Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP⁺-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP⁺ and NAD⁺ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.     

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and meso ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca²⁺. Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle.     

Chemical modification of photosystem II core complex pigments with sodium borohydride

The reaction of the irreversible chemical reduction of the 13¹-keto C=O group of pheophytin a (Pheo a) with sodium borohydride in reaction centers (RCs) of functionally active spinach photosystem II (PS II) core complexes was studied. Stable, chromatographically purified PS II core complex preparations with altered chromophore composition are obtained in which ～25% of Pheo a molecules are modified to 13¹-deoxo-13¹-hydroxy-Pheo a. Some of the chlorophyll a molecules in the complexes were also irreversibly reduced with borohydride to 13¹-deoxo-13¹-hydroxy-chlorophyll a. Based on the results of comparative study of spectral, biochemical, and photochemical properties of NaBH₄-treated and control preparations, it was concluded that: (i) the borohydride treatment did not result in significant dissociation of the PS II core complex protein ensemble; (ii) the modified complexes retained the ability to photoaccumulate the radical anion of the pheophytin electron acceptor in the presence of exogenous electron donor; (iii) only the photochemically inactive pheo-phytin Pheo_D2 is subjected to the borohydride treatment; (iv) the Q_x optical transition of the Pheo_D2 molecule in the RC of PS II core complexes is located at 543 nm; (v) in the Q_y spectral region, Pheo_D2 probably absorbs at ～680 nm.     

Deuteron NMR spectroscopy of copper(II) complexes in solution. 1. (5,7,7,12,14,14,-hexamethyl-1,4,8,1 1-tetraazacyclotetradeca-4,11-diene)-copper(II) and its derivatives in nonaqueous solvent

¹H and ²H NMR spectra of the title copper(II) complexes and its derivatives have been measured. In contrast with their ¹H NMR spectra, ²H NMR spectra gave well resolved sharp signals, and demonstrated that two diastereomers attributable to two asymmetric ligand nitrogens are readily resolved. The remarkable linewidth-narrowing was found in the peripheral methyl groups, which make ²H NMR spectra very useful even for copper(II) complexes with a long electron spin relaxation time. By using ²H NMR spectra, meso-racemate equilibrium was pursued and examined in aqueous and acetonitrile solutions.     

Tris-ruthenium(II)/copper(II) multimetallic porphyrin: Synthesis, characterization, DNA binding and supercoiled DNA photocleavage studies

The porphyrin, meso-5-(pentafluorophenyl)-10, 15, 20-tris(4-pyridyl)porphyrin has been used to synthesize two new metalloporphyrin complexes. Insertion of copper(II) into the porphyrin center gives the copper(II) porphyrin. Coordination of three [Ru(bipy)₂Cl]⁺ moieties (where bipy = 2,2′-bipyridine) to the pyridyl nitrogens of the copper(II) porphyrin gives the target complex. Electronic transitions associated with the copper(II) porphyrin and the triruthenium copper(II) porphyrin include an intense Soret band and a less intense Q-band in the visible region of the spectrum. An intense π-π∗ transition in the UV region associated with the bipyridyl groups and a metal to ligand charge transfer (MLCT) band appearing as a shoulder to the Soret band are observed for the ruthenated copper(II) porphyrin. Electrochemical properties associated with the multimetallic complex include a redox couple in the cathodic region with E_1/2 = −0.86 V versus Ag/AgCl attributed to the porphyrin and a redox couple in the anodic region E_1/2 = 0.88 V versus Ag/AgCl due to the Ru^III/II couple. DNA titrations indicate the triruthenium copper(II) porphyrin interacts with DNA potentially through a groove binding mechanism. Irradiation of aqueous solutions of the target complex and supercoiled DNA at a 10:1 base pair to complex ratio with visible light above 400 nm indicates that the complex causes nicking of the DNA helix.     

Preparation of [meso-tetraphenylchlorophinato]nickel(II) by stepwise deformylation of [meso-tetraphenyl-2,3-diformyl-secochlorinato]nickel(II): conformational consequences of breaking the structural integrity of nickel porphyrins

The stepwise, Wilkinson’s catalyst-induced decarbonylation of [meso-tetraphenyl-2,3-diformylsecochlorinato]Ni(II) (4) to produce the monoformylated pigment [meso-tetraphenyl-2-formylsecochlorinato] (5) and [meso-tetraphenylchlorophinato]Ni(II) (6) is described. Thus, we have shown how to degrade one pyrrolic unit of the starting material, [meso-tetraphenylporphyrinato]Ni(II) (2) in three steps to an aldimine linkage. The conformational changes of the porphyrinic macrocycle during the course of this degradation, as determined by comparison of the X-ray crystal structures of the compounds, are discussed. A comparative study delineates the UV-Vis spectroscopical consequences. In addition, the chemical reactivity of [meso-tetraphenylchlorophinato]Ni(II) (6) suggests the existence of an azepine-derived pyrrole-modified porphyrins (11, 12).     

Oxidase studies of some benzimidazole diamide copper(II) complexes

New bis benzimidazole diamide ligands, N,N′-bis(benzimidazolyl-2-methyl)-2,2′-thiadiethanamide (GBTAA), and N,N′-bis(benzimidazolyl-2-methyl)-3,3′-thiadipropanamide (GBTPA) have been synthesised and utilised to prepare copper(II) complexes with inner sphere ligands like Cl⁻ and . One of the ligands, GBTAA, has been structurally characterised, while the other GBTPA is characterised via an unusual tetrabenzoate bridged dicopper polymeric structure wherein the ligand GBTPA bridges the two dicopper benzoate units. The coordination environment about each copper is five coordinate, while τ value is found to be 0.32 indicating a distorted square pyramidal geometry. The copper(II) complexes catalyse the quenching of superoxide radical generated electrochemically.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Four-coordinate complexes of bis(1-diphenylphosphinoindenyl)iron(II)

Palladium, platinum and rhodium complexes of rac- and meso-bis(1-diphenylphosphinoindenyl)iron(II) (1) are reported. Both rac and meso isomers of {bis(1-diphenylphosphinoindenyl)iron(II)}palladium dichloride (rac- and meso-2) were characterized by X-ray crystallography along with the rac isomer of the Pt analogue (rac-3). NMR analysis of the rhodium complex [{bis(1-diphenylphosphinoindenyl)iron(II)}(cyclooctadiene)rhodium(I)] tetraphenylborate suggests a similar structure in solution. Coupling reactions of n- and sec-BuCl with bromobenzene in THF are catalysed by rac-2 and found to be similar to (PPh₃)₂PdCl₂ but poorer than (dppf)PdCl₂ in diethyl ether.     

Synthesis,characterization, toxicity,cytogenetic and in vivo antitumor studies of 1,1-dithiolate Cu(II) complexes with di-, tri-, tetra- amines and 1,3-thiazoles. Structure–activity correlation

A series of new mixed-ligand neutral copper(II) complexes of the general type [Cu(amine)(i-MNT)] and [Cu(tz)(i-MNT)] was prepared and characterized by elemental, spectroscopic methods, μ_eff, Λ_μ measurements and molecular modeling studies. The acute toxicity, the cytogenetic and the in vivo antitumor activity of the new complexes, is related to their chemical and physicochemical properties. Among the Cu(II) compounds tested the complex with 2-amino-5-methyl thiazole increases significantly the life span of leukemia P388 bearing mice in vivo.     

Coordination chemistry of β-ketoamides: Synthesis of copper(II) complexes,x-ray structure of bis-(1-N-benzylamino-1,3-butanedionate)copper(II) and nucleophilic behavior of the metal-β-carbonylenolate ring toward cyanogen and benzoyl cyanide

N-monosubstituted β-ketoamides react almost quantitatively with copper(II) acetate in a 1/1 ethanol-water mixture at ca. 40 °C when acetic acid and solvent are removed at reduced pressure. The novel bis(β-ketoamidate)copper(II) complexes, which can be obtained with this synthetic procedure, are O,O′-bonded species, thermally stable both in the solid state and in solution of polar solvents. The crystal structure of bis(1-N-benzylamino-1,3-butanedionate)copper(II) has been determined. Crystals are orthorhombic, space group Pbca with Z = 4, in a unit cell of dimension a = 19.506(5),b = 11.901(4),c = 8.726(3)Å. The structure was solved by the heavy atom method and refined to R = 0.048 for 915 independent reflections. The complex is monomeric and no intermolecular Cu⋯O or Cu⋯N interactions are observed. The bis(benzoylacetanilidate)copper(II) complex reacts with cyanogen or benzoyl cyanide to give addition-insertion at the methino group of the metal-β-ketoamidate ring.     

Supramolecular assemblies of a new class of nonplanar cationic metalloporphyrins and anionic metallophthalocyanines

The heteroaggregation behavior between a new class of nonplanar cationic β-octabrominated meso-alkylpyridinium zinc(II)-porphyrins (β-Br₈(ZnP)) and anionic tetrasulfonated metallophthalocyanines (MTSPc, M = Ni(II) and Cu(II)) has been studied by UV-Vis electronic spectroscopy, in dimethylsulfoxide (DMSO) solution. The heteroaggregate stoichiometry and the association constants were determined by means of Job plots. Dimers and unexpected trimers, taking into account the existence of axially coordinated DMSO molecules to the central metal in both β-Br₈(ZnP) and MTSPc complexes, are formed in solution. The spectroscopic properties of the heteroaggregates are markedly different from those observed in the correspondent planar cationic derivatives, the heteroaggregates showing major changes predominantly in the β-Br₈(ZnP) Soret band region and minor effects in the MTSPc Q bands. The observed changes in the Soret band region (red/blue shifts, decrease in the absorption intensities) depend on the nature of the alkyl substituent attached to the meso-pyridinium group. The greater versatility of the nonplanar porphyrins accommodating the meso-substituents in out-of-plane and in-plane conformations is proposed to explain the observed stoichiometries and the differences on the heteroaggregates spectroscopic properties for each β-Br₈ZnP compound. The likely conformations assumed by the meso-substituents in these β-Br₈(ZnP) compounds and its spectroscopic characteristics are in accordance with the participation of the substituents as the main factor on the extent of the observed red-shifted spectra in nonplanar porphyrins. The obtained association constants (K_IP) for the dimers and trimers are lower than those previously found for the similar planar cationic porphyrin systems, due to the lack of extensive π-π interactions and to the less effective approximation between the ionic groups, resulting in loosened heteroaggregates, particularly for the trimeric systems. Furthermore, the experimental results suggest that the NiTSPc is more distorted in DMSO solution than the CuTSPc derivative, favoring the interaction with the nonplanar β-Br₈(ZnP) compounds.     

The preparation and x-ray crystal structures of bis(4R-oxazolidine-4′-carboxylato)copper(II) (R = methyl,phenyl)

The base-catalyzed condensation reactions of formaldehyde with the copper(II) chelates of α-alanine and C-phenylglycine result in the formation of bis(4R-oxazolidine-4′-carboxylato)copper(II) where R = methyl and phenyl respectively. The 4-methyl complex, C₁₀H₂₀N₂O₈Cu, crystallizes in the monoclinic space group P2₁/n with a = 9.141(2), b = 7.335(3), c = 11.112(3) Å, β = 103.87(2)° and Z = 2. The structure has been refined to R = 0.026 and R_w = 0.031 based on 749 independent reflections collected, 651 used. The geometry about copper is essentially a (4 + 2)-elongated octahedral structure. The 4-phenyl derivative, C₂₀H₂₀N₂O₆Cu, crystallizes in the monoclinic space group P2₁/c with a = 11.939(4), b = 8.887(2), c = 8.611(3) Å, β = 95.61(3)° and Z = 2. Refinement of the structure converged to R = 0.062 and R_w = 0.071 based on 1003 reflections collected and 865 used. The structure of the 4-phenyl complex resembles that of the 4-methyl derivative and differs mainly from the latter in being anhydrous.     

Complexes of large ring macrocycles. The preparation of the C-meso- and C-racemic-diastereoisomers of Me6[18]aneN4 and characterisation of their ‘blue’ and ‘red’ copper(II) complexes

The reaction of 1,4-diaminobutane monohydroperchlorate with acetone gives trans-Me₆[18]- dieneN₄·2HClO₄·2H₂O. The diene can be reduced with NaBH₄ under basic conditions to give a mixture of C-meso Me₆[18]aneN₄ (melting point (m.p.) 119–120 °C) and C-racemic Me₆[18]aneN₄ (m.p. 79 °C) which can be separated by fractional crystallisation from xylene (Me₆[18]aneN₄=2,4,4,11,13, 13-hexamethyl-1,5,10,14-tetraazacyclooctadecane). The free base form of Me₆[18]dieneN₄ has been characterised and molecular weight measurements by vapour pressure osmometry confirm the 18-membered tetraaza structure rather than the alternative 9- membered diaza structure. Blue copper(II) complexes [CuL](ClO₄)₂ have been characterised with both C- meso and C-racemic Me₆[18] aneN₄ which have a dd band at 680 nm. These blue complexes are converted to the more thermodynamically stable red isomers (λ_max 488 nm) on stirring aqueous suspensions of the blue perchlorate salts. The red isomers are believed to have the trans III or RSSR configuration of the sec- NH centres.     

Characterization of alkaline-degradation products of some 3,4-di- and 3,4,6-tri-methyl ethers of hexoses by G.L.C.-mass spectrometry of O-trimethylsilyl derivatives

G.l.c.-mass spectrometry has been used to provide information on the O-trimethylsilyl derivatives of the products of alkaline degradation of 3,4-di- and 3,4,6-tri-O-methyl-D-glucose, and 3,4,6-tri-O-methyl-D-galactose. During reaction with sodium hydroxide-sodium borohydride mixtures, reduction occurs more rapidly than β-elimination and the only detectable products were the corresponding alditols and the epimeric 3-deoxyalditols. Extended reaction with sodium hydroxide alone, followed by treatment with sodium borohydride, gives mixtures of aldonic acids including the epimeric 3-deoxy-4-O-methylaldonic acids (metasaccharinic acids), 3-deoxyaldonic acids (with loss of the 4-O-methyl substituent), and 3,4-dideoxy-aldonic acids. Possible reaction-pathways are discussed.     

Oxyfunctionalization of unactivated C–H bonds in triterpenoids with tert-butylhydroperoxide catalyzed by meso-5,10,15,20-tetramesitylporphyrinate osmium(II) carbonyl complex

A system consisting of meso-5,10,15,20-tetramesitylporphyrinate osmium(II) carbonyl complex [Os(TMP)CO] as a precatalyst and tert-butylhydroperoxide (TBHP) as an oxygen donor is shown to be an efficient, regioselective oxidant system for the allylic oxidation, ketonization and hydroxylation of unactivated C–H bonds in a series of the peracetate derivatives of penta- and tetracyclic triterpenoids. Treatment of the substrates with this oxidant system afforded a variety of novel or scarce oxygenated derivatives in one-step. Structures of the isolated components, after chromatographic separation, were determined by spectroscopic methods including GC–MS and shift-correlated 2D-NMR techniques. Factors governing the regioselectivity and the possible mechanism for the oxyfunctionalization of the unactivated carbons are also discussed.     

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at −130°C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.     

No correlation between DNA strand breaks and HPRT mutation induced by photochemical treatment in V79 cells

DNA strand breaks, measured by alkaline elution, and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutation were studied in V79 cells after photochemical treatment (PCT) or exposure to X-rays. Cells were incubated with the photosensitizers Photofrin II (PII) and three closely related porphyrins tetra-(3-hydroxyphenyl) porphyrin (3THPP), meso-tetra-(4-sulfonatophenyl) porphine (TPPS₄) and meso-tetra-(N-methyl-4-pyridyl) porphine (TMPyPH₂). These dyes are assumed to act on cellular targets mainly via singlet oxygen when excited by light. While the hydrophilic TPPS₄ and TMPyPH₂ did not photoinduce mutants to any significant extent, both lipophilic dyes, 3THPP and PII, were significantly mutagenic when excited by light. On the other hand, TPPS₄ was the most efficient sensitizer of alkali-labile DNA strand breaks, while TMPyPH₂ did not induce any significant amount of either type of DNA damage. Surprisingly, no correlation between the two parameters was found for PCT, either after exposures inactivating 50% of the cells or after exposures inactivating 90% of them. The lack of correlation between the yields of DNA strand breaks and of mutants could not be explained by differences in the intracellular localization pattern of the dyes.     

Reactivity of ligand-swapped mutants of the SCO protein from Bacillus subtilis. Isomers of the CCH metal binding motif

The Synthesis of Cytochrome Oxidase protein, or SCO protein, is required for the assembly of cytochrome c oxidase in many mitochondrial and bacterial respiratory chains. SCOs have been proposed to deliver copper to the Cu_A site of cytochrome c oxidase. We have reported that Bacillus subtilis SCO (i.e., BsSCO) binds Cu(II) with high-affinity via a two-step process mediated by three conserved residues (i.e., two cysteines and one histidine, or the CCH motif). A remarkable feature in the reaction of reduced (i.e., di-thiol) BsSCO with copper is that it does not generate any of the disulfide form of BsSCO. This molecular aversion is proposed to be a consequence of a binding mechanism in which the initial copper complex of BsSCO does not involve cysteine, but instead involves nitrogen ligands. We test this proposal here by constructing two isomers of BsSCO in which the conserved copper binding residues (i.e., the CCH-motif) are retained, but their positions are altered. In these variants the two cysteines are exchanged with histidine, and both react transiently with copper (II) with distinct kinetic profiles. The reaction generates Cu(I) and the protein is oxidized to its disulfide form. EPR analysis supports a copper binding model in which cysteine, which is at the “histidine position” in the mutant, is part of an initial encounter complex with copper. When cysteine is the initial ligating residue an oxidation reaction ensues. In contrast initial binding to native BsSCO uses nitrogen-based ligands, and thereby avoids the opportunity for thiol oxidation.     

An epr signal from the half-reduced type 3 copper pair in Rhus vernicifera laccase

A new type of Cu(II) epr signals have been produced in native and type 2 copper depleted Rhus vernicifera laccase. They are shown to originate from one of the type 3 copper ions that are epr silent in the resting enzyme. The new epr signals show high rhombicity in g tensor and are similar to those observed in other proteins, such as superoxide dismutase and half-met hemocyanin. The half-reduced type 3 copper pair is formed by reduction with an electron from type 1 Cu(I) but only after a reoxidation of the copper pair, either by peroxide or dioxygen. It is suggested that the half-reduction of the type 3 copper pair only occurs in molecules where type 2 copper ion is either reduced or absent.