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Vol 88, No 10 (2023)
Articles
Lel A. Drachev and the direct electrometric method
Abstract
In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell’ s chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.
Biohimiâ. 2023;88(10):1717-1730
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Generation of electric potential difference by chromatophores from photosynthetic bacteria in the presence of trehalose under continuous illumination
Abstract
Measurement of the electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides, associated with the surface of a nitrocellulose membrane filter (MF), impregnated with phospholipid solution in decane, as well as immersed inside it, in the presence of exogenous mediators and disaccharide trehalose, demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The ascorbate/N,N,N′N′-tetramethyl-p-phenylenediamine pair and ubiquinone-0 were used as mediators (electron donor and acceptor), respectively. Although the stabilization of photoelectric responses under long-term continuous illumination was observed in both cases of immobilization of chromatophores, only when the samples were immersed inside the MF, the functional activity of the reaction centers was maintained for a month when they were stored in the dark at room temperature. Stable photoresponses are probably associated with the preservation of the integrity of chromatophore proteins inside MF pores. The stabilizing effect of the bioprotector trehalose may be due to both its effect on RC proteins and on the phospholipid bilayer membrane. The results obtained will expand modern ideas about the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into an electrochemical form.
Biohimiâ. 2023;88(10):1731-1741
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Electron transport in chloroplasts: regulation and alternative pathways of electron transfer
Abstract
This work represents an overview of electron transport regulation in chloroplasts as considered in the context of structure-function organization of photosynthetic apparatus in plants. A basic focus of the article is concentrated on a bifurcated oxidation of plastoquinol by the cytochrome b6f complex, which represents the rate-limiting step of electron transfer between photosystems 2 and 1. Electron transport along the chains of the noncyclic, cyclic and pseudocyclic electron flow, their relationships to generation of the trans-thylakoid difference in electrochemical potentials of protons in chloroplasts, and the pH-dependent mechanisms of regulation of the cytochrome b6f complex, are considered. Redox reactions with the participation of molecular oxygen and ascorbate, the alternative mediators of electron transport in chloroplasts, have also been discussed.
Biohimiâ. 2023;88(10):1742-1760
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Electrical signals of the plasma membrane and their effect on chlorophyll fluorescence in chara chloroplasts in vivo
Abstract
Action potentials of plant cells are involved in regulation of many cell processes, such as photosynthesis and cytoplasmic streaming. Excitable cells of characean algae submerged in media with elevated K+ content are able to generate hyperpolarizing electrical responses. This active response of plasma membrane arises upon the passage of inward electric current comparable in extent to natural currents circulating in illuminated Chara internodes. It remains currently unknown whether the hyperpolarizing electrical signals in Chara affect the photosynthetic activity. Here we show that the negative shift of cell membrane potential, which causes the K+ influx into the cytoplasm, is accompanied by a delayed decrease in the effective chlorophyll fluorescence yield (F′) and maximal yield (Fm′) under low background light (12.5 µmol m-2 s-1). The transient changes in F′ and Fm′ were evident under illumination only, which indicates their close relation to photosynthetic energy conversion in chloroplasts. The passage of inward current caused an increase in pH at the cell surface (pHo), which reflects a high H+/OH- conductance of the plasmalemma and points to the decrease in cytoplasmic pH due to H+ entry into the cell. The shifts in pHo arising in response to the first hyperpolarizing pulse disappeared upon repeated stimulations, thus indicating the long-term inactivation of plasmalemmal H+/OH- conductance. Despite the suppression of plasmalemmal H+ fluxes, the hyperpolarizing responses and the analyzed chlorophyll fluorescence changes did not disappear. The results indicate the participation of K+ flows between the outer medium, cytoplasm, and stroma in chloroplast functional changes that are reflected by the dynamics of F′ and Fm′.
Biohimiâ. 2023;88(10):1761-1774
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Ion channels in electrical signaling in higher plants
Abstract
Electrical signals (ESs) of plants occurring under the action of various external factors play an important role in adaptation to changing environmental conditions. The generation of ESs in higher plant cells is associated with the activation of Ca2+, K+, and anion fluxes, as well as changes in the activity of plasma membrane H+-ATPase. In the present review, the molecular nature of ion channels contributing to ESs transmission in higher plants is analyzed based on the comparison of data from molecular-genetic and electrophysiological studies. Based on such characteristics of ion channels as selectivity, activation mechanism, and intracellular and tissue localization, those ion channels that meet the requirements for potential participants of ESs generation were selected from a wide variety of ion channels in higher plants. Analysis of the data of experimental studies performed on mutants with suppressed or enhanced expression of a certain channel gene revealed those channels whose activation contributes to ESs formation. The channels responsible for Ca2+ flux during ESs generation include channels of GLR family, for K+ flux - GORK, for anions - MSL. Consideration of the prospects for further studies suggests the need to combine electrophysiological and genetic approaches along with the analysis of ion concentrations in intact plants within a single study.
Biohimiâ. 2023;88(10):1775-1799
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Changes in activity of H+-ATPase of plasma membrane as a link between forming electrical signals and induction of photosynthetic responses in higher plants
Abstract
Action of numerous adverse environmental factors on higher plants is spatially-heterogenous; it means that induction of a systemic adaptive response requires generation and propagation stress signals. Electrical signals (ESs) induced by local action of stressors include action potential, variation potential, and system potential and participate in forming fast physiological changes at the level of the whole plant, including photosynthetic responses. Generation of these ESs is accompanied by changes in activity of H+-ATPase which is the main system of electrogenic proton transport across the plasma membrane. Literature data show that the changes in H+-ATPase activity and related changes in intra- and extracellular pH play a key role in ESs-induced photosynthetic inactivation in non-irritated parts of plants. This inactivation is caused by both suppression of CO2 flux into mesophyll cells in leaves, which can be induced by the apoplast alkalization and, probably, cytoplasm acidification, and direct influence of acidification of stroma and lumen of chloroplasts on photosynthetic light and, probably, dark reactions. Result of the ESs-induced photosynthetic inactivation is increasing tolerance of photosynthetic machinery to action of adverse factors and probability of survive of plants.
Biohimiâ. 2023;88(10):1800-1817
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Membrane potential generation by cytochrome bd
Abstract
This treatise gives an overview of current thinking on the mechanism of generation of a transmembrane electric potential difference (Δψ) during the catalytic cycle of a bd-type triheme terminal quinol oxidase. It is assumed that the main contribution to the Δψ formation is made by the movement of H+ across the membrane along the intraprotein hydrophilic proton-conducting pathway from the cytoplasm to the active site for oxygen reduction of this bacterial enzyme.
Biohimiâ. 2023;88(10):1818-1828
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Investigation of the mechanism of membrane potential generation by heme-copper respiratory oxidases in real time mode
Abstract
Heme-copper respiratory oxidases are highly efficient molecular machines. These membrane enzymes catalyze the final stage of cellular respiration of eukaryotes and many prokaryotes: the transfer of electrons from cytochromes or quinols to molecular oxygen and the reduction of O2 to water. The free energy released in this redox reaction is converted by heme-copper respiratory oxidases into a transmembrane gradient of the electrochemical potential of hydrogen ions (ΔµH+). Heme-copper respiratory oxidases have a unique mechanism for generation ΔµH+ - a redox-coupled proton pump. The use of a combination of a direct electrometric method for measuring the kinetics of membrane potential generation with approaches and methods of prestationary kinetics and directed mutagenesis in the study of heme-copper oxidases makes it possible to obtain unique information about the movement of protons inside the protein in real time. The review summarizes the results of the use of the permitted time.
Biohimiâ. 2023;88(10):1829-1846
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Similarity and difference in the photochemistry of type I and II rhodopsins
Abstract
The diversity of retinal-containing proteins in nature is extremely large. The fundamental similarity of the structure and photochemical properties unites them into one family. However, there is still a debate about the origin of retinal-containing proteins: divergent or convergent evolution? In this review, based on the results of our own and literature data, a comparative analysis of the similarities and differences in the photoconversion of rhodopsin types I and II is carried out. The results of experimental studies of direct and reverse photoreactions of rhodopsin types I (bacteriorhodopsin) and II (visual rhodopsin) in the femto- and picosecond time interval, photo-reversible reaction of rhodopsin type II (octopus rhodopsin), photovoltaic reactions of rhodopsin types I and II, as well as quantum chemical calculations of forward photoreactions of bacteriorhodopsin and visual rhodopsin are presented. The question of the probable convergent evolution of rhodopsin types I and II is discussed.
Biohimiâ. 2023;88(10):1847-1866
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Features of proton transport mechanism in ESR, a retinal protein from Exiguobacterium sibiricum
Abstract
Retinal-containing photosensitive proteins, rhodopsins, have been detected in many microorganisms. The interest in them is largely explained by their role in storing light energy and photoregulation in microorganisms and the prospects for use in optogenetics in order to control the activity of neurons, including for the treatment of various diseases. One of the representatives of microbial rhodopsins is ESR, a retinal protein from Exiguobacterium sibiricum. The presence of a lysine residue (Lys96) as a proton donor for the Schiff base distinguishes ESR from homologous proteins. This feature, along with the hydrogen bonding of the proton acceptor Asp85 with the His57 residue, determines its functional characteristics as a proton pump. The review examines the results of ESR studies conducted using various methods, including the method of direct electrometry. Comparison of the obtained data with the results of spatial structure determination and with other retinal proteins allows drawing conclusions about the mechanisms of transport of hydrogen ions in the ESR molecule and similar retinal proteins.
Biohimiâ. 2023;88(10):1867-1879
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Channelrhodopsins: from phototaxis to optogenetics
Abstract
Channelrhodopsins stand out among other retinal proteins because of their capacity to generate passive ionic currents following photoactivation. Owing to that, channelrhodopsins are widely used in neuroscience and cardiology as instruments for optogenetic manipulation of the activity of excitable cells. Photocurrents generated by channelrhodopsins were first discovered in the cells of green algae in the 1970s. In this review we describe this discovery and discuss the current state of research in this field.
Biohimiâ. 2023;88(10):1880-1897
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Retinal-based anion pump from the cyanobacterium Tolypothrix campylonemoides
Abstract
In this work, a functional characterization of TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was obtained. Analysis of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid from the motif of the known halorodopsin chloride pump TSA. The TcaR protein was expressed in E. coli, purified and incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one membrane surface, as well as by fluorescence using voltage-dependent dye oxonol VI. We have shown that TcaR rhodopsin functions as a powerful anion pump. Our results show that the novel microbial anion transporter TcaR deserves deeper study and may be of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.
Biohimiâ. 2023;88(10):1898-1907
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Photochemical reactions of chlorophyll tetramer in water-soluble chlorophyll-binding protein BoWSCP
Abstract
The absorption dynamics of chlorophyll a in a symmetric tetrameric complex of the water-soluble chlorophyll-binding protein BoWSCP was measured by the broadband femtosecond laser pump-probe spectroscopy within the range from 400 to 780 nm with a time resolution of 20 f-200 ps. When BoWSCP was excited in the region of the Soret band at a wavelength of 430 nm, a nonradiative intramolecular conversion S3 → S1 was observed with a characteristic time of 83 ± 9 fs. When the complex was excited in the region of the Qy band at a wavelength of 670 nm, a relaxation transition between two excitonic states of the chlorophyll dimer was observed with a time of 105 ± 10 fs. Absorption spectra of excited singlet states S1 and S3 of chlorophyll a were obtained. It has been demonstrated that the delocalization of the excited state between exciton-coupled Chl molecules in the BoWSCP tetramer changes in time and depends on the excitation energy. Upon excitation of BoWSCP, an ultrafast photochemical reaction is observed in the Soret region, apparently due to the reduction of tryptophan in the vicinity of chlorophyll.
Biohimiâ. 2023;88(10):1908-1925
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Mitochondrial network: electric cable and more
Abstract
Mitochondria in a cell can unite and organize complex extended structures that extend over the entire cellular volume, ensuring a uniform supply of energy to cellular compartments with ATP synthesized in the mitochondria. In accordance with the chemiosmotic concept, the energy of oxidation of respiratory substrates is largely stored in the form of an electrical potential difference on the inner membrane of mitochondria. The theory of the functioning of extended mitochondrial structures as intracellular electrical wires suggests that mitochondria provide the fast delivery of electrical energy through the cellular volume, with following use of this energy for ATP synthesis, thereby speeding up the process of ATP delivery compared to the rather slow diffusion of ATP in the cell. The analytical review gives the history of the cable theory, solved and unsolved critical problems, the lability of the mitochondrial network and the role of oxidative stress in this process. In addition to the already proven functioning of extended mitochondrial structures as electrical cables, a few of additional functions are proposed, in particular, the hypothesis is put forward that mitochondrial networks provide a uniform distribution of redox potential throughout the cell, which can change in pathological conditions during fragmentation of the mitochondrial reticulum. A few of pathologies accompanied by a violation of the redox status and the participation of mitochondria in them are considered.
Biohimiâ. 2023;88(10):1926-1939
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Regular articles
The role and molecular mechanisms of alternative splicing of Th2-cytokines IL-4 and IL-5 in atopic bronchial asthma
Abstract
Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the respiratory tract. Allergic asthma is the most common (up to 80% of cases) phenotype developing through Th2-dependent mechanisms involving cytokines: IL-4, IL-5, IL-9 and IL-13. The genes encoding Th2-cytokines have a mosaic structure (encode exons and introns). Therefore, several mature mRNA transcripts and protein isoforms can be derived from a single mRNA precursor through alternative splicing, and they may contribute to BA pathogenesis. Analysis of published studies and databases revealed the existence of alternative mRNA transcripts for IL-4, IL-5, and IL-13. Alternative transcripts of IL-4 and IL-5 carry open reading frames and therefore can encode functional proteins. It was shown that not only alternative mRNA transcripts are exist for IL-4, but alternative protein isoforms, as well. Natural protein isoform IL-4δ2 lacking part encoded by exon-2 was identified. Similarly, alternative mRNA transcript omitting exon-2 (IL-5δ2) was also identified for IL-5. In this review, we summarize current knowledge about identified alternative mRNA transcripts and protein isoforms of Th2-cytokinins, first of all IL-4 and IL-5. We have analyzed biological properties of alternative variants of these cytokines, their possible role in the allergic asthma pathogenesis, and considered their diagnostic and therapeutic potential.
Biohimiâ. 2023;88(10):1940-1956
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Markers of end-stage renal disease progression: beyond the GFR
Abstract
Chronic kidney disease may progress to end-stage renal disease (ESRD) with a high risk of morbidity and mortality. ESRD requires immediate initiation of therapy or a decision on dialysis or kidney transplantation. Therefore, timely diagnosis of pathology progression is critical for many patients. ESRD is associated with pathological changes, including inflammation, fibrosis, endocrine disorders and following epigenetic changes in various cells, all these alterations could serve as markers for ESRD identification. This review summarizes conventional and promising biomarkers of ESRD, which can be evaluated in kidney tissue, blood, or urine. Some of them are narrowly specific to a particular pathology, while others are more versatile. We suggested several universal inflammatory, fibrotic, hormonal, and epigenetic markers indicative of severe deterioration of renal function and progression of ESRD for improvement of ESRD diagnostics.
Biohimiâ. 2023;88(10):1957-1984
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Catalytic and electrocatalytic mechanisms of cytochromes P450 in the development of biosensors and bioreactors
Abstract
Cytochromes P450 are unique family of isozymes, discovered in all kingdoms of living species (in animals, bacteria, plants, fungi, archaea). The main functional role of the cytochromes P450 is biotransformation of exogenous and endogenous compounds. This review is highlighted problem of enchasing effectivity of electrocatalysis of cytochromes P450, enzymes that have unique capabilities both for biosensors’ design and for biotechnological application. In this review paper, we summarize main methods and modern trends based on biochemical mechanism of cytochromes P450 for development of reconstructing and electrochemical catalytic systems for practical application of these enzymes.
Biohimiâ. 2023;88(10):1985-2001
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A novel two-domain laccase with a middle redox potential: physicochemical and structural properties
Abstract
The gene for a previously unexplored two-domain laccase was identified in the genome of the actinobacterium Streptomyces carpinensis VKM Ac-1300. A two-domain laccase, named ScaSL, was produced in a heterologous expression system (Escherichia coli strain M15 [pREP4]). The enzyme was purified to homogeneity using affinity chromatography. ScaSL laccase, like most two-domain laccases, exhibited activity in the homotrimer form. However, unlike most two-domain laccases, it was also active in multimeric forms. The enzyme exhibited maximum activity at 80°C and was thermally stable. The half-inactivation time of ScaSL at 80°C was 40 min. Laccase oxidized the non-phenolic organic compound ABTS at a maximum rate at pH 4.7; oxidized the phenolic compound 2,6-dimethoxyphenol at a maximum rate at pH 7.5. Laccase stability was observed in the pH range 9-11. At pH 7.5, laccase was weakly inhibited by sodium azide, sodium fluoride, and sodium chloride; at pH 4.5, laccase was completely inhibited by 100 mM sodium azide. Km and kcat of the enzyme for ABTS are 0.1 mM and 20 s-1, respectively. Km and kcat for 2,6-dimethoxyphenol are 0.84 mM and 0.36 s-1, respectively. ScaSL catalyzed the polymerization of humic acids and lignin. The redox potential of laccase was 0.472 ± 0.007 V. Thus, ScaSL laccase is the first characterized two-domain laccase with a middle redox potential. The crystal structure of ScaSL was determined with a resolution of 2.35 Å. A comparative analysis of the structures of ScaSL and other two-domain laccases suggested that the middle potential of ScaSL may be associated with conformational differences in the position of the side groups of amino acids at position 230 (in ScaSL numbering), which belong to the second coordination sphere of the copper atom of the T1 center.
Biohimiâ. 2023;88(10):2002-2013
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Proteomic approach to investigate the expression, localization, and functions of the protein product of the SOWAHD gene during granulocytic differentiation
Abstract
Cataloging human proteins, determining their level of content, cellular localization, function performed, and potential medical significance are important tasks facing the global proteomic community. At present, the localization and functions of protein products for almost half of the protein-coding genes are unknown or poorly understood. The study of the organelle proteome is a promising approach to reveal the localization and functions of human proteins. The nuclear proteome is of particular interest because many nuclear-localized proteins, such as transcription factors, perform regulatory functions that determine cell fate. According to the results of a meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated by all-trans-retinoic acid (ATRA), it was revealed that the function and localization of the protein product of the SOWAHD gene is poorly understood, in addition, there is no comprehensive information on the expression of SOWAHD at the protein level. In HL-60 cells for the protein-coding gene SOWAHD, mRNA expression was determined at the level of 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of SOWAHD protein was measured in the range of 0.27-1.25 fmol/µg of total protein. Using stable isotope pulse-chase labeling, the half-life for the protein product of the SOWAHD gene was determined to be approximately 19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the SOWAHD protein content increased during ATRA-induced granulocytic differentiation, peaking at 9 h after the addition of the inductor and followed by a decrease at later time points. The results of the study indicate for the first time the nuclear localization and involvement of the protein product of the SOWAHD gene in induced granulocytic differentiation.
Biohimiâ. 2023;88(10):2014-2031
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