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A
M.V. Lomonosov Moscow State University
 
 
 
A.N. Belozersky Institute
of Physico-Chemical Biology

 

   

ANNUAL REPORT
 
1998
 
 
 
 

 

 

 

 

 
CONTENT

 

BIOENERGETICS AND PHOTOSYNTHESIS

ENZYMOLOGY

GENE STRUCTURE, REGULATION AND EVOLUTION

MOLECULAR VIROLOGY

CELL BIOLOGY AND PHYSIOLOGY

GENEBEE-SERVER OF RUSSIAN EMBnet NODE

LIST OF MAJOR PUBLICATIONS

IN PRESS

 

 

 

 

 

BIOENERGETICS AND PHOTOSYNTHESIS

Fatty acids as natural uncouplers preventing generation of O2- and H2O2 by mitochondria in the resting state

(Skulachev V.P., Korshunov S.S., Korkina O.V., Ruuge E.K., Starkov A.A.)
 
Both natural (laurate) and artificial (m-chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O2- and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate (cAtr), an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the inhibition effect of cAtr. GDP is much less effective than cAtr 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 D y level are affected only slightly. It is suggested that (i) free fatty acids operate as natural ‘mild uncouplers’ preventing the H+ potential from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of 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.

 

Thermoregulatory uncoupling in heart muscle mitochondria: involvement of ATP/ADP antiporter and uncoupling protein.
 
(Skulachev V.P., Simonyan R.A.)

Possible involvement of ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorilation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin-treated mitochondria from cold exposed (6oC, 48h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold-exposed and non-exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold-exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. GDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr- and GDP-sensitive in the case of palmitate. The fatty acid-free bovine serum albumin enhances D y in both cold-exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the ‘mild uncoupling’ under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to increase strongly in rat heart muscle under the cold exposure conditions used).
 

  The study of the nonprotonic ion cycles in bacterial energetics
 
(Skulachev V.P., Avetisyan A.V., Bertsova Yu.V., Bogachev A.V., Muntyan M.S., Ustiyan V.S., Shestopalov A.I.)

Light-driven ATP synthesis was found in the cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin and lacking the H+-pumping bacteriorhodopsin. Photophosphorylation occurred in the cells with the cyanide-inhibited respiratory chain as well as in the untreated cells at a low chloride concentration in the incubation medium. A rise in the chloride concentration from 0.1 to 2.35 M in the incubation medium led to the inhibition of photophosphorylation. Continuous illumination increased membrane D Y when the respiration was inhibited by cyanide. DCCD, an ATPase inhibitor, stimulated this effect. To explain the data, one may suggest that halorhodopsin pumped chloride into the cells whereas chloride efflux from the cells through Cl--ATP-synthase was coupled to the ATP synthesis (chloride cycle).

Under fully reduced conditions, reassociation kinetics of CO were studied in several terminal oxidases containing copper in their binuclear center. The purified Paracoccus denitrificans ba3-type quinol oxidase was found to recombine with CO monophasically (t 25-30 ms) like the H+-translocating oxidases of bo-type from Escherichia coli, caa3-type from Bacillus halodurans FTU, and bo-type from Methylobacillus flagellatum KT. H+-translocating oxidase of the aa3-type from bovine heart recombined with CO monophasically at a higher rate (t 16-19 ms) than the studied copper-containing bacterial oxidases. After prolonged incubation in the presence of CO, oxidases of ba3- and aa3-type changed their CO-binding properties. The contribution of the slow component was diminished while new fast components arose. Measurement of the metal content in the oxidases indicated that during the incubation, the enzymes lost their copper, the process being accompanied by the appearance of a fast CO recombination rate resembling that of the non-copper, presumably Na+-translocating oxidases of bd-type from E. coli and bb-type from B. halodurans FTU. This points to a role of copper in CO-binding by terminal oxidases.
 
The study on the H+/e- ratio in B.halodurans FTU cells showed that it was about zero when the bb-type oxidase dominated in the cells. When the H+/e- ratio was studied in the cells by the oxygen pulse method in the presence of protonophorous compound CCCP, an alkalization of the incubation medium was shown to take place. The alkalization decreased in the presence of thiocyanate (the compound decreasing D Y ) and was absent in the Na+-free medium. These data support the "Na+-cycle" hypothesis and suggest that a functional Na+-pump operates in the bacterium.

 

The role of different respiratory chain enzymes of Azotobacter vinelandii in the respiratory protection
 
(Bertsova Yu.V., Bogachev A.V., Skulachev V.P.)

It was found that two NADH:ubiquinone oxidoreductases operate in the A.vinelandii respiratory chain: proton-motive NADH dehydrogenase (NDH-I) and enzyme non-coupled to the energy conservation (NDH-II). The level of these oxidoreductases strongly depends upon [O2] and [NH3] in the growth medium. Increase in [O2] results in lowering of the coupled enzyme level and in rise of the non-coupled one. Exclusion of NH3 from the growth medium increases the level of the non-coupled enzyme whereas that of the coupled enzyme remains constant.

It was found that A.vinelandii bd-type oxidase can produce D pH and D Y . The H+/e- ratio for this enzyme was established as being close to 1. It is concluded that the bd-type oxidase of A.vinelandii is competent in generating a protonic potential but its efficiency is lower than that of the o-type oxidase. It was also shown that Q-cycle does operate in the o-type cytochrome oxidase terminated branch of the A.vinelandii respiratory chain and does not in the bd-type quinoloxidase terminated branch.

Summarising the above observations one can assume that the respiratory protection of nitrogenase could be carried out by co-operation of the non-coupled NADH:ubiquinone oxidoreductase and the bd-type quinoloxidase. Efficiency of this chain seems to be five-fold lower than that of the usual proton-motive chain, which is also present in A.vinelandii and operates at low [O2].

 

Molecular mechanism of proton translocation by cytochrome oxidase (COX)
 
(Konstantinov A.A., Arutjunyan A.M., Atsarkina N.V., Borisov V.B.,
Kirichenko A.V., Siletsky S.A., Vygodina T.V.)

Crystal structure of COX reveals two potential input proton channels denoted as D- and K-channels. We have compared wild type R. sphaeroides COX and mutants with amino acid replacement in these channels: E286, D132 (D-channel) and K362, T359 (K-channel). Effects of the mutations on the spectra, ligand reactivity and peroxidase activity of COX were studied. In the dithionite-reduced E286Q, MCD spectra in the Soret indicate transition of 70-80% of heme a3 from the high to low spin state. MCD of the oxidized E286Q is consistent with hexacoordinated ferric state of heme a3. Accordingly this mutant COX does not react with KCN and H2O2 in the ‘oxidised’ state. Presumably heme a3 in E286Q is trapped at the unprotonated iron(III)-peroxy complex with spectrum of the ferrous-oxy state. CD and MCD data together suggest that replacement E286Q perturbs significantly surrounding symmetry of the both hemes a and heme a3. D132N COX showes decreased reactivity towards H2O2. E286Q COX did not react with H2O2 at all. Reaction of K362M COX with H2O2 at pH above 8 resembles reaction of WT enzyme at acidic pH. Formation of compound P during aerobic CO treatment of the oxidized K362M COX is decreased. Some K362M COX preparations have significant catalase activity. The K-channel mutants although unable to turnover with dioxygen, can utilize H2O2 as electron acceptor with either ferrocyanide or cytochrome c as electron donors. Their peroxidase activity is similar to that of the WT COX and is coupled to D y generation and proton pumping. No peroxidase activity is revealed in the D-channel mutant COX, D132N and E286Q. Reduction by dithionite is severely inhibited in K362M, but not in D132N COX. Mutations in the D-channel arrest COX turnover by inhibiting proton uptake in the peroxidase part of the catalytic cycle while the K-channel is required for the initial reduction of binuclear center in the eu-oxidase phase. Electrometric technique applied earlier in the studies of F->Ox transition has been used to resolve charge translocation coupled to P->F transition. P was generated by aerobic CO bubbling through suspension of COX proteoliposomes in the presence of ferricyanide. As in the case of F->Ox transition, the electric response includes a rapid KCN insensitive electrogenic phase with t of 40-50 m s (reduction of heme a by CuA) and a slower multiphasic cyanide-sensitive part (vectorial proton transfer). The protonic part of the P->F electric response is faster than in the F->Ox transition but much slower than reported for the P->F transition during oxidation of the reduced COX by oxygen. This difference is due to different states of CuB in compounds P formed under these conditions. We also made preliminary experiments on D y generation in the E286D mutant COX. In contrast to the ‘dead’ E286Q mutant, the E286D COX is about 50% active and first single-turnover experiments reveal retardation of the protonic electrogenic phases.

 
 
Comparative study on uncoupling liver and skeletal muscle mitochondria by laurate and lauryl sulfate
 
(Samartsev V.N., Simonyan R.A., Markova O.V., Mokhova E.N., Skulachev V.P.)

Uncoupling effects of laurate and lauryl sulfate have been studied in isolated rat liver and skeletal muscle mitochondria. It is found that two-fold stimulation of respiration of the oligomycin-treated liver mitochondria, accompanied by a membrane potential decrease, is caused by 2x10-5 M laurate or 1.6x10-4 M lauryl sulfate. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the action of laurate and lauryl sulfate on respiration rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Cations neutralizing negative charges of the membrane surface, such as tetraphenyl phosphonium and cetyltrimethyl ammonium, cause an alkaline shift of these pH dependencies. A small amount of lauryl sulfate, increasing the membrane negative charge, induce the opposite shift. ADP but not GDP induce partial recoupling with both laurate and lauryl sulfate. It is concluded that in liver mitochondria uncoupling by lauryl sulfate, like that by laurate, is mediated by the ATP/ADP and glutamate/aspartate antiporters. The antiporters are assumed to be involved not only in translocation of the uncoupler anions but also in their protonation by extramitochondrial H+ ions.

In skeletal muscle mitochondria uncoupled by laurate, it is found that GDP added before CAtr causes partial recoupling which becomes complete after subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP entails larger recoupling than after GDP and prevents the GDP recoupling. ADP but not CDP substitutes for GDP. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP resistant but sensitive to CAtr, glutamate and serum albumin. It is suggested that a skeletal muscle mitochondria, besides the ATP/ADP and aspartate/glutamate antiporters, a GDP-sensitive mechanism, presumably uncoupling proteins (UCP) 3L and 2, is involved in uncoupling by laurate. Some of these UCPs, in contrast to UCP1, seem to be sensitive to CAtr but, like UCP1, cannot protonate the transported anions such as lauryl sulfate.

 

Mitochondrial benzodiazepine receptor and permeability transition pore. Possible involvement in the nucleic acid transport
 
(Zorov D.B., Isaev N.K., Krasnikov B.F., Kuzminova A.E., Zorova L.D.,
Pristchepova A.E., Vyssokikh M.Yu., Belousov V.V.)

During the period covering 1996-1998, different aspects of the mitochondrial permeability transitions (PT), which, from our point of view, represent the functional state of the mitochondrial benzodiazepine receptor, were under study. This study has been split into different projects as follows:

The Project 1 was dealing with the exploring of functional parameters, determining the severity of PT as well as the ways of its proper regulation. Among the factors reasonably capable to regulate the activity of the mitochondrial megachannel, first, the oxygen was chosen. We have demonstrated that in spite of a common view on the obligatory role of oxygen in PT, mediated by reactive oxygen species, this factor seems to be essential but can be bypassed by other stimuli. Nucleic acids were another factor under our decent analysis. According to the hypothesis we put forward, the activity of the mitochondrial benzodiazepine receptor is modulated by nucleic acids. In this project we were able to prove that first, mitochondrial DNA or its fragments are capable of penetrating through the megachannel and second, the structural analogs of nucleic acids as well as viral RNA, both interact with cytochrome c, resulting in its release from mitochondria for possible activation of the apoptotic cascade.

In the Project 2 we were trying to isolate the proteinaceous complex from the mitochondrial contact sites, which is responsible for PT. By the electrophoresis (under native conditions) of the extracts after mitochondria being treated with low Triton X-100, we isolated the proteinaceous supercomplex tentatively containing porin, hexokinase, cyclophilin, adenine nucleotide carrier and creatine kinase. We found the tight functional coupling existing between all components of the complex. The reconstitution of the complex into bilayer membrane resulted in the channel formation, resembling the megachannel observed when PT was activated.

The Project 3 was focusing on the study of PT in a living cell. We were first who demonstrated that in the primary culture of the rat brain granule cells glutamate induces the depolarization of the inner mitochondrial membrane assigned to PT pore opening. The role of Ca and Na ions in the glutamate toxicity was analyzed. The conclusion has been made that Ca/Na exchanger when being activated prevents the cell death probably induced by PT pore opening in neuronal mitochondria.
 

Study of the kinetics of gramicidin channel functioning in bilayer lipid membranes by the method of sensitized photoinactivation
 
(Antonenko Yu.N., Krylov A.V., Rokitskaya T.I., Kotova E.A., Evtodienko V.Yu.)

Effect of a cationic polymer, poly(L-lysine), on the kinetic properties of ionic channels formed by neutral gramicidin A and its negatively charged analogue O-pyromellitylgramicidin in a bilayer lipid membrane is studied using a method of sensitized photoinactivation. This newly developed method is based on the analysis of transmembrane current transients induced by a flash in the presence of a photosensitizer. It has been shown previously that the time course of the flash-induced current decrease in most cases follows a single exponential decay with an exponential factor (t , the characteristic time of photoinactivation) that correlates well with the single-channel lifetime. Addition of polylysine does not affect t for gramicidin A channels, but causes a substantial increase in t for O-pyromellitylgramicidin channels. This effect is reversed by addition of polyacrylic acid. The deceleration of the photoinactivation kinetics is ascribed to electrostatic interaction of polylysine with O-pyromellitylgramicidin probably resulting in O-pyromellitylgramicidin clustering. The latter can stabilize the channel state by reducing the rotational and lateral mobility of O-pyromellitylgramicidin monomers and dimers, and thus increase the single channel lifetime.

 

Energy delivery and trapping from the bulk chlorophylls to reaction centers in purple bacteria and plants. Physico-chemical mechanism.
 
(Borisov A.Yu., Kotova E.A., Chamorovsky S.K., Samuilov V.V., Mohov V.V., Bezryadnov D.V., Gusev O.S., Gorshkov N.C.)
 
1) Optico-mechanical developments were conducted in the quvette compartments of phase fluorometer and differential spectrophotometer as well as the light chopper of the latter instrument, which ensures synchronous modulating of both the exciting and measuring light beams. A time resolution about 50 ps was achieved in phase fluorometer.
 
2) The PC-program was further developed. Now it enables to model the sequence of primary photoprocesses of energy migration in the bulk chlorophylls, its trapping and conversion in reaction centers of purple bacteria and oxygen-evolving photosystem-2 of plants.
 
3) The parallel experiments were performed with particles enriched in plant photosystem-2 with phase fluorometer and spectrofluorometer. The efficiency of wasteful energy quenching in bulk chlorophyll-a both by natural and exogenous quinones was estimated. An article published in cooperation with Prof. V.D. Samuilov' group.
 
4) The experiments were conducted in modeling of energy migration from light-absorbing bulk chlorophyll-a of plant photosystem-2 to its reaction centers and its trapping in them. On this basis the criterion was formulated and grounded how to distinguish between two alternative mechanisms of energy trapping in reaction centers.
 
5) The PC analysis conducted has confirmed that the polaron model of energy trapping in reaction centers (was developed in the course of fulfilling the program of our preceding RFFI grant 95-04-12699a) can noncontroversially accommodate all experimental data accumulated during last decade for purple bacteria and two plant photosystems as well. Published in BAMBI (1999) V. 47, P 117-125.
 
 
Development of new quantitative approach to describe regulatory properties of oscillatory metabolic systems and its application to study regulation of photosynthesis in chloroplasts.
 
(Demin, O.V., Lebedeva, G.V., Riznichenko, G.Yu., Pljusnina, T.Yu., Belyaeva, N.E.)
 
For metabolic systems under condition of forced oscillation regime on the basis of package SCAMP program which allows to calculate periodic control coefficients for arbitrary periodic influence and any metabolic system has been created. Complete system of algebraic equations which allows calculating control coefficients of averaged (on period) fluxes using kinetic properties of enzymes and periodic solution, has been obtained. Application of the relationships to describe regulatory properties of membrane associated enzyme which is influenced with (weak) electromagnetic field, has revealed differences in regulatory properties of the enzyme under different dynamic regimes: steady state and forced oscillation. To describe regulatory properties of autooscillation metabolic systems logarithmic derivatives of period of oscillation and Fourier coefficients of fluxes and concentrations of intermediates with respect to enzyme concentrations has been employed. It was shown that these derivatives - depending on time control coefficients of autooscillation characteristics with respect to enzymes of system considered - satisfy linear relationships which in framework of metabolic control analysis usually names as «connectivity theorems». To study effects of external periodic influence on different biochemical/biophysical systems mathematical models of energetic metabolism of plant and animal cells (oxidative phosphorylation in hepatocite mitochondria and photosynthesis in plant chloroplasts) have been developed. In framework description of photosynthesis we modelled dependence of chlorophyll fluorescence on time - induction fluorescence curve. The dependence has been calculated using two different mathematical models: detailed model of photosystem II and model, which takes into consideration both generators (photosystem II, b6f complex, photosystem I) and consumers (ATP-synthetase, proton, chloride and potassium leaks) of electrochemical potential. It was shown that local minima in beginning of induction fluorescence curve could not be obtained using the model of photosystem II only. However, the curve calculated on basis of the model including kinetic description both generators and consumers of electrochemical potential has the minima. It was found that possible reason of such nonmonotonous behaviour was nonmonotonous change of components of electrochemical potential (electric potential difference and delta pH) with time.
 
 
Structure-function relationships in photosynthetic antenna of green bacteria
 
(Fetisova Z.G., Taisova A.S., Novoderezhkin V.I.)

The concept of strict functional optimization of light-harvesting photosynthetic antenna structure was put forward several years ago. Using simulations of excitation energy transfer from antenna to reaction center, some guiding principles for organization of an optimal antenna model were identified. Targeted searches for theoretically identified structural optimization criteria have subsequently allowed one to recognize them in natural photosynthetic antennae, in particular, in chlorosomal superantennae of green bacteria.
 
Oligomerization of light-harvesting antenna pigments in vivo was predicted to be biologically expedient being one of efficient strategies for light harvesting. The direct experimental proof for oligomeric organization of chlorosomal pigments in vivo was demonstrated by hole burning in fluorescence and fluorescence excitation spectra of intact cells. Spectral hole-burning studies allowed one to determine the fine structure of electronic spectra of chlorosomal pigments in vivo, that provided us with an exciton level structure criterion for theoretical evaluation of any model for pigment aggregation in the chlorosome. Using the standard approach to exciton-phonon problem in molecular crystals, we discovered that none of the models of pigment aggregation in Cf.aurantiacus chlorosome, proposed for last 20 years, displays the in vivo exciton level structure of the aggregate revealed by hole-burning studies, and, moreover, may serve as prototypes for optimal antennae. Our pump-probe experiments yielded information about Cf.aurantiacus antenna pigments kinetics, absorption difference spectra evolution on femto- and picosecond time scales, and pigment aggregation. We demonstrated that two methods of nonlinear spectroscopy -hole-burning and ultrafast pump-probe spectroscopies, used in combination - allow one to discover key data on both electronic and three-dimensional structure of the antenna aggregates in vivo.

A model of pigment aggregation in the membrane B808-866 antenna of the green bacterium Cf.aurantiacus is proposed. The size of a unit building block of this antenna has been determined.

 

Investigation of the light-dependent H+ and Cl- transport by bacteriorhodopsin and halorhodopsin
 
(Kaulen A.D., Zaspa A.A., Kalaidzidis I.V., Radionov A.N., Khitrina L.V.)

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. The two-fold decrease in magnitude of the microsecond photovoltage generation coupled to the M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable with that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of the M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bR ground state and transition of the purple neutral form into the blue acid form. Thus, pK of E204 in the M state is close to pK of D85 in the bacteriorhodopsin ground state (<3) and far below pK of the terminal proton release group (~6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.

Flash-induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. First component with t~1 ms does not exceed 4% of the overall response amplitude and is probably associated with the formation of L (hR520) intermediate. The second main component w