversibly controls the generation of ROS as signals involved in mitochondrial-nuclear communication. During aging, there is a progressive loss of control of ROS generation so that their production is irreversibly enhanced, inducing a vicious circle in which signaling is altered and structural damage takes place. FUTURE DIRECTIONS: A better understanding on the forces affecting SC association would allow the manipulation of ROS generation, directing these species to their physiological signaling role.

Redox-Based Flagging of the Global Network of Oxidative Stress Greatly Promotes Longevity.

Despite more than 50 years of investigations into the free radical theory, the direct role of oxidative stress (OS) in aging and age-related diseases remains unproven. Little progress in identifying antioxidant drugs promoting longevity has been made, likely due to selectivity toward one or few radical species, variable efficacy in vivo, inherent pro-oxidant behavior of such drugs, or lack of synergism with metabolic redox homeostasis. Silencing the wide range of reactive free radicals has a great impact on OS-linked outcomes and age-related disorders. Here we show that an innovative, redox-active, multi-radical-scavenger catalytic drug delays the age-associated decline in physiological processes and markedly prolongs the mean lifespan of the adult freshwater annelids Aeolosoma viride by 170%. This unprecedented extension is associated with a decreased OS status. Consistently, treatment of annelids increases their natural resistance to oxygen-derived damage without affecting mitochondrial respiration or reproductive activity. Conversely, the superoxide dismutase (SOD)-mimetic EUK 134 that we selected as a positive control led to an increase in lifespan of ~50%, the same increase previously observed in nematodes. Our results show that reduction of the global network of OS has a profound impact on aging, prompting the development of a possible redox-based therapeutic intervention to counteract the progression of aging.

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.

Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I.

AIMS: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. RESULTS: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I. INNOVATION: Dissociation of supercomplexes may link oxidative stress and energy failure in a vicious circle. CONCLUSION: Our findings support a central role of mitochondrial supramolecular structure in the development of the aging process and in the etiology and pathogenesis of most major chronic diseases.

A critical appraisal of the role of respiratory supercomplexes in mitochondria.

Substantial evidence exists that the mitochondrial respiratory chain is organized in supramolecular units called supercomplexes or respirasomes. While the structural evidence of the supercomplexes is overwhelming, fewer studies have focused on their functional relevance. Although the presence of coenzyme Q channeling between complexes I and III has been ascertained, no such clear demonstration has been carried out for cytochrome c between complexes III and IV, at least in mammalian mitochondria. This review also discusses the implications concerning the number of respiratory complexes organized in supercomplexes and the possibility that they represent associations in dynamic equilibrium with the individual complexes.

Supramolecular organisation of the mitochondrial respiratory chain: a new challenge for the mechanism and control of oxidative phosphorylation.

Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based on specific interactions between individual respiratory complexes. These supercomplexes are detected by blue-native electrophoresis and are found to be functionally relevant by flux control analysis; moreover, they have been isolated and characterised by single-particle electron microscopy. 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. Supercomplex association confers several new properties with respect to the non-associated respiratory complexes to the respiratory chain: the most obvious is substrate channelling, specifically addressing Coenzyme Q and cytochrome c to interact directly with the partner enzymes without the need of a less efficient random diffusion step; in addition, supramolecular association may provide a further rate advantage by conferring long-range conformational changes to the individual complexes. Additional properties are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or Complex IV, and prevention of excessive generation of reactive oxygen species. On the basis of the properties described above, we hypothesise that an oxidative stress acts primarily by disassembling supercomplex associations thereby establishing a vicious circle of oxidative stress and energy failure, ultimately leading to cell damage and disease. We provide evidence that in physiological ageing and in some disease states, characterised by oxidative stress and mitochondrial damage, such as heart failure, neurodegenerative disorders and cancer, a loss of supercomplex association occurs, in line with our working hypothesis.

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.

Mitochondrial respiratory chain super-complex I-III in physiology and pathology.

Recent investigations by native gel electrophoresis showed the existence of supramolecular associations of the respiratory complexes, confirmed by electron microscopy analysis and single particle image processing. Flux control analysis demonstrated that Complex I and Complex III in mammalian mitochondria kinetically behave as a single unit with control coefficients approaching unity for each component, suggesting the existence of substrate channeling within the super-complex. The formation of this supramolecular unit largely depends on the lipid content and composition of the inner mitochondrial membrane. The function of the super-complexes 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, particularly Complex I, and in preventing excess oxygen radical formation. There is increasing evidence that disruption of the super-complex organization leads to functional derangements responsible for pathological changes, as we have found in K-ras-transformed fibroblasts.

Structure and organization of mitochondrial respiratory complexes: a new understanding of an old subject.

The enzymatic complexes of the mitochondrial respiratory chain have been extensively investigated in their structural and functional properties. A clear distinction is possible today between three complexes in which the difference in redox potential allows proton translocation (complexes I, III, and IV) and those having the mere function to convey electrons to the respiratory chain. We also have a clearer understanding of the structure and function of most respiratory complexes, of their biogenesis and regulation, and of their capacity to generate reactive oxygen species. Past investigations led to the conclusion that the complexes are randomly dispersed and functionally connected by diffusion of smaller redox components, coenzyme Q and cytochrome c. More-recent investigations by native gel electrophoresis and single-particle image processing showed the existence of supramolecular associations. Flux-control analysis demonstrated that complexes I and III in mammals and I, III, and IV in plants kinetically behave as single units, suggesting the existence of substrate channeling. This review discusses conditions affecting the formation of supercomplexes that, besides kinetic advantage, have a role in the stability and assembly of the individual complexes and in preventing excess oxygen radical formation. Disruption of supercomplex organization may lead to functional derangements responsible for pathologic changes.

Control of respiration by cytochrome c oxidase in intact cells: role of the membrane potential.

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Delta psi) and chemical (Delta pH) component of the mitochondrial transmembrane proton electrochemical gradient (Delta mu(H(+))) was investigated. The overall O(2) consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Delta psi and Delta pH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C(IV) = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C(IV) = 0.85) was also measured in the presence of nigericin, i.e. when Delta psi is maximal, and in the presence of nigericin and valinomycin (C(IV) = 0.77), when Delta mu(H(+)) is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C(IV) = 0.30) upon addition of valinomycin, when Delta psi is converted into Delta pH. These results show that Delta psi is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.

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.

Mobility and function of coenzyme Q (ubiquinone) in the mitochondrial respiratory chain.

The kinetic analysis by Kröger and Klingenberg on electron transfer in the Coenzyme Q region led to the conclusion that the quinone behaves kinetically as a homogeneous pool freely diffusing in the lipid bilayer, thus setting the basis for the widely accepted random diffusion model of electron transfer. The recent description of supramolecular complexes of the respiratory chain enzymes, in particular Complex I-III supercomplexes, has reopened the problem of electron transfer in the Coenzyme Q region. Flux control analysis has revealed that Complexes I and III indeed function as a single unit indicating substrate channelling by Coenzyme Q in transferring electrons from Complex I to Complex III. In this review we analyse in detail the reasons that suggested Coenzyme Q pool behaviour; although electron transfer between Complexes I and III indeed appears to be effected by substrate channelling, the Coenzyme Q pool is in equilibrium with bound quinone and is required to fill the site(s) within the supercomplex. In addition, the pool equation of Kröger and Klingenberg still describes in the most adequate way the electron transfer from Complex II and other Coenzyme Q-reducing enzymes to Complex III, besides the energy-dependent reverse electron transfer from Complex II to Complex I.

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.

Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling.

Recent evidence, mainly based on native electrophoresis, has suggested that the mitochondrial respiratory chain is organized in the form of supercomplexes, due to the aggregation of the main respiratory chain enzymatic complexes. This evidence strongly contrasts the previously accepted model, the Random Diffusion Model, largely based on kinetic studies, stating that the complexes are randomly distributed in the lipid bilayer of the inner membrane and functionally connected by lateral diffusion of small redox molecules, i.e., coenzyme Q and cytochrome c. This review critically examines the experimental evidence, both structural and functional, pertaining to the two models and attempts to provide an updated view of the organization of the respiratory chain and of its kinetic consequences. The conclusion that structural respiratory assemblies exist is overwhelming, whereas the expected functional consequence of substrate channeling between the assembled enzymes is controversial. Examination of the available evidence suggests that, although the supercomplexes are structurally stable, their kinetic competence in substrate channeling is more labile and may depend on the system under investigation and the assay 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.