Biochemistry (Moscow) (v.80, #6)
Different effects of identical symmetry-related mutations near the bacteriochlorophyll dimer in the photosynthetic reaction center of Rhodobacter sphaeroides by L. G. Vasilieva; T. Y. Fufina; A. G. Gabdulkhakov; V. A. Shuvalov (647-653).
In the bacterial photosynthetic reaction center (RC), asymmetric protein environment of the bacteriochlorophyll (BChl) dimer largely determines the photophysical and photochemical properties of the primary electron donor. Previously, we noticed significant differences in properties of Rhodobacter sphaeroides RCs with identical mutations in symmetry-related positions — I(M206)H and I(L177)H. The substitution I(L177)H resulted in covalent binding of BChl PA with the L-subunit, as well as in 6-coordination of BChl BB, whereas in RC I(M206)H no such changes of pigment-protein interactions were found. In addition, the yield of RC I(M206)H after its isolation from membranes was significantly lower than the yield of RC I(L177)H. This study shows that replacement of amino acid residues in the M203–M206 positions near BChls PB and BA by symmetry-related residues from the L-subunit near BChls PA and BB leads to further decrease in RC amount in the membranes associated obviously with poor assembly of the complex. Introduction of a new hydrogen bond between BChl PB and its protein environment by means of the F(M197)H mutation stabilized the mutant RC but did not affect its low yield. We suggest that the mutation I(M206)H and substitution of amino acid residues in M203–M205 positions could disturb glycolipid binding on the RC surface near BChl BA that is important for stable assembly of the complex in the membrane.
Keywords: bacterial photosynthesis; Rhodobacter sphaeroides ; photosynthetic reaction center; site-directed mutagenesis; bacteriochlorophyll; magnesium atom coordination; pigment-protein interactions
Electron transfer in photosystem I containing native and modified quinone acceptors by A. Yu. Semenov; A. A. Petrova; M. D. Mamedov; V. A. Nadtochenko (654-661).
The pigment-protein complex of photosystem I (PS I) catalyzes light-driven oxidation of plastocyanin or cytochrome c 6 and reduction of ferredoxin or flavodoxin in oxygenic photosynthetic organisms. In this review, we describe the current state of knowledge of the processes of excitation energy transfer and formation of the primary and secondary ion-radical pairs within PS I. The electron transfer reaction involving quinone cofactor in the A1 site and its role in providing asymmetry of electron transport as well as interaction with oxygen and ascorbate in PS I are discussed.
Keywords: photosystem I; reaction center; electron transfer; asymmetry; primary reactions; quinone acceptors; interaction with oxygen and ascorbate
The green alga Chlamydomonas reinhardtii as a tool for in vivo study of site-directed mutations in PsbO protein of photosystem II by A. V. Pigolev; V. V. Klimov (662-673).
The photosynthetic water oxidation in photosystem II (PS II) takes place in a special water-oxidizing complex (WOC) that consists of a catalytic center, Mn4CaO5 cluster, and also includes a group of extrinsic proteins needed for its stability. The most important of these is PsbO, which binds to the donor side of PS II near the Mn cluster and is directly involved in the regulation of its stability and activity. However, the molecular mechanism of PsbO involvement in photosynthetic water oxidation remains unclear. One of the main approaches to solving this problem is site-directed mutagenesis. Until recently, the effect of mutations in PsbO in vivo has been studied only in cyanobacteria (prokaryotes). In eukaryotic organisms, such studies (site-directed mutagenesis of PsbO) have not been carried out, though it is known that the role of PsbO protein in plants and cyanobacteria may be different. In this review, we consider the possibility of using for this purpose the unicellular green alga Chlamydomonas reinhardtii, a eukaryotic organism with a set of extrinsic proteins of the WOC similar to that of the higher plants. However, in contrast to higher plants, the ΔpsbO mutant of C. reinhardtii is viable. Another reason to use this alga is that the ΔpsbO strain of C. reinhardtii grown in the dark (heterotrophically) is able to build the minimal photochemically active complex of PS II, allowing investigation of the role of individual amino acid substitutions in PsbO in vivo without damaging PS II due to photoinactivation.
Keywords: photosystem II; water-oxidizing complex; PsbO protein; Chlamydomonas reinhardtii ; site-directed mutagenesis
Carbonic anhydrases in photosynthetic cells of higher plants by N. N. Rudenko; L. K. Ignatova; T. P. Fedorchuk; B. N. Ivanov (674-687).
This review presents information about carbonic anhydrases, enzymes catalyzing the reversible hydration of carbon dioxide in aqueous solutions. The families of carbonic anhydrases are described, and data concerning the presence of their representatives in organisms of different classes, and especially in the higher plants, are considered. Proven and hypothetical functions of carbonic anhydrases in living organisms are listed. Particular attention is given to those functions of the enzyme that are relevant to photosynthetic reactions. These functions in algae are briefly described. Data about probable functions of carbonic anhydrases in plasma membrane, mitochondria, and chloroplast stroma of higher plants are discussed. Update concerning carbonic anhydrases in chloroplast thylakoids of higher plants, i.e. their quantity and possible participation in photosynthetic reactions, is given in detail.
Keywords: carbonic anhydrase; carbonic anhydrase families; plants; photosynthesis; chloroplasts; thylakoids
ESR — A retinal protein with unusual properties from Exiguobacterium sibiricum by L. E. Petrovskaya; S. P. Balashov; E. P. Lukashev; E. S. Imasheva; I. Yu. Gushchin; A. K. Dioumaev; A. B. Rubin; D. A. Dolgikh; V. I. Gordeliy; J. K. Lanyi; M. P. Kirpichnikov (688-700).
This review covers the properties of a retinal protein (ESR) from the psychrotrophic bacterium Exiguobacterium sibiricum that functions as a light-driven proton pump. The presence of a lysine residue at the position corresponding to intramolecular proton donor for the Schiff base represents a unique structural feature of ESR. We have shown that Lys96 successfully facilitates delivery of protons from the cytoplasmic surface to the Schiff base, thus acting as a proton donor in ESR. Since proton uptake during the photocycle precedes Schiff base reprotonation, we conclude that this residue is initially in the uncharged state and acquires a proton for a short time after Schiff base deprotonation and M intermediate formation. Involvement of Lys as a proton donor distinguishes ESR from the related retinal proteins — bacteriorhodopsin (BR), proteorhodopsin (PR), and xanthorhodopsin (XR), in which the donor function is performed by residues with a carboxyl side chain. Like other eubacterial proton pumps (PR and XR), ESR contains a histidine residue interacting with the proton acceptor Asp85. In contrast to PR, this interaction leads to shift of the acceptor’s pK a to more acidic pH, thus providing its ability to function over a wide pH range. The presence of a strong H-bond between Asp85 and His57, the structure of the proton-conducting pathways from cytoplasmic surface to the Schiff base and to extracellular surface, and other properties of ESR were demonstrated by solving its three-dimensional structure, which revealed several differences from known structures of BR and XR. The structure of ESR, its photocycle, and proton transfer reactions are discussed in comparison with homologous retinal proteins.
Keywords: retinal protein; proteorhodopsin; Exiguobacterium sibiricum ; Schiff base; proton acceptor; proton donor; photocycle
Bioluminescence assay for cell viability by G. Yu. Lomakina; Yu. A. Modestova; N. N. Ugarova (701-713).
Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin–luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.
Keywords: bioluminescence; firefly luciferase; ATP; intracellular ATP; cell lysis; extracellular ATP; lyophilized vaccines
Coelenterazine-dependent luciferases by S. V. Markova; E. S. Vysotski (714-732).
Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature — the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.
Keywords: bioluminescence; luciferase; luciferin; coelenterazine
Photobiosensors containing luminescent bacteria by A. D. Ismailov; L. E. Aleskerova (733-744).
The scientific basis for producing luminescent biosensors containing free and immobilized luminescent bacteria is discussed. Modern technologies for engineering target objects, procedures used to immobilize bacteria in different carriers, as well as procedures for integral and specific biodetection of toxins are presented. Data regarding generation and application of biomonitoring for ecotoxicants derived from natural and genetically engineered photobacterial strains are analyzed. Special attention is given to immobilization of photobacteria in polyvinyl alcohol-containing cryogel. The main physicochemical, biochemical, and technological parameters for stabilizing luminescence in immobilized bacteria are described. Results of the application of immobilized photobacterial preparations both during discrete and continuous biomonitoring for different classes of ecotoxicants are presented.
Keywords: bioluminescence; biomonitoring; luminescent bacteria; biosensors; immobilization; gels; polyvinyl alcohol
Photodynamic inactivation of gramicidin channels in bilayer lipid membranes: Protective efficacy of singlet oxygen quenchers depends on photosensitizer location by T. I. Rokitskaya; A. M. Firsov; E. A. Kotova; Y. N. Antonenko (745-751).
The impact of double bonds in fatty acyl tails of unsaturated lipids on the photodynamic inactivation of ion channels formed by the pentadecapeptide gramicidin A in a planar bilayer lipid membrane was studied. The presence of unsaturated acyl tails protected gramicidin A against photodynamic inactivation, with efficacy depending on the depth of a photosensitizer in the membrane. The protective effect of double bonds was maximal with membrane-embedded chlorin e6-monoethylenediamine monoamide dimethyl ester, and minimal — in the case of water-soluble tri-sulfonated aluminum phthalocyanine (AlPcS3) known to reside at the membrane surface. By contrast, the protective effect of the hydrophilic singlet oxygen scavenger ascorbate was maximal for AlPcS3 and minimal for amide of chlorin e6 dimethyl ester. The depth of photosensitizer position in the lipid bilayer was estimated from the quenching of photosensitizer fluorescence by iodide. Thus, the protective effect of a singlet oxygen scavenger against photodynamic inactivation of the membrane-inserted peptide is enhanced upon location of the photosensitizer and scavenger molecules in close vicinity to each other.
Keywords: photodynamic action; gramicidin A; photosensitizer; unsaturated lipids; ascorbate; singlet oxygen; bilayer lipid membrane
Development of bacteriochlorophyll a-based near-infrared photosensitizers conjugated to gold nanoparticles for photodynamic therapy of cancer by I. V. Pantiushenko; P. G. Rudakovskaya; A. V. Starovoytova; A. A. Mikhaylovskaya; M. A. Abakumov; M. A. Kaplan; A. A. Tsygankov; A. G. Majouga; M. A. Grin; A. F. Mironov (752-762).
We report the synthesis and characterization of a new sulfur-containing derivative of bacteriochlorophyll a. The latter was isolated from biomass of the nonsulfur purple bacterium Rhodobacter capsulatus strain B10. The developed photosensitizer is N-aminobacteriopurpurinimide with an exocyclic amino group acylated with a lipoic acid moiety, which is a biogenic substance that acts as a cofactor of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes in the body. The disulfide moiety of lipoic acid confers the compound aurophilicity, thus allowing its conjugation with gold nanoparticles (NP-Au) via S—Au bonds. The shape and the size of the resulting nanoconjugate with immobilized photosensitizer (PS—Au) were assessed by dynamic light scattering and transmission electron microscopy. The conjugated nanoparticles are spherical with hydrodynamic diameter of 100–110 nm. The PS—Au conjugate absorbs light at 824 nm and emits strong fluorescence at 830 nm, which allowed in vivo study of its dynamic biodistribution in rats bearing sarcoma M-1. Compared to the free photosensitizer, PS loaded on the gold nanoparticles (PS—Au) showed extended circulation time in the blood and enhanced tumor uptake due to nonspecific passive targeting when the drug accumulates in tumor sites through the leaky tumor neovasculature and does not return to the circulation.
Keywords: bacteriochlorophyll a ; bacteriopurpurinimide; photosensitizers; photodynamic therapy; lipoic acid; gold nanoparticles; sarcoma M-1
On the mechanism of erythrocyte hemolysis induced by photooxidized psoralen by E. V. Nevezhin; N. V. Vlasova; I. A. Pyatnitskiy; E. P. Lysenko; M. V. Malakhov (763-768).
Contemporary concepts on a possible mechanism of erythrocyte hemolysis induced by photooxidized psoralen — the medicinal photosensitizing furocoumarin — are reviewed. The hypothesis on the mechanochemical mechanism of hemolysis is considered in view of recent data on photoinduced aggregation in photooxidized psoralen solutions. Appropriate chemical structures of photoproduct hemolysins and aggregating photoproducts are discussed.
Keywords: psoralen; photooxidized psoralen; photoproducts; erythrocyte hemolysis; photoinduced aggregation; chemical regulation of hemolysis
Effect of laser optoperforation of the zona pellucida on mouse embryo development in vitro by E. O. Zakharchenko; A. D. Zalessky; A. A. Osychenko; A. S. Krivokharchenko; A. K. Shakhbazyan; A. V. Ryabova; V. A. Nadtochenko (769-775).
The effect of laser optical perforation of the zona pellucida on the viability and development of mouse embryos has been studied. Operations of zona pellucida thinning and single or double perforation were carried out on 2-cell embryo, morula, and blastocyst stages with a laser pulse (wavelength 1.48 µm, pulse duration 2 ms). Embryo development up to the blastocyst stage and hatching efficiency were statistically analyzed. It was found that 2-cell or morula stage embryo zona pellucida thinning or single perforation did not affect development to the blastocyst stage and number of hatched embryos, but it accelerated embryo hatching compared to control groups one day earlier in vitro. Double optoperforation on 2-cell embryo or morula stage did not significantly affect development to the blastocyst stage, but it strongly decreased the number of hatched embryos. Also, zona pellucida perforation at the blastocyst stage had a negative effect: hatching did not occur after this manipulation. Blastocyst cell number calculation after single zona pellucida perforation at 2-cell and morula stages showed that cell number of hatching or hatched blastocysts did not differ from the same control groups. This fact points out that the laser single optoperforation method is a useful and safe experimental tool that allows further manipulations within the zona pellucida.
Keywords: biophotonics; photobiology; laser optoperforation; embryo; laser hatching
Participation of intracellular and extracellular pH changes in photosynthetic response development induced by variation potential in pumpkin seedlings by O. N. Sherstneva; V. A. Vodeneev; L. A. Katicheva; L. M. Surova; V. S. Sukhov (776-784).
Electrical signals presented in plants by action potential and by variation potential (VP) can induce a reversible inactivation of photosynthesis. Changes in the intracellular and extracellular pH during VP generation are a potential mechanism of photosynthetic response induction; however, this hypothesis requires additional experimental investigation. The purpose of the present work was to analyze the influence of pH changes on induction of the photosynthetic response in pumpkin. It was shown that a burning of the cotyledon induced VP propagation into true leaves of pumpkin seedlings inducing a decrease in the photosynthetic CO2 assimilation and an increase in non-photochemical quenching of fluorescence, whereas respiration was activated insignificantly. The photosynthetic response magnitude depended linearly on the VP amplitude. The intracellular and extracellular concentrations of protons were analyzed using pH-sensitive fluorescent probes, and the VP generation was shown to be accompanied by apoplast alkalization (0.4 pH unit) and cytoplasm acidification (0.3 pH unit). The influence of changes in the incubation medium pH on the non-photochemical quenching of fluorescence of isolated chloroplasts was also investigated. It was found that acidification of the medium stimulated the nonphotochemical quenching, and the magnitude of this increase depended on the decrease in pH. Our results confirm the contribution of changes in intracellular and extracellular pH to induction of the photosynthetic response caused by VP. Possible mechanisms of the influence of pH changes on photosynthesis are discussed.
Keywords: Cucurbita pepo ; pH; CO2 assimilation; variation potential; non-photochemical quenching; chloroplasts
Photoinduced changes in subcellular structures of the retinal pigment epithelium from the Japanese quail Coturnix japonica by P. P. Zak; N. B. Serezhnikova; L. S. Pogodina; N. N. Trofimova; T. S. Gur’eva; O. A. Dadasheva (785-789).
Fifteen-week-old sexually mature female Japanese quails (Coturnix japonica) grown under various lighting conditions were used in the study. It was found that the number of mitochondria and phagosomes was increased by 1.5-fold in the retinal pigment epithelium from birds reared for 95 days under blue light (440–470 nm) vs. reduced blue light component conditions. Also, it was found that egg production was increased by 15% in birds reared under blue light compared to other lightning conditions. Thus, we concluded that blue light conditions resulted in elevating metabolic activity and accelerating pace of life in Japanese quails. It is assumed that the blue light-induced effects are probably due to inhibition of melatonin synthesis.
Keywords: retinal pigment epithelium; blue light; mitochondria; Japanese quail; electron microscopy
Expression of neuronal and signaling proteins in penumbra around a photothrombotic infarction core in rat cerebral cortex by S. V. Demyanenko; S. N. Panchenko; A. B. Uzdensky (790-799).
Photodynamic impact on animal cerebral cortex using water-soluble Bengal Rose as a photosensitizer, which does not cross the blood-brain barrier and remains in blood vessels, induces platelet aggregation, vessel occlusion, and brain tissue infarction. This reproduces ischemic stroke. Irreversible cell damage within the infarction core propagates to adjacent tissue and forms a transition zone — the penumbra. Tissue necrosis in the infarction core is too fast (minutes) to be prevented, but much slower penumbral injury (hours) can be limited. We studied the changes in morphology and protein expression profile in penumbra 1 h after local photothrombotic infarction induced by laser irradiation of the cerebral cortex after Bengal Rose administration. Morphological study using standard hematoxylin/eosin staining showed a 3-mm infarct core surrounded by 1.5–2.0 mm penumbra. Morphological changes in the penumbra were lesser and decreased towards its periphery. Antibody microarrays against 224 neuronal and signaling proteins were used for proteomic study. The observed upregulation of penumbra proteins involved in maintaining neurite integrity and guidance (NAV3, MAP1, CRMP2, PMP22); intercellular interactions (N-cadherin); synaptic transmission (glutamate decarboxylase, tryptophan hydroxylase, Munc-18-1, Munc-18-3, and synphilin-1); mitochondria quality control and mitophagy (PINK1 and Parkin); ubiquitin-mediated proteolysis and tissue clearance (UCHL1, PINK1, Parkin, synphilin-1); and signaling proteins (PKBα and ERK5) could be associated with tissue recovery. Downregulation of PKC, PKCβ1/2, and TDP-43 could also reduce tissue injury. These changes in expression of some neuronal proteins were directed mainly to protection and tissue recovery in the penumbra. Some upregulated proteins might serve as markers of protection processes in a penumbra.
Keywords: stroke; neurodegeneration; penumbra; proteomics