Biochemistry (Moscow) (v.77, #9)

Transmembrane electric potential difference in the protein-pigment complex of photosystem 2 by M. D. Mamedov; V. N. Kurashov; I. O. Petrova; A. Yu. Semenov (947-955).
The protein-pigment complex of photosystem 2 (PS2) localized in the thylakoid membranes of higher plants, algae, and cyanobacteria is the main source of oxygen on Earth. The light-induced functioning of PS2 is directly linked to electron and proton transfer across the membrane, which results in the formation of transmembrane electric potential difference (ΔΨ). The major contribution to ΔΨ of the PS2 reaction center is due to charge separation between the primary chlorophyll donor P680 and the quinone acceptor QA, accompanied by re-reduction of P 680 + by the redox-active tyrosine residue YZ. The processes associated with the uptake and release of protons on the acceptor and donor sides of the enzyme, respectively, are also coupled with ΔΨ generation. The objective of this work was to describe the mechanisms of ΔΨ generation associated with the S-state transitions of the water-oxidizing complex in intact PS2 complex and in PS2 preparation depleted of Mn4Ca cluster in the presence of artificial electron donors. The findings elucidate the mechanisms of electrogenic reactions on the PS2 donor side and may be a basis for development of an effective solar energy conversion system.
Keywords: photosystem 2; reaction center; S-state transitions; proteoliposomes; electron donors; direct electrometric method; photoelectric response; electrogenicity; channels

The review focuses on the energetic and regulatory role of proton potential in the activity of chloroplasts, the light energy-converting organelles of plant cells. Mechanisms of generation of the transmembrane difference of electrochemical potentials of hydrogen ions $$left( {Delta ilde mu _{{ m H}^ + } } ight)$$ in the chloroplast thylakoid membranes are considered. Methods for measuring the intrathylakoid pH in chloroplasts are described. It is shown that under conditions of phosphorylation in chloroplasts, the pH of the intrathylakoid space decreases moderately (pHin ⩾ 6.0–6.2, at the stroma pHout ∼ 7.8–8.0), with a corresponding concentration component of $$Delta ilde mu _{{ m H}^ + }$$ equal to ΔpH ⩽ 1.6–2.0. On analyzing the energy and structural features of ATP synthase of chloroplasts, we conclude that the energy stored as the concentration component of the proton potential ΔpH is sufficient to sustain ATP synthesis. The mechanisms of pH-dependent regulation of electron transport in chloroplasts (photosynthetic control of electron transport, enhancement of non-photochemical quenching of chlorophyll excitation in the light-harvesting antenna, light-induced activation of the Calvin-Benson cycle reactions, activation of ATP synthase) are considered briefly.
Keywords: bioenergetics; chloroplasts; proton potential; regulation of photosynthesis

Boronated derivatives of chlorin e 6 and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes by T. I. Rokitskaya; A. V. Zaitsev; V. A. Ol’shevskaya; V. N. Kalinin; M. M. Moisenovich; I. I. Agapov; Y. N. Antonenko (975-982).
Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e 6 and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e 6 by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec−1, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.
Keywords: penetrating anions; monocarborane; bilayer lipid membrane; photosensitizer; chlorin e 6 ; porphyrin

Novel mitochondria-targeted compounds composed of natural constituents: Conjugates of plant alkaloids berberine and palmatine with plastoquinone by B. V. Chernyak; Y. N. Antonenko; E. R. Galimov; L. V. Domnina; V. B. Dugina; R. A. Zvyagilskaya; O. Yu. Ivanova; D. S. Izyumov; K. G. Lyamzaev; A. V. Pustovidko; T. I. Rokitskaya; A. G. Rogov; I. I. Severina; R. A. Simonyan; M. V. Skulachev; V. N. Tashlitsky; E. V. Titova; T. A. Trendeleva; G. S. Shagieva (983-995).
Novel mitochondria-targeted compounds composed entirely of natural constituents have been synthesized and tested in model lipid membranes, in isolated mitochondria, and in living human cells in culture. Berberine and palmatine, penetrating cations of plant origin, were conjugated by nonyloxycarbonylmethyl residue with the plant electron carrier and antioxidant plastoquinone. These conjugates (SkQBerb, SkQPalm) and their analogs lacking the plastoquinol moiety (C10Berb and C10Palm) penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria in living human cells in culture. Reduced forms of SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In isolated mitochondria and in living cells, the berberine and palmatine moieties were not reduced, so antioxidant activity belonged exclusively to the plastoquinol moiety. In human fibroblasts, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. At higher concentrations, conjugates of berberine and palmatine induced proton transport mediated by free fatty acids both in model and in mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. As an example of application of the novel mitochondria-targeted antioxidants SkQBerb and SkQPalm to studies of signal transduction, we discuss induction of cell cycle arrest, differentiation, and morphological normalization of some tumor cells. We suggest that production of oxygen radicals in mitochondria is necessary for growth factors-MAP-kinase signaling, which supports proliferation and transformed phenotype.
Keywords: mitochondria-targeted antioxidants; SkQ; berberine; palmatine; phospholipid membranes; mitochondria; tumor cells

Mitochondria-targeted plastoquinone antioxidant SkQR1 decreases trauma-induced neurological deficit in rat by N. K. Isaev; S. V. Novikova; E. V. Stelmashook; I. V. Barskov; D. N. Silachev; L. G. Khaspekov; V. P. Skulachev; D. B. Zorov (996-999).
A protective effect of a mitochondria-targeted antioxidant, a cationic rhodamine derivative linked to a plastoquinone molecule (10-(6′-plastoquinonyl)decylrhodamine-19, SkQR1) was studied in the model of open focal trauma of rat brain sensorimotor cortex. It was found that daily intraperitoneal injections of SkQR1 (100 nmol/kg) for 4 days after the trauma improved performance in a test characterizing neurological deficit and decreased the volume of the damaged cortical area. Our results suggest that SkQR1 exhibits profound neuroprotective effect, which may be explained by its antioxidative activity.
Keywords: brain trauma; neuroprotection; mitochondria-targeted antioxidants; SkQR1

Changes in respiratory activity, transmembrane electric potential, and ATP synthesis as induced by additions of limited amounts of ADP and Pi to tightly coupled inverted (inside-out) Paracoccus denitrificans plasma membrane vesicles were traced. The pattern of the changes was qualitatively the same as those observed for coupled mitochondria during the classical State 4-State 3-State 4 transition. Bacterial vesicles devoid of energy-dependent permeability barriers for the substrates of oxidation and phosphorylation were used as a simple experimental model to investigate two possible mechanisms of respiratory control: (i) in State 4 phosphoryl transfer potential (ATP/ADP × Pi) is equilibrated with proton-motive force by reversibly operating F1·Fo-ATPase (thermodynamic control); (ii) in State 4 apparent “equilibrium” is reached by unidirectional operation of proton motive force-activated F1·Fo-ATP synthase. The data support the kinetic mechanism of the respiratory control phenomenon.
Keywords: FoF1-H+-ATP synthase; oxidative phosphorylation; bacterial plasma membrane; Paracoccus denitrificans

On the photocycle of 4-ketobacteriorhodopsin by L. V. Khitrina (1008-1010).
The artificial pigment 4-ketobacteriorhodopsin is an interesting analog of bacteriorhodopsin. Arguments concerning the scheme of the photocycle of 4-ketobacteriorhodopsin are discussed.
Keywords: chromophore; retinal analogs; 13-cis-cycle; M-intermediate; M419 ; K570

Primary steps of electron and energy transfer in photosystem I: Effect of excitation pulse wavelength by A. Yu. Semenov; I. V. Shelaev; F. E. Gostev; M. D. Mamedov; V. A. Shuvalov; O. M. Sarkisov; V. A. Nadtochenko (1011-1020).
Time-resolved differential spectra of photosystem I complex were obtained by the “pump-probe” technique with 25-fs pulses with maxima at 670, 700, and 720 nm. The ratio between the number of excited chlorophyll molecules of the antenna and of the reaction center was shown to depend on spectral characteristics of the pump pulses. In all cases, an ultrafast (<150 fs) formation of the primary radical pair P700+A 0 was recorded. However, on excitation by pulses with maxima at 670 or 700 nm, detection of the charge separation was masked by the much more intensive bleaching at the chlorophyll Qy band due to excitation of the bulk antenna chlorophylls. We show that triggering the charge separation by 25-fs pulses centered at 720 nm allows to detect more clearly kinetics of formation of the primary and secondary ion-radical pairs. The findings help to explain possible reasons for discrepancies of kinetics of primary steps of electron transfer detected in different laboratories.
Keywords: femtosecond absorption spectroscopy; photosystem I; reaction center; electron transfer; primary reactions

Interaction of tetraphenylphosphonium and dodecyltriphenylphosphonium with lipid membranes and mitochondria by T. A. Trendeleva; A. G. Rogov; D. A. Cherepanov; E. I. Sukhanova; T. M. Il’yasova; I. I. Severina; R. A. Zvyagilskaya (1021-1028).
The permeability of a planar lipid membrane (composed of diphytanoylphosphatidylcholine) for tetraphenylphosphonium (TPP) was investigated. The observed level of the diffusion potential generated as a function of the TPP concentration gradient differed from the theoretically expected value, possibly due to proton leakage of the membrane mediated by the traces of fatty acids in the phospholipid forming the membrane. Using the molecular dynamics approach to study movement of TPP and dodecyltriphenylphosphonium (C12TPP) with different affinity to the lipid bilayer through a bilayer lipid membrane, it was found that C12TPP has a greater affinity to the membrane surface than TPP. However, the two cations have the same activation energy for transmembrane transfer. Interaction of TPP and C12TPP with tightly-coupled mitochondria from the yeast Yarrowia lipolytica was also investigated. At low, micromolar concentrations, both cations are “relatively weak, mild uncouplers”, do not shunt electron transfer along the respiratory chain, do not disturb (damage) the inner mitochondrial membrane, and profoundly promote the uncoupling effect of fatty acids. At higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their detergent action.
Keywords: TPP; C12TPP; diffusion potential; movement through lipid bilayer; yeast; Yarrowia lipolytica ; mitochondria; fatty acids

Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family by E. Y. Plotnikov; D. N. Silachev; S. S. Jankauskas; T. I. Rokitskaya; A. A. Chupyrkina; I. B. Pevzner; L. D. Zorova; N. K. Isaev; Y. N. Antonenko; V. P. Skulachev; D. B. Zorov (1029-1037).
It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C12R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C12R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C12R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation.
Keywords: ischemia; rhabdomyolysis; kidney; brain; stroke; oxidative stress; mitochondria-targeted compounds; SkQ; mitochondria

Substitution of ether linkage for ester bond in phospholipids increases permeability of bilayer lipid membrane for SkQ1-type penetrating cations by T. M. Il’yasova; T. I. Rokitskaya; I. I. Severina; Y. N. Antonenko; V. P. Skulachev (1038-1043).
Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed.
Keywords: BLM; phospholipid; proton conductivity; dipole potential; SkQ1

Solid tumor growth is dependent on the development of an adequate blood supply. For years, sprouting angiogenesis has been considered as the exclusive mechanism of tumor vascularization. However, in recent years, another mechanism of tumor vascularization has been identified that does not involve endothelial cells, a process called vasculogenic mimicry (VM). VM describes the unique ability of highly aggressive tumor cells to form vessel-like networks by virtue of their high plasticity. VM has been observed in several tumor types, and its occurrence is strongly associated with poor prognosis. This review focuses on signaling molecules and cascades involved in VM. In addition, the clinical significance of VM regardless of anti-angiogenesis treatment modalities is described.
Keywords: highly aggressive tumor; vasculogenic mimicry; signaling pathway; prognosis

Organization of chloroplast psbA-trnH intergenic spacer in dicotyledonous angiosperms of the family umbelliferae by G. V. Degtjareva; M. D. Logacheva; T. H. Samigullin; E. I. Terentieva; C. M. Valiejo-Roman (1056-1064).
Chloroplast intergenic psbA-trnH spacer has recently become a popular tool in plant molecular phylogenetic studies at low taxonomic level and as suitable for DNA barcoding studies. In present work, we studied the organization of psbA-trnH in the large family Umbelliferae and its potential as a DNA barcode and phylogenetic marker in this family. Organization of the spacer in Umbelliferae is consistent with a general pattern evident for angiosperms. The 5′-region of the spacer situated directly after the psbA gene is more conserved in length compared to the 3′-region, which has greater sequence variation. This pattern can be attributed to the maintenance of the secondary structural elements in the 5′-region of the spacer needed for posttranscriptional regulation of psbA gene expression. In Umbelliferae only, the conserved region contains a duplication of the fragment corresponding to the loop of the stem-loop structure and an independent appearance of identical sequence complementarities (traits) necessary to stabilize the stem-loop structure in different lineages. The 3′-region of the spacer nearest to trnH ranges greatly in size, mainly due to deletions, and the decrease in spacer length is a general trend in the evolution psbA-trnH in Umbelliferae. The features revealed in spacer organization allow us to use it as phylogenetic marker, and indels seem to be more informative for analyses than nucleotide substitutions. However, high conservation among closely related taxa and occurrence of homoplastic inversions in the stem-loop structure limit its application as DNA barcode.
Keywords: psbA-trnH; secondary structure; phylogeny; DNA barcoding; Umbelliferae

Separation and study of the range of plasminogen isoforms in patients with prostate cancer by E. F. Shin; D. I. Vodolazhsky; A. Yu. Golikov; T. N. Belova; N. V. Boyko; D. V. Zimakov; E. N. Cherkasova; M. I. Kogan; M. B. Chibichian; S. A. Moshkovskii; V. A. Tarasov; D. G. Matishov (1065-1071).
Using affinity chromatography, two-dimensional electrophoresis, and MALDI-TOF mass spectrometry, plasminogen isoforms were separated and identified in blood plasma. Healthy donors and patients with prostate cancer in various stages of development were included in the studied sample. With the development of prostate cancer, four additional specific plasminogen isoforms are registered in blood plasma; they are characterized by lower molecular weights and higher pI values compared to isoforms found in the control group.
Keywords: plasminogen isoforms; two-dimensional electrophoresis; MALDI-TOF-MS; prostate cancer

In vitro phosphorylation of the N-terminal half of hordeivirus movement protein by V. V. Makarov; A. Y. Iconnikova; M. A. Guseinov; V. K. Vishnichenko; N. O. Kalinina (1072-1081).
The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions.
Keywords: hordeivirus; TGB1 movement protein; domain phosphorylation; RNA-binding activity