Vladimir P. Skulachev

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

Formation of H2O2 has been studied in rat heart mitochondria, pretreated with H2O2 and aminotriazole to lower their antioxidant capacity. It is shown that the rate of H2O2 formation by mitochondria oxidizing 6 mM succinate is inhibited by a protonophorous uncoupler, ADP and phosphate, malonate, rotenone and myxothiazol, and is stimulated by antimycin A. The effect of ADP is abolished by carboxyatractylate and oligomycin. Addition of uncoupler after rotenone induces further inhibition of H2O2 production. Inhibition of H2O2 formation by uncoupler, malonate and ADP+Pi is shown to be proportional to the ΔΨ decrease by these compounds. A threshold ΔΨ value is found, above which a very strong increase in H2O2 production takes place. This threshold slightly exceeds the state 3 ΔΨ level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169–202] that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation.

Role of uncoupled and non-coupled oxidations in maintenance of safely low levels of oxygen and its one-electron reductants

To proceed at a high rate, phosphorylating respiration requires ADP to be available. In the resting state, when the energy consumption is low, the ADP concentration decreases so that phosphorylating respiration ceases. This may result in an increase in the intracellular concentrations of O 2 as well as of one-electron O 2 reductants such as These two events should dramatically enhance non-enzymatic formation of reactive oxygen species, i.e. of , and OH, and, hence, the probability of oxidative damage to cellular components. In this paper, a concept is put forward proposing that non-phosphorylating (uncoupled or non-coupled) respiration takes part in maintenance of low levels of both O 2 and the O 2 reductants when phosphorylating respiration fails to do this job due to lack of ADP. In particular, it is proposed that some increase in the H + leak of mitochondrial membrane in State 4 lowers , stimulates O 2 consumption and decreases the level of which otherwise accumulates and serves as one-electron O 2 reductant. In this connection, the role of natural uncouplers (thyroid hormones), recouplers (male sex hormones and progesterone), non-specific pore in the inner mitochondrial membrane, and apoptosis, as well as of non-coupled electron transfer chains in plants and bacteria will be considered.

Cytochrome c in the apoptotic and antioxidant cascades

Recent progress in studies on apoptosis has revealed that cytochrome c is a pro‐apoptotic factor. It is released from its places on the outer surface of the inner mitochondrial membrane at early steps of apoptosis and, combining with some cytosolic proteins, activates conversion of the latent apoptosis‐promoting protease pro‐caspase‐9 to its active form. Cytochrome c release can be initiated by the pro‐apoptotic protein Bax. This process is blocked by the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL. The role of cytochrome c in apoptosis may be understood within the framework of the concept assuming that the evolutionary primary function of apoptosis was to purify tissues from ROS‐overproducing cells. In this context, the pro‐apoptosis activity of cytochrome c might represent one of the anti‐oxidant functions inherent in this cytochrome. Among other cytochrome c‐linked antioxidant mechanisms, the following systems can be indicated. (1) Cytochrome c released from the inner mitochondrial membrane to the intermembrane space can operate as an enzyme oxidizing O−⋅ 2 back to O2. The reduced cytochrome c is oxidized by cytochrome oxidase (or in yeasts and bacteria, by cytochrome c peroxidase). (2) The intermembrane cytochrome c can activate the electron transport chain in the outer mitochondrial membrane. This bypasses the initial and middle parts of the main respiratory chain, which produce, as a rule, the major portion of ROS in the cell. (3) The main respiratory chain losing its cytochrome c is inhibited in such a fashion that antimycin‐like agents fail to stimulate ROS production.

Reactive oxygen species, mitochondria, apoptosis and aging

In this paper, we shall review various antioxygen defense systems of the cell paying particular attention to those that prevent superoxide formation rather than scavenge already formed superoxide and its products. The role of uncoupled, decoupled and non-coupled respiration, mitochondrial pore, mitochondrion-linked apoptosis will be considered. Mitochondrial theory of aging will be regarded in context of reactive oxygen species-induced damage of mitochondrial DNA. (Mol Cell Biochem 174: 305–319, 1997)

Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation

Free fatty acids, natural uncouplers of oxidative phosphorylation, are shown to differ from artificial ones in that they fail to increase conductance of phospholipid bilayers which are permeable for the protonated form of fatty acids but impermeable for their anionic form. Recent studies have revealed that uncoupling by fatty acids in mitochondria is mediated by the ATP/ADP antiporter and, in brown fat, by thermogenin which is structurally very similar to the antiporter. It is suggested that both the ATP/ADP antiporter and thermogenin facilitate translocation of the fatty anions through the mitochondrial membrane.

Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide‐producing mitochondria and cell

Petit and co‐authors have recently summarized results of their studies on the involvement of mitochondria in apoptosis [Petit et al. (1996) FEBS Lett. 396, 7–13]. The mechanism consists in the release to the cytosol of a protein (presumably a protease) that is normally sequestered in the intermembrane space of mitochondria. This protein, when added to isolated nuclei, caused typical apoptotic changes. Its release from mitochondria was shown to occur as a result of disruption of the outer mitochondrial membrane due to swelling of mitochondria caused by opening of so‐called permeability transition pores in their inner membranes. Increase in the level of products of the one‐electron reduction of O2 (reactive oxygen species, ROS) is known to induce the mitochondrial pores. The hypothesis described here assumes that pore formation and apoptosis are involved in the organization of a defense system preventing ROS formation. It is proposed that ROS‐induced pore opening lowers ROS production due to (a) maximal stimulation of mitochondrial O2 consumption and, hence, intracellular [O2] lowering and (b) complete dissipation of mitochondrial membrane potentials and, as a consequence, maximal oxidation of such respiratory chain carriers as CoQ.– which serve as one‐electron O2 reductants. ROS decrease allows pore closure. If, nevertheless, ROS are still accumulating in a mitochondrion, long‐lived pores cause degradation of the organelle which cannot import and synthesize proteins due to the absence of the membrane potential. In this way, ROS‐producing mitochondria can be eliminated (mitochondrial selection). Another result of the long‐lived pores is mitochondrial swelling. This disrupts the outer mitochondrial membrane and releases the apoptosis‐inducing protein. Apoptosis eliminates ROS‐producing cells (cell selection).

Mitochondrial filaments and clusters as intracellular power-transmitting cables

Mitochondria exist in two interconverting forms; as small isolated particles, and as extended filaments, networks or clusters connected with intermitochondrial junctions. Extended mitochondria can represent electrically united systems, which can facilitate energy delivery from the cell periphery to the cell core and organize antioxidant defence of the cell interior when O2 is consumed by mitochondrial clusters near the the outer cell membrane, and protonic potential is transmitted to the cell core mitochondria to form ATP. As to small mitochondria, they might represent a transportable form of these organelles.

Bioenergetic aspects of apoptosis, necrosis and mitoptosis

In this review I summarize interrelations between bioenergetic processes and such programmed death phenomena as cell suicide (apoptosis and necrosis) and mitochondrial suicide (mitoptosis). The following conclusions are made. (I) ATP and rather often mitochondrial hyperpolarization (i.e. an increase in membrane potential, ΔΨ) are required for certain steps of apoptosis and necrosis. (II) Apoptosis, even if it is accompanied by ΔΨ and [ATP] increases at its early stage, finally results in a ΔΨ collapse and ATP decrease. (III) Moderate (about three-fold) lowering of [ATP] for short and long periods of time induces apoptosis and necrosis, respectively. In some types of apoptosis and necrosis, the cell death is mediated by a ΔΨ-dependent overproduction of ROS by the initial (Complex I) and the middle (Complex III) spans of the respiratory chain. ROS initiate mitoptosis which is postulated to rid the intracellular population of mitochondria from those that are ROS overproducing. Massive mitoptosis can result in cell death due to release to cytosol of the cell death proteins normally hidden in the mitochondrial intermembrane space.

An attempt to prevent senescence: A mitochondrial approach

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

Conversion of biomembrane-produced energy into electric form. I. Submitochondrial particles

The hypothesis of an electric membrane potential generated by respiration or ATP hydrolysis in submitochondrial particles has been verified. To this end a number of synthetic ions penetrating lipid membranes were used. Penetrating anions of phenyl dicarbaundecaborane (PCB−), tetraphenyl boron and picrate were shown to accumulate in sonicated submitochondrial particles in an energy-dependent manner. The process was inhibited by rotenone, antimycin and cyanide if supported by respiration, and by oligomycin, if ATP was used as the energy source. Uncouplers were inhibitory in both cases. The following oxidation reactions were found to support the energy-dependent accumulation of PCB−: oxidation of NADH by oxygen or fumarate; oxidation of succinate or ascorbate by oxygen; oxidation of NADPH by NAD+. In the latter case, which is the reverse of the energy-requiring transhydrogenase reaction, ion transport was inhibited by NADH and NADP+ as well as by uncouplers. Oxidation of NADH by NADP+ in the energy-requiring transhydrogenase reaction was accompanied by an efflux of PCB− anions which had accumulated during succinate oxidation. The redox ‘succinate-ferricyanide’ couple could not be used as a supply of energy for the accumulation of PCB− Particles deprived of the coupling factor F1 showed a decreased ability for respiration-dependent anion uptake, the process being stimulated by oligomycin. ATP-driven PCB− accumulation was completely absent in F1-deprived particles but could be reconstituted after preincubation with F1. The active accumulation of anions penetrating into particles was readily distinguished from passive anion absorption, since the latter did not require energy and could be demonstrated both in native particles and in those deprived of F1, as well as in phospholipid micelles. The energy-dependent accumulation of anions penetrating into submitochondrial particles was accompanied by alkalinization of the incubation medium. The efflux of ions upon the cessation of the energy supply induced acidification. Anion accumulation was followed by the suppression of other energy-linked functions of submitochondrial particles. Under the same conditions the penetrating cations, dibenzyl dimethyl ammonium, tetrabutyl ammonium and triphenyl methyl phosphonium, did not affect either the pH of the medium or energy-linked functions. It was concluded that a mechanism for ion accumulation in submitochondrial particles is specific for the sign of the charge but not for other features of the penetrating compounds. This mechanism operates in such a way that anions, but not cations, are pumped into the particle as if the process were supported by an electric field, orientated across the membrane, being positive inside the particles.