Synthesis of 17α-substituted ethynylestradiols: Potential ligands for drug vectors

17α-substituted ethynylestradiols, derived from estrone, were converted to their corresponding 17α-(bromo- or iodo-propargyl)estrone intermediates. Nucleophilic substitution onto these moieties with malonate diester followed by hydrolysis and complexation with cis-Pt(Me₂en)I₂ (Me₂en = N,N-dimethylethylenediamine) gave cis-Pt(Me₂en)(2-(3-(17β-estradiol-17α-yl)-prop-2-ynyl)malonato) 7, thus demonstrating that these estrogen-derived compounds can be used to synthesize stable Pt(II) complexes. The 3-(17β-estradiol-17α-yl)-prop-2-ynyl-1-sulfanylethylthiol 23 was also prepared.     

Exploring the effect of chain length of bridging ligands in coordination complexes and polymers derived from mixed ligand systems of pyridylnicotinamides and dicarboxylates

The supramolecular structural diversities in mixed ligand systems derived from a series of dicarboxylate anions with varying chain lengths and N-donor exo-bidentate ligand equipped with hydrogen bonding capable amide backbone with Co(II)/Zn(II) metal centers are analyzed. In this context, two complexes namely (Co(L1)₂(malonate)(H₂O)₂} (1a), {Zn(L1)₂(malonate)(H₂O)₂} (1b) and one coordination polymer namely {[Co(μ-L1)(μ-glutarate)(H₂O)] · H₂O}_n (4) (where L1 = N-(4-pyridyl)nicotinamide) have been synthesized and crystallographically characterized. The main aim of this work is to explore the effects of chain lengths of the anionic carboxylate ligands such as malonate, succinate, maleate, and glutarate, in determining the final architecture of coordination compounds based on the mixed ligands. Analyses of the structures revealed that the length of the bridging ligands have prominent effect in the formation of hierarchical structures.     

Effect of succinate on aminopyrine N-demethylase activity

Succinate stimulated the aminopyrine N-demethylase activity in the presence of rate-limiting concentrations of NADPH in the crude mitochondrial preparation, as well as in a reconstituted system containing microsomes and microsome-free mitochondria. The increase in enzyme activity was accompanied by a decrease of the apparent K_m of NADPH.The stimulating effect of succinate was counteracted by malonate, rotenone and pentachlorophenol. No significant formaldehyde oxidation was exhibited by the crude mitochondrial preparation under the conditions of N-demethylase assay.Enhancement of the N-demethylase activity by succinate is supposed to be due to a mitochondrial-microsomal interaction which may play a role in the regulation of drug metabolism.     

Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO₂ in Echinochloa and rice is lower and the peak rate of CO₂ evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO₂] N₂/[CO₂] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.

Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N₂, but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

     

Synthesis and spectroscopic studies of platinum(II) complexes of N,N′-dicyclohexylethylenediamine

The platinum(II) complexes of the formula [Pt(DCHEDA)X₂], where DCHEDA is N,N′-dicyclohexylethylenediamine and X⁻ is CL⁻, Br⁻, I⁻, 0.5C₂O₄²⁻ (oxalate), 0.5C₃H₂O₄²⁻ (malonate), 0.5C₉H₄O₆²⁻ (4-carboxyphthalate), 0.5S₂O₃²⁻ or 0.5SO₄²⁻, have been synthesized and characterized by UVVis, IR, and ¹H NMR spectral techniques. All the above complexes are non-electrolytes in DMF/H₂O, except the sulphate complex which becomes a 1:1 electrolyte after incubation for 24 h at 28 °C. The halide complexes were also studied by X-ray photoelectron spectroscopy and these data suggest that there is π-bonding from platinum to halide in these complexes. The oxalate, malonate and sulphate bind in their complexes as bidentate ligands to platinum through two oxygen atoms whereas the thiosulphate in its complex binds as a bidentate ligand to platinum through one oxygen atom and one sulphur atom.     

Identification of an Na+-Dependent Malonate Transporter of Malonomonas rubra and Its Dependence on Two Separate Genes

Two membrane proteins encoded by the malonate fermentation gene cluster of Malonomonas rubra, MadL and MadM, have been synthesized in Escherichia coli. MadL and MadM were shown to function together as a malonate transport system, whereas each protein alone was unable to catalyze malonate transport. Malonate transport by MadLM is Na⁺ dependent, and imposition of a ΔpNa⁺ markedly enhanced the rate of malonate uptake. The kinetics of malonate uptake into E. coli BL21(DE3) cells synthesizing MadLM at different pH values indicated that Hmalonate⁻ is the transported malonate species. The stimulation of malonate uptake by Na⁺ ions showed Michaelis-Menten kinetics, and a K_m for Na⁺ of 1.2 mM was determined. These results suggest that MadLM is an electroneutral Na⁺/Hmalonate⁻ symporter and that it is dependent on two separate genes.     

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis,rather than enhancing striatal damage,protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

The cannabinoid CB₂ receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington''s disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E₂ glyceryl ester (PGE₂-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE₂-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE₂-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE₂-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE₂-G aggravates the malonate toxicity.     

Laccase-Catalyzed Oxidation of Mn2+ in the Presence of Natural Mn3+ Chelators as a Novel Source of Extracellular H2O2 Production and Its Impact on Manganese Peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn²⁺ to Mn³⁺, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn³⁺ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H₂O₂ formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H₂O₂ production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn²⁺ oxidation and abiotic decomposition of these organic chelators by the resulting Mn³⁺, which leads to formation of superoxide and its subsequent reduction to H₂O₂. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn³⁺ complexes in assays containing 1 mM Mn²⁺ and 100 mM oxalate or malonate, but omitting an additional H₂O₂ source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn²⁺ oxidation in providing H₂O₂ for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Substitution of carbonate by non-physiological synergistic anion modulates the stability and iron release kinetics of serum transferrin

Serum transferrin (sTf) is a bi-lobal protein. Each lobe of sTf binds one Fe³⁺ ion in the presence of a synergistic anion. Physiologically, carbonate is the main synergistic anion but other anions such as oxalate, malonate, glycolate, maleate, glycine, etc. can substitute for carbonate in vitro. The present work provides the possible pathways by which the substitution of carbonate with oxalate affects the structural, kinetic, thermodynamic, and functional properties of blood plasma sTf. Analysis of equilibrium experiments measuring iron release and structural unfolding of carbonate and oxalate bound diferric-sTf (Fe₂sTf) as a function of pH, urea concentration, and temperature reveal that the structural and iron-centers stability of Fe₂sTf increase by substitution of carbonate with oxalate. Analysis of isothermal titration calorimetry (ITC) scans showed that the affinity of Fe³⁺ with apo-sTf is enhanced by substituting carbonate with oxalate. Analysis of kinetic and thermodynamic parameters measured for the iron release from the carbonate and oxalate bound monoferric-N-lobe of sTf (Fe_NsTf) and Fe₂sTf at pH 7.4 and pH 5.6 reveals that the substitution of carbonate with oxalate inhibits/retards the iron release via increasing the enthalpic barriers.     

Plasmalemma dicarboxylate transporter of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is involved in citrate and succinate influx and is modulated by pH and cations

Succinate and citrate transport into yeast (Saccharomyces cerevisiae) cells was studied by measuring substrate oxidation rates in the presence and in the absence of effective impermeable oxidation inhibitors O-palmitoyl-L-malate and 2-undecyl malonate. Linearity of the Dixon plot for 2-undecyl malonate suggests that this inhibitor blocked the rate-limiting step upon oxidation of both substrates, which was, most probably, transport of these substrates across the plasma membrane (due to inability of the inhibitor to penetrate into the membrane). This approach allowed fast (within 30–40 min) measurement of kinetic parameters of the transporter in individual samples without losing control over limiting conditions. In case of succinate transport, the limiting rate of succinate oxidation (V _max) depended on pH and increased monotonously from near-zero at pH 4.5 to the maximum level at pH 7.5. At pH 5.5, succinate and citrate transport was insensitive to the protonophore FCCP, being activated by Na⁺ ions and competitively inhibited by 2-undecyl malonate and K⁺ ions. Values of K _i for 2-undecyl malonate were similar for both substrates. These data suggest that citrate and succinate influx is mediated by a common plasma membrane transporter. This is not typical of fungi. At pH 6.5, Tris⁺, K⁺ and Na⁺ had no effect on succinate oxidation. In monosodium media pH increase was accompanied by a decrease of succinate K _m due to higher proportion of the dianionic form of the substrate. Atypical substrate specificity and mechanisms of functional activity of the dicarboxylate transporter in plasma membrane of S. cerevisiae are discussed.     

The effects of N-substitution of chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus

N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH₃ > CH₃CH₂ > H > CH₃CH₂CH₂ > (CH₃)₂CH > NH₂CH₂ > ClCH₂. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C₅C₁₈) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.     

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES*

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N²,N²-dimethyl (Me₂)-substituted guanine (N²,N²-Me₂G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N²,N²-Me₂G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N²,N²-Me₂G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N²,N²-Me₂G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3′-terminal dideoxycytosine (C_dd) opposite template N²,N²-Me₂G in a post-insertion position showed C_dd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal C_dd as follows: (i) flipped into the minor groove and (ii) a long pairing with N²,N²-Me₂G in which one hydrogen bond exists between the O-2 atom of C_dd and the N-1 atom of N²,N²-Me₂G, with a second water-mediated hydrogen bond between the N-3 atom of C_dd and the O-6 atom of N²,N²-Me₂G. A crystal structure of Dpo4 with dTTP opposite template N²,N²-Me₂G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N²,N²-Me₂G compared with mono-substituted N²-alkyl G adducts.     

Isolation and properties of isocitrate lyase from Lupinus seed

Isocitrate lyase (threo-d_s-isocitrate glyoxylate-lyase, EC 4.1.3.1) was purified from cotyledons of Lupinus seedlings. The final preparation showed two bands after polyacrylamide-gel electrophoresis. The optimum pH using phosphate, Tris or imidazole buffer was at pH 7.5; with triethanolamine (TRA) it was at pH 7. The enzyme required Mg²⁺ for maximal activity, and N-ethylmaleimide (NEM) inactivated the enzyme. Activity was increased by incubation with the reducing agents, glutathione (GSH), acetylcysteine (acetylcys), dithionite (Na₂S₂O₄), thioglycolate (TG) or 1,4-dithioerythritol (DTE). Na₂S₂O₄ and DTE were the most active among the tested substances and DTE prevented much of the inactivation by NEM. The apparent K_m value for isocitrate was ca 1 mM in phosphate buffer at pH 6.8 or 7.5 but was substantially lower (0.1–0.2 mM) using Tris, TRA or imidazole buffers. Glyoxylate, oxalate and malonate were competitive inhibitors of the enzyme. Synthase activity of the enzyme (i.e. formation of isocitrate from succinate and glyoxylate) was demonstrated. The K_m values for glyoxylate and succinate were 0.05 and 0.2 mM, respectively. The addition of glyoxylate to the culture medium in which Lupinus seeds germinate resulted in a reduced development of isocitrate lyase activity during germination.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1–C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K_Is of 7.3–8.7 μM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K_Is in the range of 42.8–88.6 μM. Propionate, butyrate, malonate, l-malate, pyruvate, l-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225–1267 μM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K_Is in the range of 8.6–26.9 μM. l-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K_Is of 87.7–94.0 μM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K_Is in the range of 35.1–61.6 μM). Notably the inhibition profiles of the two fungal β-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the α-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective β-CA inhibitors.     

Synthesis, spectral and crystallographic characterization of manganese(II) coordination polymers based on aliphatic dicarboxylate and flexible bis(imidazolyl) derivatives

Two structurally related flexible imidazolyl ligands, bis(N-imidazolyl)methane (L₁) and 1,4-bis(N-imidazolyl)butane (L₂) reacted with Mn(II) salts of aliphatic dicarboxylic acids resulted in the formation of a number of novel metal-organic coordination architectures. All complexes have been structurally characterized by X-ray diffraction analysis. The different coordination modes of dicarboxylate anions due to their chain length, rigidity and diimidazolyl functionality lead to a range of different coordination structures. The coordination polymers exhibit 1D single chain, 2D sheet and 3D network structures. The aliphatic dicarboxylates can adopt chelating μ₂, bridging μ₂, and chelating-bridging μ₃ coordination modes, or act as uncoordinated counter anions. The central metal ions are coordinated in N₂O₄ and N₄O₂ fashions depending on the ancillary ligands. The topology of [Mn(male)(L₁)(H₂O)₂] (1, male = maleate) gives rise to singly bridged 1D chains, whereas compound [Mn(mal)(L₁)(H₂O)] · H₂O (2, mal = malonate) exhibits 2D sheets in which the metal centers are bridged by both imidazolyl ligands and dicarboxylates. Compounds [Mn(L₁)₂(H₂O)₂](suc) · 6H₂O (3, suc = succinate) and [Mn(L₁)₂(H₂O)₂](fum) · 6H₂O (4, fum = fumarate) show doubly bridged 1D chains, and the dicarboxylate groups are not coordinated but form 2D corrugated sheets with water molecules intercalated between the cationic layers. Compound [Mn(suc)(L₂)(H₂O)₂] (5, suc = succinate) was built from very flexible succinate and 1,4-bis(N-imidazolyl)butane which yielded three-dimensional interpenetrate networks, both succinate anion and the imidazolyl ligand act as bidentate bridging.     

Syntheses and structures of four- and five-coordinate bio-related zinc complexes containing dithiolate-diamine ligands

Two new zinc complexes, namely, [{Zn(N₂H₂S₂)}₂] (3) [N₂H₂S₂²⁻ = N,N-bis(2-mercaptophenyl)ethylendiamine (2−)] and [Zn(N₂Me₂S₂)] (4) [N₂Me₂S₂²⁻ = N,N′-dimethyl-N,N′-bis(2-mercaptophenyl)ethylendiamine) (2−)] have been synthesized and structurally characterized by X-ray structure analyses. The structure of 3 consists of a bis(μ-thiolato) binuclear unit, in which each zinc center was found to reside in an N₂S₃ array between square-pyramidal and trigonal-bipyramidal environment. The two zinc centers are bridged by one of the two thiolates of an [N₂S₂] ligand. In the crystal packing, the neighboring binuclear units interact with each other by H-bonding interaction, which extends the binuclear unit into a 3D network. In contrast to 3 complex 4 is mononuclear, where each zinc center now was found to reside in an N₂S₂ distorted tetrahedral environment with a large S-Zn-S bite angle. The relevance of these compounds in biological systems is discussed. Unlike 3, the formation of hydrogen bridges in 4 is no longer possible and instead the molecular packing is determined by π-stacking between the phenyl rings.     

Evidence for Involvement of Gut-Associated Denitrifying Bacteria in Emission of Nitrous Oxide (N2O) by Earthworms Obtained from Garden and Forest Soils

Earthworms (Aporrectodea caliginosa, Lumbricus rubellus, and Octolasion lacteum) obtained from nitrous oxide (N₂O)-emitting garden soils emitted 0.14 to 0.87 nmol of N₂O h⁻¹ g (fresh weight)⁻¹ under in vivo conditions. L. rubellus obtained from N₂O-emitting forest soil also emitted N₂O, which confirmed previous observations (G. R. Karsten and H. L. Drake, Appl. Environ. Microbiol. 63:1878–1882, 1997). In contrast, commercially obtained Lumbricus terrestris did not emit N₂O; however, such worms emitted N₂O when they were fed (i.e., preincubated in) garden soils. A. caliginosa, L. rubellus, and O. lacteum substantially increased the rates of N₂O emission of garden soil columns and microcosms. Extrapolation of the data to in situ conditions indicated that N₂O emission by earthworms accounted for approximately 33% of the N₂O emitted by garden soils. In vivo emission of N₂O by earthworms obtained from both garden and forest soils was greatly stimulated when worms were moistened with sterile solutions of nitrate or nitrite; in contrast, ammonium did not stimulate in vivo emission of N₂O. In the presence of nitrate, acetylene increased the N₂O emission rates of earthworms; in contrast, in the presence of nitrite, acetylene had little or no effect on emission of N₂O. In vivo emission of N₂O decreased by 80% when earthworms were preincubated in soil supplemented with streptomycin and tetracycline. On a fresh weight basis, the rates of N₂O emission of dissected earthworm gut sections were substantially higher than the rates of N₂O emission of dissected worms lacking gut sections, indicating that N₂O production occurred in the gut rather than on the worm surface. In contrast to living earthworms and gut sections that produced N₂O under oxic conditions (i.e., in the presence of air), fresh casts (feces) from N₂O-emitting earthworms produced N₂O only under anoxic conditions. Collectively, these results indicate that gut-associated denitrifying bacteria are responsible for the in vivo emission of N₂O by earthworms and contribute to the N₂O that is emitted from certain terrestrial ecosystems.     

Transition metal derivatives of 1,10-phenanthroline-5,6-dione: Controlled growth of coordination polynuclear derivatives

The synthesis and the characterization of several mono- and polymetallic derivatives of 1,10-phenanthroline-5,6,-dione (1) are presented.The reaction of 1 with M(CO)₆ (M = Cr, Mo) gives compounds of general formula M(O,O′-C₁₂H₆N₂O₂)₃, M = Cr (2), Mo (3).Compound 3 is also obtained starting from Mo(η⁶-CH₃C₆H₅)₂, whereas the reaction of Cr(η⁶-CH₃C₆H₅)₂ with 1 affords the ionic derivative [Cr(η⁶-CH₃C₆H₅)₂][C₁₂H₆N₂O₂] (4), which has been studied by EPR spectroscopy and DFT calculations.FeCl₂(N,N′-C₁₂H₆N₂O₂)₂ (6), is obtained by thermal decomposition of [Fe(N,N′-C₁₂H₆N₂O₂)₃]Cl₂ (5).Polymetallic compounds of general formula Cr[O,O′-C₁₂H₆N₂O₂-N,N′-MCl₄]₃,containing chromium and a Group 4 element M = Ti (7), Zr (8), Hf (9), are prepared from Cr(O,O′-C₁₂H₆N₂O₂)₃ and the corresponding MCl₄ or MCl₄DME. Polynuclear derivatives of iron and chromium of formula [Fe(N,N′-C₁₂H₆N₂O₂-O,O′-CrCl₂(THF)₂)₃][PF₆]₂ (10), and Cr[O,O′-C₁₂H₆N₂O₂-N,N′-FeCl₂(THF)]₃ (11), are obtained by the reaction of [Fe(N,N′-C₁₂H₆N₂O₂)₃][PF₆]₂ with three equivalents of CrCl₂(THF)₂ and from Cr(O,O′-C₁₂H₆N₂O₂)₃ and FeCl₂(THF)_1.5, respectively. Compound 11 reacts with 1 (3 equivalents in sym-C₂H₂Cl₄ or 6 equivalents in ethanol) to give Cr[O,O′-C₁₂H₆N₂O₂-N,N′-FeCl₂(N,N′-C₁₂H₆N₂O₂)]₃ (12), and [Cr(O,O′-C₁₂H₆N₂O₂-N,N′-Fe(N,N′-C₁₂H₆N₂O₂)₂)₃]Cl₆ (13), respectively.     

Symbiotic Bradyrhizobium japonicum Reduces N2O Surrounding the Soybean Root System via Nitrous Oxide Reductase

N₂O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N₂O uptake and conversion of ¹⁵N-N₂O into ¹⁵N-N₂. Free-living cells of USDA110 showed N₂O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N₂O reductase activity. When detached soybean nodules formed with USDA110 were fed with ¹⁵N-N₂O, they rapidly emitted ¹⁵N-N₂ outside the nodules at a ratio of 98.5% of ¹⁵N-N₂O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N₂O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N₂O (0.34 ppm). These results indicate that the conversion of N₂O to N₂ depends exclusively on the respiratory N₂O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N₂O.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis