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Bacteriorhodopsin-mediated photopotential generation has been studied in two kinds of lipid/water systems: (1) decane solution of asolectin was used as the lipid phase; (2) a mixture of bacteriorhodopsin sheets and hexane solution of phosphatidyl choline was applied onto a water surface to form a monolayer, and then the monolayer was covered with a 0.3-mm decane layer. In both cases, illumination was found to induce formation of an electric potential difference, with the bulk water phase being found negative when measured with a vibrating electrode. In the latter, but not in the former, system small amounts of a protonophorous uncoupler were found to stimulate the photoresponse. Large amounts of the uncoupler proved depressing in both systems. Phenyldicarbaundecaborane anion (PCB−) was shown to substitute for the uncoupler, being much more potent both as an activator and as an inhibitor of the photoresponse. In both studied systems, gramicidin A inhibits the photoresponse, the effect being greatly potentiated by K+, Na+ or H+ ions.

The kinetics of 3 photoinduced responses of bacteriorhodopsin have been compared: spectral changes, pH shifts in a suspension of open purple membrane sheets and electric potential generation by the sheets incorporated into a lipid-impregnated collodion film. In the presence of a pH-buffer, the H + release by bacteriorhodops in was shown to correlate with the formation of the M412 intermediate and the microsecond phase of the potential generation. The H + /M412 ratio is equal to 0.7±0.1 if the ionic strength of the solution is high. In the absence of the buffer, the H + release proved to be much slower than spectral and electric responses. The kinetics of H + uptake by bacteriorhodopsin is close to M412 decay and to the electrogeneous millisecond phase in both the presence and absence of the pH buffer. The bacteriorhodopsin-induced proton release phase accounts for about 20%, and the uptake phase for about 80% of the overall potential. This is compatible with the model assuming that the proton start-out point - possibly, the protonated Schiff base connecting lysine 216 with retinal - is closer to the outer rather than the inner (cytoplasmic) surface of the bacterial membrane.

Novel observations related to the Na+-linked energy transduction in bacterial membranes are considered. It is concluded that besides the well-known systems based on the circulation of protons, there are those based on the circulation of Na+. In some cases, H+ and Na+ cycles co-exist in one and the same membrane. Representatives of the 'sodium world', i.e. cells possessing primary Na+ pumps (delta mu Na generators and consumers) are found in many genera of bacteria. Among the delta mu Na generators, one should mention Na+-NADH-quinone reductase and Na+-terminal oxidase of the respiratory chain, Na+-decarboxylases and Na+-ATPases. For delta mu Na consumers, there are Na+-ATP-synthases, Na+-metabolite symporters and Na+ motors. Sometimes, one and the same enzyme can transport H+ or, alternatively, Na+. For instance, an Na+-ATP-synthase of the F0F1 type translocates H+ when Na+ is absent. Employment of the Na+ cycle, apart from or instead of the H+ cycle, increases the resistance of bacteria to alkaline or protonophore-containing media and, apparently, to some other unfavourable conditions.

DCCD and cyanide, the H+-ATPase and respiratory chain inhibitors, increase the sensitivity of Halobacteriwn halobium to the attractant effect of light. This phenomenon requires the presence of bacteriorhodopsin and occurs at high light intensities (about 100 W/m2). Strains lacking both bacteriorhodopsin and halorhodopsin exhibit an increased sensitivity of aerotaxis. These observations are in good agreement with the hypothesis that Δg̃mH reception is one of the mechanisms responsible for phototaxis and aerotaxis in H. halobium.

It has been found that bacteriorhodopsin containing 13- cis -retinal as the chromophore (13- cis -bR) is competent in a one-photon photocycle which involves an M-type intermediate. Such an effect can be shown in 13- cis -Br obtained by means of 13- cis -retinal treatment of white bacterioopsin membrane sheets, as well as in dark-adapted purple sheets, solubilized Br and Br proteoliposomes. The 13- cis -Br photocycle with the M intermediate is found to be proton-motive, the electrogenic phases resembling those of the all- trans -Br photocycle. All of these effects are observed only when the Ph is above some critical value, namely Ph 8.5–9.5 for the Br sheets, 7.6 for proteoliposomes and 7.0 for the solubilized Br.

15 min cold exposure of rats adapted to cold results in switching on a pathway of the fast oxidation of extramitochondrial NADH in the isolated liver mitochondria. This pathway is sensitive to mersalyl and cyanide, resistant to amytal and antimycin A, and can be stimulated by dinitrophenol. A portion of the endogenous cytochrome c pool can easily be removed by washing mitochondria of the cold-exposed rats. A scheme is discussed, postulating desorption of the inner membrane-bound cytochrome c into intermembrane space of mitochondria, resulting in formation of a link between the non-phosophorylating NADH-cytochrome c reductase in the outer mitochondrial membrane and cytochrome c oxidase in the inner membrane. It is suggested that such an oxidative pathway is involved in the urgent heat production in liver in response to the cold treatment.

Rhodospirillum rubrum reaction centre (RC) complexes with or without antenna bacteriochlorophyll have been incorporated into liposomes; the latter were associated with a phospholipid-impregnated collodion film, and flash-induced electric responses were measured. It is shown that in the antenna-free RC-proteoliposomes a very rapid phase of charge separation (less than 200 ns) due to P-870 → QA electron transfer has a polarity (negative inside) opposite to that of the intact chromatophores or of the antenna-containing RC-proteoliposomes. In the antenna-free RC-proteoliposomes (but not in the chromatophores), P-870 is readily accessible to membrane-impermeable cationic electron donors like cytochrome c and Ru(NH3)2+6, which indicates localization of P-870 near the outer surface of the membrane. Oxidation of externally added cytochrome c2+ by P-870+ in these proteoliposomes initiates an additional slower phase of ΔΨ generation comprising about 25% of the total photoelectric response. The dependence of the slower phase rate on the cytochrome c concentration is characteristic of a saturating reaction with Km of 0.9 μM and kmax of approx. 6 · 103 s−1 at low ionic strength. A similar electrogenic phase can be observed without cytochrome c in the presence of sufficiently high concentrations of artificial redox dyes, e.g., 2 mM TMPD or more than approx. 50 μM PMS. The new phase of ΔΨ generation is tentatively ascribed to e− transfer within reaction centre molecule from the protein surface to the porphyrin ring of P-870+.

Regeneration of bacteriorhodopsin (bR) from the M intermediate via a new intermediate P has been studied. In the purple sheets treated with 0.015% Triton X-100 (the P → bR transition is suppressed), the α-maximum of absorption of P is located at 560 ± 5 nm, the extinction coefficient being equal to 0.7 ± 0.1 of that of bR. Besides, there is a small but measurable absorbance increase at 330–350 nm, which seems to be due to a β-maximum of 13-cis-retinal residue in P. The similarity of the α-maximum of P and bR suggests the Schiff base nitrogen to be protonated at the M → P transition. The kinetics of P → bR transition measured at the 335 nm absorbance decrease coincides with that of proton uptake accompanying bR regeneration after flash. A scheme is proposed assuming that H+ absorption from the water phase is coupled to the 13-cis → all-trans isomerization of retinal residue in which the Schiff base is already protonated by a proton-donor group of the protein.

The present state of the chemiosmotic hypothesis of oxidative phosphorylation is considered. It is pointed out that the available data testify to the validity of the following postulates of this hypothesis: (1) Energization of coupling membranes results in formation of a transmembrane electric potential and/or a pH difference whose values prove to be of the same order of magnitude as standard free energy of ATP hydrolysis. (2) The redox chain can generate a membrane potential independently of whether or not high-energy intermediates are formed. (3) ATPase can generate a membrane potential independently of whether or not mechanisms of electron transfer via coupling sites are operative. (4) Energy accumulated in the form of transmembrane electric and osmotic gradients can be utilized for ATP synthesis (“ion transfer phosphorylation”).

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS − ) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K + , or tetrachlorotri-fluoromethylbenzimidazole for H + ) under conditions where there existed K + (or H + ) gradients across the mitochondrial membrane. The ANS − fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS − responses. A hypothesis is put forward to explain the energy-dependent ANS − responses in mitochondria and particles by the membrane potential-induced redistribution of ANS − between the membrane and water phases.

The intramembrane arrangement of the respiratory chain generating electric potential difference across the mitochondrial membrane has been studied. The accessibility of various respiratory carriers to the non-penetrating electron donors and acceptors, such as ferri-and ferrocyanide, cytochrome c. fumarate and nicotinamide nuclcotides has been used as a test for surface localization of the carrier in the membrane of mitochondria and “inside-out” (sonicated) submitochondrial particles. Membrane potential formation was detected by measuring the transmembrane flows of the penetrating anion, phenyl dicarbaundecaborne (PCB−). It is shown that ferricyanide reduction can support PCB− movement if this electron acceptor interacts with intact mitochondria in the region localized on the oxygen site of the antimycin-sensitive point. The same region is accessible for ferrocyanide whose oxidation by O2 can be also coupled with PCB− translocation. Added nicotinamide nuclcotides cannot be utilized by mitochondria for supporting PCB− movement. PCB− movement in the “inside-out” submitochondrial particles can be supported by reduction of ferricyanide or fumarate by NADH, and of NAD+ by NADPH, the former process being sensitive to rotenone but not to antimycin. Antimycin-insensitive reduction of feericyanide or of CoQ6 by succinate is not coupled with PCB− transport. Neither ferrocyanide nor ferrocytochromec can be used as electron donors in the particles. Penetrating electron donors (TMPDH2, succinate) and acceptors (menadione) are effective both in mitochondria and particles. It is coucluded that flavin and transhydrogenase regions of the potential-generating redox chain are localized near the inner surface, cytochromec region-near the outers surface of the internal membrane of intact mitochondria. It means that the redox chain includes at least one act of the transmembrane transfer of reducing equivalents between flavins and cytochromec.

The mechanisms of energy coupling and ion transport in Rhodospirillum rubrum chromatophores have been studied. Photoreduction of NAD + and photophosphorylation have been measured under anaerobic conditions in the presence of N,N,N ′,N′-tetramethyl- p -phenylenediamine (TMPD), ascorbate and antimycin A. The uncoupler p -trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP), as well as ADP + phosphate, has been found to inhibit the photoreduction of NAD + in this system. Addition of an electron acceptor, such as methylviologen, fumarate or O 2 , to antimycin-treated chromatophores initiates the process of photophosphorylation. Chromatophores of R. rubrum accumulate the penetrating anions, phenyl dicarboundecaborane and tetraphenyl boron, as well as iodide, if the I − carrier, di-(pentafluorophenyl) mercury, is added. The anion accumulation can be supported by light-induced cyclic electron flow (NADH → O 2 , succinate → ferricyanide), by hydrolysis of ATP or inorganic pyrophosphate, as well as by reversal of the energy-requiring transhydrogenase reaction (NADPH → NAD + ). The type of energy source influences only the extent of the anion accumulation process. Cessation of the energy supply ( e.g. by exhaustion of the energy source or poisoning of the system by specific inhibitors or an uncoupler) brings about an efflux of the accumulated anions. Uptake of anions is accompanied by alkalinization of the outer solution; release of anions is accompanied by acidification. It is concluded that there is an energy-dependent charge-specific mechanism for anion accumulation in the chromatophore membrane resembling that found in the membrane of submitochondrial particles. It is stated that the electric field (the “ plus ” inside the chromatophore) is the motive force for ion transfer through the chromatophore membrane against a concentration gradient. The data on NAD + photoreduction, noncyclic photophosphorylation and energy-dependent anion transport are summarized as the concept of four sites of energy coupling in the chromatophore redox chain localized at the same steps as in animal mitochondria (NADPH → NAD + , NADH → cytochrome b , cytochrome b → cytochrome c , and the region after cytochrome c ). Each of these coupling sites can provide energy for generation of a membrane potential.

Fission of the mitochondrial reticulum (the thread–grain transition) and following gathering of mitochondria in the perinuclear area are induced by oxidative stress. It is shown that inhibitors of the respiratory chain (piericidin and myxothiazol) cause fission of mitochondria in HeLa cells and fibroblasts, whereas a mitochondria-targeted antioxidant (MitoQ) inhibits this effect. Hydrogen peroxide also induced the fission, which was stimulated by the inhibitors of respiration and suppressed by MitoQ. In untreated cells, the mitochondrial reticulum consisted of numerous electrically-independent fragments. Prolonged treatment with MitoQ resulted in drastic increase in size and decrease in number of these fragments. Local photodamage of mitochondria caused immediate depolarization of a large fraction of the mitochondrial network in MitoQ-treated cells. Our data indicate that the thread–grain transition of mitochondria depends on production of reactive oxygen species (ROS) in initial segments of the respiratory chain and is a necessary step in the process of elimination of mitochondria (mitoptosis).

A novel intermediate (P) of the bacteriorhodopsin (bR) photocycle, appearing between M412 and bR is described. Like bR, intermediate P shows an absorption maximum at 560–570 nm. However, the extinction coefficient of P is somewhat lower than that of bR. Moreover, there are some differences in spectra of bR and P at wavelengths shorter than 450 nm. The P → bR transition correlates with the absorption of H+ from the water medium. The following conditions proved to be favourable for the detection of the new intermediate: a high salt concentration, low light intensity and low temperature (0.5°C). The P → bR transition is strongly decelerated by a small amount of Triton X-100. Illumination of P does not produce M412 before bR is formed. It is assumed that M412 converts to P when the Schiff base is protonated by a proton transferred from a protein protolytic group which participates in the inward H+-conductivity pathway. Reprotonation of this group results in the conversion of P to bR. No more than 1 H+ is transported per bR photocycle.

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 delta psi decrease by these compounds. A threshold delta psi value is found, above which a very strong increase in H2O2 production takes place. This threshold slightly exceeds the state 3 delta psi level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169-2021 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.

Halobacterium halobium swims by rotating its polarly inserted flagellar bundle. The cells are attracted by green-to-orange light which they can use for photophosphorylation but flee damaging blue or ultraviolet light. It is generally believed that this kind of ‘colour vision’ is achieved by the combined action of two photoreceptor proteins, sensory rhodopsins-I and -II, that switch in the light the rotational sense of the bundle and in consequence the swimming direction of a cell. By expressing the bacteriorhodopsin gene in a photoreceptor-negative background we have now demonstrated the existence of a proton-motive force sensor (protometer) and the function of bacteriorhodopsin as an additional photoreceptor covering the high intensity range. When the bacteriorhodopsin-generated proton-motive force drops caused by a sudden decrease in light intensity, the cells respond by reversing their swimming direction. This response does not occur when the proton-motive force is saturated by respiration or fermentation.

During recent years, several pieces of indirect evidence of a programmed death in yeast have been published. Among them there are observations that some mammalian pro- or anti-apoptotic proteins induce or prevent the death of yeast; some toxic compounds kill yeast at lower concentrations if protein synthesis is operative; this death, as well as the death due to certain mutations, shows some apoptotic markers. In April 2002, the yeast programmed death concept received direct support. Madeo et al. [Madeo et al., Mol. Cell 9 (2002) 911–917] disclosed a caspase which is activated by H2O2 or aging and is required for the protein-synthesis-dependent death of yeast. Thus, a specific apoptosis-mediating protein was identified for the first time in Saccharomyces cerevisiae. Independently, Severin and Hyman [Severin, F.F., Hyman, A.A., Curr. Biol. 12 (2002) R233–R235] discovered that death of yeast, induced by a high level of a pheromone, is programmed. In particular, the death was found to be prevented by cycloheximide and cyclosporin A. It required mitochondrial DNA, cytochrome c and the pheromone-initiated protein kinase cascade. When haploids of opposite mating types were mixed, some cells died, the inhibitory pattern being the same as in the case of the killing by pheromone. Inhibition of mating proved to be favorable for death. Thus, pheromone not only activates mating but also eliminates yeast cells failing to mate. Such an effect should (i) stimulate switch of the yeast population from vegetative to sexual reproduction, and (ii) shorten the life span and, hence, accelerate changing of generations. As a result, the probability of appearance of new traits could be enhanced when ambient conditions turned for the worse.

Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na+] or by monensin-induced increase in intracellular [Na+]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no Δ μ - Na to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) Δ μ - Na rather than Δ μ - NH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na+-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of Δ μ Na generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na+ can replace that of H+ in V. alginolyticus, Δ μ - Na being formed by the Na+-motive respiratory chain and utilized by Na+-solute symporters, the Na+-driven flagellar motor and maybe by a reverse ion-motive ATPase.

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.

Electron transfer QA → QB has been reconstituted with added Q-10 in Rhodospirillum rubrum chromatophores associated with a phospholipid-impregnated collodion film. Rapid kinetics measurements of laser flash-induced ΔΨ generated in the chromatophores show that whereas electron transfer from Qa− to QB upon the first flash is not electrogenic in dark-adapted chromatophores, reduction of Q−B to Qbh2 induced by the second flash gives rise to an electrogenic phase with τ = 250 μs at pH 7.5 which contributes about 10% to the total ΔΨ generated upon the flash. The electrogenic phase is ascribed to vectorial protonation of Q2−B.

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.

The pH effect on carboxyatractylate-, glutamate- and aspartate-induced recoupling of palmitate-uncoupled rat liver mitochondria has been studied. Stimulation of respiration by low palmitate concentrations (5–20 μM) in the presence of 3 mM MgCl2 is shown to be pH-independent within the 7.0–7.8 range. The recoupling effect of glutamate (or aspartate) decreases and that of carboxyatractylate increases with increase in pH. The recoupling effect of a combination of carboxyatractylate and glutamate (aspartate) appears to be constant at these pH values, being as high as about 80%. It is concluded that uncoupling by low palmitate in liver mitochondria is mediated mainly by ATP/ADP and aspartate/glutamate antiporter.

Growth of E. coli in the presence of the protonophorous uncoupler pentachlorophenol is shown to strongly enhance levels of cytochrome d, a putative Na(+)-motive oxidase. This effect was found to be arrested by chloramphenicol and stimulated by high Na+ concentration in the growth medium. The induction of cytochrome d takes place in a mutant deficient in the F0F1 ATP-synthase but does not occur in mutants deficient in either of two different components of the Arc system. Similar relationships were revealed when pentachlorophenol was replaced by ferricyanide and phenazine methosulfate, agents oxidizing the respiratory chain. Induction of cytochrome d is also shown to occur in riboflavin-deficient mutants growing in the presence of such low riboflavin concentrations as to be insufficient to maintain a high respiration rate. It is suggested (i) that it is delta mu H+ decrease rather than reduction of the respiratory chain that is the signal for the induction of cytochrome d, and (ii) the Arc system is involved in this type of metabolic regulation.

In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10–35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H2O2-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular “ATP-meter(s)” generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).

Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed (“dark”) accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+). The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O(2-*) to O(2). A possible role of cytochrome c in the antioxidant defence is discussed.

Light-dependent Na+ and H+ transports, membrane potential (delta psi) and motility have been studied in the cells of the marine cyanobacterium Oscillatoria brevis. In the presence of a protonophorous uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, the intracellular Na+ level is shown to increase in the dark and decrease in the light. The Na+/H+ antiporter, monensin, stimulates the dark CCCP-dependent [Na+]in increase and abolishes the light-dependent [Na+]in decrease. Na+ ions are necessary for the fast light-induced delta psi generation and H+ uptake by the cells. This uptake is inhibited by monensin being resistant to CCCP. Monensin sensitizes the delta psi level and the motility rate to low CCCP concentrations. The obtained data are consistent with the assumption that O. brevis possesses a primary Na+ pump which utilizes (directly or indirectly) the light energy.

Both natural (laurate) and artificial (m-chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O⋅−2 and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate, an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the releasing effect of carboxyatractylate. GDP is much less effective than carboxyatractylate in releasing the laurate inhibition of reactive oxygen species (ROS) formation. Micromolar laurate concentrations arresting the ROS formation cause strong inhibition of reverse electron transfer from succinate to NAD+, whereas State 4 respiration and the transmembrane electric potential difference (ΔΨ) level are affected only slightly. It is suggested that (i) free fatty acids operate as natural `mild uncouplers' preventing the transmembrane electrochemical H+ potential difference ( Δ μ H + ) from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of the respiratory chain, and (ii) it is the ATP/ADP-antiporter, rather than uncoupling protein 2, that is mainly involved in this antioxidant mechanism of heart muscle mitochondria.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C 4 R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C 12 R1). Surprisingly, C 12 R1 increased H + conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C 4 R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C 4 R1. In experiments with HeLa cells, C 4 R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C 4 R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C 12 R1. Thus, C 4 R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

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 H2O2-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 H2O2 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/kg × day 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.

Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies aposition at the vertices of a triangular antiprism with a 32 point group symmetry.

Phototaxis in Halobacterium halobium is governed by two distinct photosysterns1. Cells are attracted by green light, which is absorbed by bacteriorhodopsin, effectively a light-driven proton pump2,3, and repelled by UV and blue light absorbed by a retinal-dependent pigment and by carotenoids1,4–10. One possibility is that the bacterium detects changes in light intensity directly by the interaction of excited absorbent molecules with the tactile system, in which case the phototactic receptors would be analogous to bacterial chemoreceptors11. However, H. halobium may sense light changes indirectly as changes in the chemical potential of hydrogen ions (ΔμH+)12,13; the coincidence of the action spectra for positive phototaxis and photosynthesis14,15 indeed support the suggestion that the cells may possess a specific ΔμH+ receptor, called a ‘protometer’. We now describe experiments which indicate that positive phototaxis in H. halobium is governed by ΔμH+ sensing, but that negative phototaxis is mediated by a specific photoreceptor system.

In 1966, Mitchell [l] put forward the idea of electrogenesis in coupling membrane systems. According to this concept, there are molecular electric generators incorporated in membranes of mitochondria, chloroplasts, photosynthetic and respiring bacteria. These generators were postulated to represent enzyme complexes charging the membranes by electron or proton transport across hydrophobic barriers. According to Mitchell, the transmembrane electric potential and pH differences generated by enzymes of respiratory or photosynthetic redox chains are utilized by a reversible ATPase to form ATP. Several years ago, Tupper and Tedeschi [2] tried to detect membrane potentials in mitochondria, using microelectrodes, but failed. The size of the mitochondrion is apparently too small to allow an electrode to be introduced into the matrix space without a sharp decrease in the very high electric resistance of the mitochondrial membrane [3]. Some light-dependent electric responses of a complex character were demonstrated by Bulychev et al. [4] with microelectrodes in isolated chloroplast. The electric phenomena in coupling membranes were also studied by other methods. To measure the membrane potential in mitochondria, Mitchell and Moyle determined K+ distribution across the membrane of energized mitochondria whose K+ permeability was increased by valinomycin [5] . Our group, has described the antiport of synthetic penetrating anions and cations across the membranes of energized mitochondria [6], submitochondrial particles [7,8], subchloroplast particles [9] , chromatophores of photo

A method is proposed to estimate the stoichiometries of primary Na + -pumps in intact bacterial cells. It is based on technique when the H + /e − stoichiometry is measured in the presence of protophorous uncoupler and in the absence of penetrating ions other than H + . Under these conditions, the H + influx discharges membrane potential generated by the Na + pump so the Na + /e − and H + /e − ratios become equal. Using this approach it is shown that the Na + /e − ratio for the Na + -motive NADH : quinone oxidoreductase of Vibrio alginolyticus is equal to 0.71±0.06. The Na + /e − stoichiometry appears to be ≈1, provided that the contribution of the non-coupled NADH : quinone oxidoreductase, which is resistant to low HQNO concentrations, is taken into account.

The concept of transmembrane electron flow in the coupling sites of redox chains was put forward by Mitchell virtually simultaneously with the general chemiosmotic principle of energy coupling via protonmotive force (l).Whereas the general formulation of the chemiosmotic theory proved to be accepted by majority of bioenergeticists, there are still considerable debates on the electrogenic transmembrane electron transfer as a mechanism of ΔµH+ generation.

Electrogenic and redox events in the reaction-centre complexes from Rhodopseudomonas viridis have been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochrome c have different Em values (-60, +20, +310 and +380 mV). The first three hemes reveal α absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380-mV heme displays a split α band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non-degenerated Qx and Qy transitions in the iron-porphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high-potential hemes c-559 and c-556 are reduced, heme c-559 appears to be the electron donor to P-960+ (τ= 0.32 μs) whereas heme c-556 serves to rereduce c-559 (τ= 2.5 μs). Upon reduction of the third heme (c-552), the P-960+ reduction rate increases twofold (τ= 0.17 μs) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 μs after the flash. The following sequence of the redox centers is tentatively suggested: c-554, c-556, c-552, c-559, P-960. To study electrogenesis, the reaction-centre complexes from Rps. viridis were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid-impregnated film. The electrical difference induced by a single 15-ns flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QA reduction by P-960+ (II) reduction of P-960+ by heme c-559; (III) reduction of c-559 by c-556 and (IV) protonation of Q2−B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X-ray data of Deisenhofer et al. [J. Mol. Biol. 180, 385–398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved. The efficiency is higher, the deeper these groups are immersed in the membrane.