The effect of phospholipid vesicles on the kinetics of reduction of cytochrome c.

The rate of reduction of cytochrome c by ascorbate and by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of ionic strength and of binding to phospholipid vesicles (liposomes). Binding of cytochrome c to liposomes, which occursat low ionic strength, decreases the rate of reduction by ascorbate by a factor of up to 100, which can be primarily explained on electrostatic grounds. In the absence of liposomes, kinetics of reduction by the neutral pteridine derivative showed no ionic strength dependence. Binding of cytochrome c to liposomes increased the rate of reduction by pteridine. An estimation of the binding constant of cytochrome c to liposomes at 0.06 M ionic strength, pH 7, is given.     

Photoinduced charge separation in liposomes containing chlorophyll a. I. Photoreduction of copper(II) by potassium ascorbate through liposome bilayer containing purified chlorophyll a.

Photosensitivity of dispersion of phosphatidylcholine bilayer liposomes containing purified chlorophyll alpha was examined. The reduction of Cu(II) in the solution outside liposomes was observed upon illumination with visible light under anaerobic condition by means of ESR. The rate of photoreduction was significantly increased by a reductant, potassium ascorbate, localized in the solution of the opposite side of the membrane. The aciton spectrum of the reduction agreed with the absorption spectrum of chlorphyll a in the dispersion. The amount of bleach chlorophyll a was negligible compared with that of reduced (Cu(II). These facts lead to the conclusion that the potoinduced redox reactions at both the membrane-solution interfaces are coupled with each other through the bilayer of each liposome. Kinetic analysis of the reactions based on a possible reaction scheme was carried out and some of the kinetic parameters were determined.     

Photoinduced charge separation in liposomes containing chlorophyll a. I. Photoreduction of copper(II) by potassium ascorbate through liposome bilayer containing purified chlorophylla a

Photosensitivity of a dispersion of phosphatidylcholine bilayer liposomes containing purified chlorophyll a was examined. The reduction of Cu(II) in the solution outside liposomes was observed upon illumination with visible light under anaerobic condition by means of ESR. The rate of photoreduction was significantly increased by a reductant, potassium ascorbate, localized in the solution of the opposite side of the membrane. The action spectrum of the reduction agreed with the absorption spectrum of chlorophyll a in the dispersion. The amount of bleached chlorophyll a was negligible compared with that of reduced Cu(II).These facts lead to the conclusion that the photoinduced redox reactions at both the membrane-solution interfaces are coupled with each other through the bilayer of each liposome.Kinetic analysis of the reactions based on a possible reaction scheme was carried out and some of the kinetic parameters were determined.     

Identification of the 120 μs phase in the decay of delayed fluorescence in spinach chloroplasts and subchloroplast particles as the intrinsic back reaction. The dependence of the level of this phase on the thylakoids internal pH

After a 500 μs laser flash a 120 μs phase in the decay of delayed fluorescence is visible under a variety of circumstances in spinach chloroplasts and subchloroplast particles enriched in Photosystem II prepared by means of digitonin. The level of this phase is high in the case of inhibition of oxygen evolution at the donor side of Photosystem II. Comparison with the results of Babcock and Sauer (1975) Biochim. Biophys. Acta 376, 329–344, indicates that their EPR signal II_f which they suppose to be due to Z⁺, the oxidized first secondary donor of Photosystem II, is well correlated with a large amplitude of our 120 μs phase. We explain our 120 μs phase by the intrinsic back reaction of the excited reaction center in the presence of Z⁺, as predicted by Van Gorkom and Donze (1973) Photochem. Photobiol. 17, 333–342. The redox state of Z⁺ is dependent on the internal pH of the thylakoids. The results on the effect of pH in the μs region are compared with those obtained in the ms region.     

Protective effect of bicarbonate against extraction of the extrinsic proteins of the water-oxidizing complex from Photosystem II membrane fragments

A protective effect of bicarbonate (BC) against extraction of the extrinsic proteins, predominantly the Mn-stabilizing protein (PsbO protein), during treatment of Photosystem II (PS II) membrane fragment from pea with 2 M urea, and at low pH (using incubation in 0.2 M glycine-HCl buffer, pH 3.5 or 0.5 M citrate buffer, pH 4.0-4.5) was detected. It was shown that the extraction of the proteins with Mw 24 kDa (PsbP protein) and 18 kDa (PsbQ protein) by the use of highly concentrated solutions of NaCl does not depend on the presence of BC in the medium. An optimal concentration of BC at which it produces the maximum protecting effect was shown to be between 1 mM and 10 mM. The addition of formate did not influence the protein extraction but it reduced the stabilizing effect of BC. Independence of the stabilizing effect on the presence of the functionally active Mn within the water-oxidizing complex indicates that the protecting effect of BC is not related to its interaction with Mn ions. The fact that there is a preferable sensitivity of the PsbO protein to the absence of BC in the medium during all the treatments makes it possible to suggest that either BC interacts directly with the PsbO protein or it binds to some other sites within PS II and this binding facilitates the preservation of the native structure of this protein.     

Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.     

Synthesis and crystal structure of a tetranuclear five-coordinated copper(II) complex with Schiff-base of N-(3-carboxylsalicylidene)-4-(2-iminoethyl)-morpholine

A tetranuclear copper(II) complex [Cu₄L₂(CH₃COO)₂(OH)₂]·6H₂O, in which L stands for the dianion of N-(3-carboxylsalicylidene)-4-(2-iminoethyl)morpholine, was synthesized and characterized by elemental analysis, IR, UV-Vis, TGA and X-ray single crystal diffraction. The crystal structure shows that the coordination unit is centrosymmetric with all the Cu(II) ions in square pyramidal coordination geometry. The coordination unit consists of two equivalent parts [Cu₂L(CH₃COO)(OH)], each containing two Cu(II) ions, a tetradentate N₂O₂ Schiff base dianion L²⁻, a CH₃COO⁻, and a OH⁻ anion. In [Cu₂L(CH₃COO)(OH)], the six coordination atoms (N₂O₄) are nearly coplanar, with Cu(1) and Cu(2) enchased in between; the phenolate oxygen and the OH⁻ oxygen as bridging atoms bind the two Cu(II) ions in close proximity; both O₄ around Cu(1) and N₂O₂ around Cu(2) form the basal plane of the coordination square pyramids. The two parts are connected by sharing two μ₃-OH⁻ oxygens and two μ₂-CH₃COO⁻ oxygens from each other, forming four edge-sharing coordination square pyramids around the four Cu(II) ions. A 3D network is formed through hydrogen bonding along a and c axis, and π-π interaction along b axis.     

Reactions of hybrid organotellurium ligands 1-(4-methoxyphenyl telluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L) and 1-ethylthio-2-[2-thienyltelluro]ethane (L) with mercury (II) bromide: formation of complexes and their decomposition

Two tellurium ligands 1-(4-methoxyphenyltelluro)-2-[3-(6-methyl-2-pyridyl)propoxy]ethane (L¹) and 1-ethylthio-2-[2-thienyltelluro]ethane (L²) have been synthesized by reacting nucleophiles [4-MeO-C₆H₄Te⁻] and [C₄H₃S-2-Te⁻] with 2-[3-(6-methyl-2-pyridyl)propoxy]ethylchloride and chloroethyl ethyl sulfide, respectively. Both the ligands react with HgBr₂ resulting in complexes of stoichiometry [HgBr₂ · L¹/L²] (1/4), which show characteristic NMR (¹H and ¹³C{¹H}). On crystallization of 1 from acetone-hexane (2:1) mixture, the cleavage of L¹ occurs resulting in 4-MeOC₆H₄HgBr (2) and [RTe⁺→HgBr₂]Br⁻ (3) (where R = -CH₂CH₂OCH₂CH₂CH₂-(2-(6-CH₃-C₅H₃N))). The 2 is characterized by X-ray diffraction on its single crystal. It is a linear molecule and is the first such system which is fully characterized structurally. The Hg-C and Hg-Br bond lengths are 2.085(6) and2.4700(7) Å. The distance of four bromine atoms (3.4041(7)-3.546(7) Å) around Hg (cis to C) is greater than the sum of van der Waal’s radii 3.30 Å. This mercury promoted cleavage is observed for an acyclic ligand of RArTe type for the first time and is unique, as there appears to be no strong intramolecular interaction to stabilize the cleavage products. The 4 on crystallization shows the cleavage of organotellurium ligand L² and formation of a unique complex [(EtS(CH₂)₂SEt)HgBr(μ-Br)Hg(Br)(μ-Br)₂Hg(Br)(μ-Br)BrHg(EtS(CH₂)₂SEt)] · 2HgBr₂ (5), which has been characterized by single crystal structure determination and ¹H and ¹³C{¹H} NMR spectra. The elemental tellurium and [C₄H₃SCH₂]₂ are the other products of dissociation as identified by NMR (proton and carbon-13). The cleavage appears to be without any transmetalation and probably first of its kind. The centrosymmetric structure of 5 is unique as it has [HgBr₃]⁻ unit, one Hg in distorted tetrahedral geometry and one in pseudo-trigonal bipyramidal one. The molecule of 5 may also be described as having [(EtSCH₂CH₂SEt)HgBr]⁺ [HgBr₃]⁻ units, which dimerize and co-crystallize with two HgBr₂ moieties. There are very weak Hg?Br interactions between co-crystallized HgBr₂ units and rest of the molecule. [Hg(3)-Br(1)/Hg(3)-Br(4) = 3.148(1)/3.216(1) Å]. The bridging Hg?Br distances, Hg(2)-Br(4)′, Hg(2)′-Br(4) and Hg(1)-Br(2), are from 2.914(1) to 3.008(1) Å.     

The site of inhibition of photosystem II by 3-(3,4-dichlorophenyl)-N-N'-dimethylurea in thylakoids of the cyanobacterium Anabaena cylindrica.

Thylakoids isolated from the cyanobacterium $\text{Anabaena}\text{cylindrica}$ exhibit Photosystem II activity. Photosynthetic electron transfer from water to ferricyanide and to 2,6-dichlorophenolindophenol is inhibited by 3-(3,4-dichlorophenyl)-N-N′-dimethyl urea. Diphenylcarbazide stimulates ferricyanide and 2,6-dichlorphenolindophenol photoreduction, whilst inhibiting oxygen evolution. Diphenylcarbazide-supported Photosystem II activity is completely insensitive to 3-(3,4-dichlorophenyl)-N-N′-dimethyl urea, indicating that the site of action of this inhibitor lies on the donor side of Photosystem II in $\text{A}\text{.}\text{cylindrica}$, before the site of electron donation by diphenylcarbazide.     

The mechanism of alkylation of DNA by 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC), a metabolite of DIC (NSC-45388). Non-involvement of diazomethane

Comparison of the nuclear magnetic resonance spectra of chemically synthesized methyl-d₁-methanol with the methanol produced in the solvolytic decompostion of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MIC) in D₂O under acidic, basic or neutral conditions indicated that no deuterium was exchanged for the hydrogens on the methyl group. Diazomethane can therefore be ruled out as an intermediate in this reaction.The methyl-d₃-guanine isolated after incubation of methyl-d₃-MIC with calfthymus DNA in vitro displayed, on chemical ionization mass spectrometry, a quasimolecular ion (MH⁺) at m/e 169, which was 3 mass units higher than the quasimolecular ion for an undeuterated 7-methylguanine standard. The major fragment ions for 7-methyl-d₃-guanine on electron impact mass spectrometry likewise were situated at positions 3 mass units higher than the fragment ions for 7-methylguanine itself.These data indicate that the methylation of biological macromolecules by MIC must involve the transfer of an intact methyl group.     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: II. Effects of High Potassium, Adenylate Cyclase Activators, and N-n-Propyl-3-(3-Hydroxyphenyl) Piperidine

The modulation of 3,4-dihydroxyphenylethylamine (dopamine, DA) synthesis and release in rabbit retina in vitro by high K+; adenylate cyclase activators such as forskolin, 2-chloroadenosine, vasoactive intestinal polypeptide (VIP); and the putative DA autoreceptor agonist N-n-propyl-3-(3-hydroxyphenyl) piperidine (3-PPP) has been investigated. Incubation of retinas in 50 mM K+ resulted in the activation of tyrosine hydroxylase (TH). Activation did not require the presence of extracellular Ca2+. K+ 50 mM also induced a Ca2+-dependent release of DA. Forskolin 50 microM stimulated TH but 100 microM 2-chloroadenosine and 650 nM VIP did not. Individually, (+)-3-PPP, (-)-3-PPP, and (+/-)-3-PPP reduced DA synthesis and increased its release. The effects of (+/-)-3-PPP were dose-dependent and did not require the presence of extracellular Ca2+. The activation of TH induced by 50 mM K+, but not that induced by 50 microM forskolin, was abolished by 100 microM (+/-)-3-PPP.     

Investigation of the insulin-like properties of zinc(II) complexes of 3-hydroxy-4-pyridinones: identification of a compound with glucose lowering effect in STZ-induced type I diabetic animals

Results from an investigation in an in vivo model of STZ-induced diabetic rats demonstrate that compound bis(1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate)zinc(II), Zn(dmpp)₂, significantly lowers the blood glucose levels of individuals, thus showing evidence of glucose lowering activity.The compound was selected from a set of eight zinc(II) complexes of 3-hydroxy-4-pyridinones with diverse lipophilicity that were prepared and characterized in our laboratory. Assessment of insulin-like activity of the complexes was firstly performed in vitro by measuring the inhibition of FFA release in isolated rat adipocytes. The results indicate that compounds bis(2-methyl-3-hydroxy-4-pyridinonate)zinc(II), Zn(mpp)₂ and Zn(dmpp)₂ display significantly higher activity than that of the respective positive control thus suggesting its selection for in vivo tests.Safety evaluation of the active zinc(II) compounds was performed in freshly isolated rat hepatocytes. The results support that cell viability is not significantly different from the control set after 1 and 2 h of incubation with both zinc(II) complexes.     

The stimulating effect of 3′,5′-(cyclic)adenosine monophosphate and lipolytic hormones on 3-O-methylglucose transport and Ca release in adipocytes and skeletal muscle of the rat

(1) In order to assess the possible role of 3′,5′-(cyclic)adenosine monophosphate (cAMP) in the control of glucose transport, the effect of the nucleotide or agents known to increase its intracellular concentration on sugar transport or ⁴⁵Ca²⁺ washout were characterized in epididymal fat pads, free fat cells and soleus muscles of the rat. (2) When added to the incubation medium, cAMP (0.1–2.0 mM) stimulated 3-O-[¹⁴C]methylglucose washout from fat pads. This effect was abolished by cytochalasin B, and additive to that induced by submaximal (10–25 μU/ml), but not by supramaximal (10 mU/ml) concentrations of insulin. (3) cAMP (2 mM) stimulated the conversion of [U-¹⁴C]glucose into CO₂ and triacylglycerols. This effect was additive to that of insulin (100 μU/ml). (4) ACTH, glucagon, adrenaline, noradrenaline and salbutamol, which are all known to increase the cAMP content of adipose tissue, stimulated the washout of 3-O-[¹⁴C]methylglucose and ⁴⁵Ca²⁺ from preloaded fat pads. The fractional losses of the two isotopes were significantly correlated (P < 0.001, r = 0.73). (5) In free fat cells, adrenaline (10⁻⁶ M) and salbutamol (10⁻⁵ M) stimulated the uptake of 3-O-[¹⁴C]methylglucose, and salbutamol (10⁻⁵ M) did not interfere with the stimulating effect of insulin (25 μU/ml) on sugar uptake. (6) In rat soleus muscles, adrenaline and salbutamol produced a dose-dependent stimulation of the washout of 3-O-[¹⁴C]methylglucose and ⁴⁵Ca²⁺. The effect of adrenaline on sugar efflux was abolished by propranolol. (7) It is concluded that the activation of the glucose transport system by insulin is unlikely to be mediated by a drop in the cellular concentration of cAMP. An increase in cAMP brought about by β-adrenoceptor agonists or lipolytic hormones may induce a mobilization of calcium ions from cellular pools into the cytoplasm, which in turn leads to the activation of the glucose transport system demonstrated in the present as well as in several earlier studies.     

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of Tyr_Z were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the Tyr_Z/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of Tyr_Z by P₆₈₀^+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn₄CaO₅ cluster in the S₂ and S₃ states could weaken the strength of the H-bond interaction between Tyr_Z^● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around Tyr_Z.     

Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroA_A.sp. A phylogenetic analysis revealed that AroA_A.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The K_M for PEP and IC₅₀ [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroA_A.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC₅₀ [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroA_A.sp. The specific activity of the wild-type AroA_A.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity.