Radioimmunoassay for leukotriene B4

A rabbit immunized with leukotriene B₄ [LTB₄; (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (K_a) for [14,15-³H]LTB₄ of 3.2 × 10⁹ M^-1 at 37°C and in a concentration of 0.37 μg/ml of the immune plasma. When 10 μl of anti-LTB₄ and 3.9 nCi of [14,15-³H]LTB₄ (28 Ci/mmol; 1 Ci = 3.7 × 10¹⁰ becquerels) were incubated in a volume of 250 μl, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB₄ and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB₄. The sulfidopeptide leukotrienes, LTC₄ and LTD₄, displaced the radioligand from this antibody with less than 1/100th the activity of LTB₄, and the diastereoisomers of 6-trans-LTB₄, 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB₄ and quantitatively correlated with the physical measurement by integrated A₂₆₉ in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB₄ generated averaged 4.6 ng per 10⁶ rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB₄, a highly potent chemotactic factor, to unfractionated biologic fluids.     

A New Homogeneous Catalyst for the Dehydrogenation of Dimethylamine Borane Starting with Ruthenium(III) Acetylacetonate

The catalytic activity of ruthenium(III) acetylacetonate was investigated for the first time in the dehydrogenation of dimethylamine borane. During catalytic reaction, a new ruthenium(II) species is formed in situ from the reduction of ruthenium(III) and characterized using UV-Visible, Fourier transform infrared (FTIR), ¹H NMR, and mass spectroscopy. The most likely structure suggested for the ruthenium(II) species is mer-[Ru(N₂Me₄)₃(acac)H]. Mercury poisoning experiment indicates that the catalytic dehydrogenation of dimethylamine-borane is homogeneous catalysis. The kinetics of the catalytic dehydrogenation of dimethylamine borane starting with Ru(acac)₃ were studied depending on the catalyst concentration, substrate concentration and temperature. The hydrogen generation was found to be first-order with respect to catalyst concentration and zero-order regarding the substrate concentration. Evaluation of the kinetic data provides the activation parameters for the dehydrogenation reaction: the activation energy E_a = 85 ± 2 kJ·mol⁻¹, the enthalpy of activation ∆H^# = 82 ± 2 kJ·mol⁻¹ and the entropy of activation; ∆S^# = −85 ± 5 J·mol⁻¹·K⁻¹. The ruthenium(II) catalyst formed from the reduction of ruthenium(III) acetylacetonate provides 1700 turnovers over 100 hours in hydrogen generation from the dehydrogenation of dimethylamine borane before deactivation at 60 °C.     

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA₄ and transfer it to cells expressing LTA₄ hydrolase (LTA₄H) or LTC₄ synthase (LTC₄S) to produce LTB₄ or LTC₄. This process has been demonstrated in vivo for LTB₄, but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB₄ and cysLTs (LTC₄, LTD₄, and LTE₄). 5LO^−/− mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO^−/− bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA₄H^−/− and WT mice, but were reduced in LTC₄S^−/− animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA₄H^−/− or LTC₄S^−/− mice injected into 5LO^−/− mice restored the ability to synthesize LTB₄ and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA₄ for the synthesis of LTs mediating biological activities during inflammatory events in vivo.     

Interaction between the intermediary electron acceptor (pheophytin) and a possible plastoquinone-iron complex in photosystem II reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Pheo), in photosystem II reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and redox potential E_h = -450 mV produces an EPR doublet centered at g = 2.00 with a splitting of 52 G at 7 K in addition to a narrow signal attributed to Pheo^[unk] (g = 2.0033, ΔH ≈ 13 G). The doublet is eliminated after extraction of lyophilized TSF-II with hexane containing 0.13-0.16% methanol but is restored by reconstitution with plastoquinone A (alone or with β-carotene) although not with vitamin K₁. TSF-II and TSF-IIa are found to contain ≈2 nonheme Fe atoms per reaction center. Incubation with 0.55 M LiClO₄ plus 2.5 mM o-phenanthroline (but not with 0.55 M LiClO₄ alone) decreases this value to ≈0.6 and completely eliminates the EPR doublet, but photoreduction of Pheo is not significantly affected. Partial restoration of the doublet (about 25%) was achieved by subsequent incubation with 0.2 mM Fe²⁺, but not with either Mn²⁺ or Mg²⁺. The Fe removal results in the development of a photoinduced EPR signal (g = 2.0044 ± 0.0003, ΔH = 9.2 ± 0.5 G) at E_h = 50 mV, which is not observed after extraction with 0.16% methanol in hexane. It is ascribed to plastosemiquinone no longer coupled to Fe in photosystem II reaction centers. The results show that a complex of plastoquinone and Fe can act as the stable “primary” electron acceptor in photosystem II reaction centers and that the interaction of its singly reduced form with the reduced intermediary acceptor, Pheo^[unk], is responsible for the EPR doublet.     

Low Temperature Electronic Absorption Spectra of Oxidized and Reduced Spinach Ferredoxins. Evidence for Nonequivalent Iron(III) Sites

The electronic absorption spectra of oxidized and reduced spinach ferredoxins have been measured between 1200 and 600 nm at low temperature in D₂O/ethylene glycol glasses. Relatively weak absorption bands are observed at 720, 820, and 920 nm in oxidized ferredoxin, and at 652, 820, and 920 nm in reduced ferredoxin. The spectral results show that the two Fe(III) centers in oxidized ferredoxin are not equivalent, and that the 820- and 920-nm bands are associated with the nonreducible site. Assignment of the reducible site as tetrahedral Fe(III) is indicated. The 720-nm (13.9 kcm^-1) band in oxidized ferredoxin is attributed to an intensity-enhanced ⁶A₁ → ⁴T₁d-d transition, whereas the 652-nm (15.3 kcm^-1) feature of reduced ferredoxin could be due either to ⁵E → ³T₁ in tetrahedral Fe(II)S₄ or an Fe(II) → Fe(III) intervalence excitation.     

Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear Fe^II enzyme family. Many steps in the O₂ activation and reaction cycle of Fe^II-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O₂ to the H200N-4NC complex is trapped and characterized using EPR and Mössbauer (MB) spectroscopies. Int-1 is a high-spin (S₁ = 5/2) Fe^III antiferromagnetically (AF) coupled to an S₂ = 1/2 radical (J ≈ 6 cm^-1 in ). It exhibits parallel-mode EPR signals at g = 8.17 from the S = 2 multiplet, and g = 8.8 and 11.6 from the S = 3 multiplet. These signals are broadened significantly by hyperfine interactions (A_¹⁷O ≈ 180 MHz). Thus, Int-1 is an AF-coupled species. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O₂. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled Fe^III-radical complex. Int-2 exhibits an EPR signal at g = 8.05 arising from an S = 2 state. The signal is only slightly broadened by (< 3% spin delocalization), suggesting that Int-2 is a peroxo-Fe^III-4NC semiquinone radical species. Our results demonstrate facile electron transfer between Fe^II, O₂, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism.     

Picosecond photochemistry of a cofacial diporphyrin containing iron(III) and zinc(II): Mimicking electron transfer between cytochrome c and the primary electron donor in reaction centers of photosynthetic bacteria

Comparison of picosecond kinetic and spectroscopic data for Zn octaethylporphine and Fe(III)Cl octaethylporphine with that for Zn—Fe(III)Cl, a cofacial diporphyrin composed of a Zn porphyrin covalently bound to an Fe(III)Cl porphyrin with two chains of five atoms each, supports the assignment of a light-driven electron transfer (k > 10¹¹s^-1) within Zn—Fe(III)Cl to form [Zn⁺·—Fe(II)]Cl. The kinetics (k ≈ 10¹⁰s^-1) and thermodynamics of the reverse electron transfer are compared to those of a similar electron transfer in bacterial photosynthesis, the reduction of an oxidized bacteriochlorophyll dimer, (BChl)₂⁺·, by Fe(II) cytochrome c.     

Alternating electron and proton transfer steps in photosynthetic water oxidation

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel–production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese–calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S₂ → S₃ transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein–water interface is characterized by a high activation energy (E_a = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S₀ → S₁ transition are similar (τ, approximately 100 µs; E_a = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.     

Picosecond kinetics of fluorescence and absorbance changes in photosystem II particles excited at low photon density

Oxygen-evolving photosystem II particles (from Synechococcus) with about 80 chlorophyll molecules per primary electron donor (P₆₈₀) were used for a correlated study of picosecond kinetics of fluorescence and absorbance changes, detected by the single-photon-timing technique and by a pump-probe apparatus, respectively. Chlorophyll fluorescence decays were biexponential with lifetimes τ₁ = 80 ± 20 ps and τ₂ = 520 ± 120 ps in open reaction centers and τ₁ = 220 ± 30 ps and τ₂ = 1.3 ± 0.15 ns in closed reaction centers. The corresponding fluorescence yield ratio F_max/F_o was 3-4. Absorbance changes were monitored in the spectral range of 620-700 nm after excitation at 675 nm with 10-ps pulses sufficiently weak (<7 × 10¹² photons/cm² per pulse) to avoid singlet-singlet annihilation. With open reaction centers, the absorbance changes could be fit to the sum of three exponentials. The associated absorbance difference spectra were attributed to (i) exciton trapping and charge separation (τ = 100 ± 20 ps), (ii) the electron-transfer step P₆₈₀⁺ I^- Q_A → P₆₈₀⁺ I Q_A^- (where I is the primary electron acceptor and Q_A is the first quinone acceptor) (τ = 510 ± 50 ps), and (iii) the reduction of P₆₈₀⁺ by the intact donor side (τ > 10 ns). With closed reaction centers, the absorbance changes were biexponential with lifetimes τ₁ = 170-260 ps and τ₂ = 1.6-1.75 ns. The results are explained in terms of a kinetic model that assumes P₆₈₀ to constitute a shallow trap. The results show that Q_A reduction in these photosystem II particles decreases both the apparent rate and the yield of the primary charge separation by a factor of 2-3 and increases the mean lifetime of excitons in the antenna by a factor of 3-4. Thus, we conclude that the long-lived, nanosecond chlorophyll fluorescence is not charge-recombination luminescence but rather emission from equilibrated excited states of antenna chlorophylls.     

Molecular and Polymer Ln2M2 (Ln = Eu,Gd, Tb,Dy; M = Zn,Cd) Complexes with Pentafluorobenzoate Anions: The Role of Temperature and Stacking Effects in the Structure; Magnetic and Luminescent Properties

Varying the temperature of the reaction of [{Cd(pfb)(H₂O)₄}⁺_n·n(pfb)⁻], [Ln₂(pfb)₆(H₂O)₈]·H₂O (Hpfb = pentafluorobenzoic acid), and 1,10-phenanthroline (phen) in MeCN followed by crystallization resulted in the isolation of two type of products: 1D-polymers [LnCd(pfb)₅(phen)]_n·1.5nMeCN (Ln = Eu (I), Gd (II), Tb (III), Dy (IV)) which were isolated at 25 °C, and molecular compounds [Tb₂Cd₂(pfb)₁₀(phen)₂] (V) formed at 75 °C. The transition from a molecular to a polymer structure becomes possible because of intra- and intermolecular interactions between the aromatic cycles of phen and pfb from neighboring tetranuclear Ln₂Cd₂ fragments. Replacement of cadmium with zinc in the reaction resulted in molecular compounds Ln₂Zn₂ [Ln₂Zn₂(pfb)₁₀(phen)₂]·4MeCN (Ln = Eu (VI), Tb (VIII), Dy (IX)) and [Gd₂Zn₂(pfb)₁₀(H₂O)₂(phen)₂]·4MeCN (VII). A new molecular EuCd complex [Eu₂Cd₂(pfb)₁₀(phen)₄]·4MeCN (X)] was isolated from a mixture of cadmium, zinc, and europium pentafluorobenzoates (Cd:Zn:Ln = 1:1:2). Complexes II-IV, VII and IX exhibit magnetic relaxation at liquid helium temperatures in nonzero magnetic fields. Luminescent studies revealed a bright luminescence of complexes with europium(III) and terbium(III) ions.     

Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution

We have measured directly the rate of formation of the oxidized chlorophyll a electron donor (P680⁺) and the reduced electron acceptor pheophytin a^- (Pheoa^-) following excitation of isolated spinach photosystem II reaction centers at 4°C. The reaction-center complex consists of D₁, D₂, and cytochrome b-559 proteins and was prepared by a procedure that stabilizes the protein complex. Transient absorption difference spectra were measured from 440 to 850 nm as a function of time with 500-fs resolution following 610-nm laser excitation. The formation of P680⁺-Pheoa^- is indicated by the appearance of a band due to P680⁺ at 820 nm and corresponding absorbance changes at 505 and 540 nm due to formation of Pheoa^-. The appearance of the 820-nm band is monoexponential with τ = 3.0 ± 0.6 ps. The time constant for decay of ^1*P680, the lowest excited singlet state of P680, monitored at 650 nm, is τ = 2.6 ± 0.6 ps and agrees with that of the appearance of P680⁺ within experimental error. Treatment of the photosystem II reaction centers with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of Pheoa^-, results in formation of a transient absorption spectrum due to ^1*P680. We find no evidence for an electron acceptor that precedes the formation of Pheoa^-.     

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of (CO)3Fe(μ-StBu)3Ni{SC6H3-2,6-(mesityl)2} and reversible CO addition at the Ni site

A [NiFe] hydrogenase model compound having a distorted trigonal-pyramidal nickel center, (CO)₃Fe(μ-S^tBu)₃Ni(SDmp), 1 (Dmp = C₆H₃-2,6-(mesityl)₂), was synthesized from the reaction of the tetranuclear Fe-Ni-Ni-Fe complex [(CO)₃Fe(μ-S^tBu)₃Ni]₂(μ-Br)₂, 2 with NaSDmp at -40 °C. The nickel site of complex 1 was found to add CO or CN^tBu at -40 °C to give (CO)₃Fe(S^tBu)(μ-S^tBu)₂Ni(CO)(SDmp), 3, or (CO)₃Fe(S^tBu)(μ-S^tBu)₂Ni(CN^tBu)(SDmp), 4, respectively. One of the CO bands of 3, appearing at 2055 cm^-1 in the infrared spectrum, was assigned as the Ni-CO band, and this frequency is comparable to those observed for the CO-inhibited forms of [NiFe] hydrogenase. Like the CO-inhibited forms of [NiFe] hydrogenase, the coordination of CO at the nickel site of 1 is reversible, while the CN^tBu adduct 4 is more robust.     

Synthesis,Crystal Structure and Luminescent Property of Mg(II) Complex with N-Benzenesulphonyl-L-Leucine and 1,10-Phenanthroline

A new complex [Mg(L)₂(phen)(H₂O)₂](phen)(H₂O)₂ [L= N-benzenesulphonyl-L-leucine] was synthesized by the reaction of magnesium chloride hexahydrate with N-benzenesulphonyl-L-leucine and 1,10-phenanthroline in the CH₃CH₂OH/H₂O (v:v = 5:1). It was characterized by elemental analysis, IR and X-ray single crystal diffraction analysis. The crystal of the title complex [Mg(L)₂(phen)(H₂O)₂](phen)(H₂O)₂ belongs to triclinic, space group P-1 with a = 0.72772(15) nm, b = 1.4279(3) nm, c = 1.4418(3) nm, α = 63.53(3)°, β = 79.75(3)°, γ = 81.83(3)°, V = 1.3163(5) nm³, Z =1, D_c= 1.258 μg·m⁻³, μ = 0.177 mm⁻¹, F(000) = 526, and final R₁ = 0.0506, ωR₂ = 0.1328. The complex comprises a six-coordinated magnesium(II) center, with a N₂O₄ distorted octahedron coordination environment. The molecules are connected by hydrogen bonds and π-π stacking to form one dimensional chain structure. The luminescent property of the Mg(II) complex has been investigated in solid.     

Synthesis,Crystal Structure and Luminescent Property of Cd (II) Complex with N-Benzenesulphonyl-L-leucine

A new trinuclear Cd (II) complex [Cd₃(L)₆(2,2-bipyridine)₃] [L = N-phenylsulfonyl-L-leucinato] has been synthesized and characterized by elemental analysis, IR and X-ray single crystal diffraction analysis. The results show that the complex belongs to the orthorhombic, space group P2₁2₁2₁ with a = 16.877(3) Å, b = 22.875(5) Å, c = 29.495(6) Å, α = β = γ = 90°, V = 11387(4) ų, Z = 4, D_c= 1.416 μg·m⁻³, μ = 0.737 mm⁻¹, F (000) = 4992, and final R₁ = 0.0390, ωR₂ = 0.0989. The complex comprises two seven-coordinated Cd (II) atoms, with a N₂O₅ distorted pengonal bipyramidal coordination environment and a six-coordinated Cd (II) atom, with a N₂O₄ distorted octahedral coordination environment. The molecules form one dimensional chain structure by the interaction of bridged carboxylato groups, hydrogen bonds and π-π interaction of 2,2-bipyridine. The luminescent properties of the Cd (II) complex and N-Benzenesulphonyl-L-leucine in solid and in CH₃OH solution also have been investigated.     

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4]

The all-ferric [Fe₄S₄]⁴⁺ cluster [Fe₄S₄{N(SiMe₃)₂}₄] 1 and its one-electron reduced form [1]^- serve as convenient precursors for the synthesis of 3∶1-site differentiated [Fe₄S₄] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)₂C₆H₃, mesityl = 2,4,6-Me₃C₆H₂) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe₄S₄(SDmp)₃(THF)₃] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)₃(O)₃ coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1]^- with four equiv of HSDmp gave [Fe₄S₄(SDmp)₄]^- 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me₄Im) to give [Fe₄S₄(SDmp)₃(Me₄Im)] 4 modeling the [Fe₄S₄(Cys)₃(His)] cluster in hydrogenases, and its one-electron reduced form [4]^- was synthesized from the reaction of 3 with Me₄Im. The reversible redox couple between 3 and [3]^- was observed at E_1/2 = -820 mV vs. Ag/Ag⁺, and the corresponding reversible couple for 4 and [4]^- is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe₄S₄]⁴⁺ cluster, [Fe₄S₄(SDmp)₄].     

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4 degrees C: Characterizations of two additional chlorophyll a-protein complexes

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4°C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of β-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (M_r 50,000) and that of CP IV is polypeptide 6 (M_r 47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtii CP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtii complexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196°C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA 72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.     

Organometallic mechanism of action and inhibition of the 4Fe-4S isoprenoid biosynthesis protein GcpE (IspG)

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe₄S₄ cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using ²H, ¹³C and ¹⁷O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η²-alkenyl π- (or π/σ) complex with the 4th Fe in the Fe₄S₄ cluster, and a similar “metallacycle” also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K_i ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a ¹³C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π/σ metallacycle, an η²-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.     

Similarities of artificial photosystems by ruthenium oxo complexes and native water splitting systems

The nature of chemical bonds of ruthenium(Ru)–quinine(Q) complexes, mononuclear [Ru(trpy)(3,5-t-Bu₂Q)(OH₂)](ClO₄)₂ (trpy = 2,2^′:6^′,2^′′-terpyridine, 3,5-di-tert-butyl-1,2-benzoquinone) (1), and binuclear [Ru₂(btpyan)(3,6-di-Bu₂Q)₂(OH₂)]²⁺ (btpyan = 1,8-bis(2,2^′:6^′,2^′′-terpyrid-4^′-yl)anthracene, 3,6-t-Bu₂Q = 3,6-di-tert-butyl-1,2-benzoquinone) (2), has been investigated by broken-symmetry (BS) hybrid density functional (DFT) methods. BS DFT computations for the Ru complexes have elucidated that the closed-shell structure (2b) Ru(II)–Q complex is less stable than the open-shell structure (2bb) consisting of Ru(III) and semiquinone (SQ) radical fragments. These computations have also elucidated eight different electronic and spin structures of tetraradical intermediates that may be generated in the course of water splitting reaction. The Heisenberg spin Hamiltonian model for these species has been derived to elucidate six different effective exchange interactions (J) for four spin systems. Six J values have been determined using total energies of the eight (or seven) BS solutions for different spin configurations. The natural orbital analyses of these BS DFT solutions have also been performed in order to obtain natural orbitals and their occupation numbers, which are useful for the lucid understanding of the nature of chemical bonds of the Ru complexes. Implications of the computational results are discussed in relation to the proposed reaction mechanisms of water splitting reaction in artificial photosynthesis systems and the similarity between artificial and native water splitting systems.     

One-Pot Self-Assembly of Dinuclear,Tetranuclear, and H-Bonding-Directed Polynuclear Cobalt(II), Cobalt(III), and Mixed-Valence Co(II)/Co(III) Complexes of Schiff Base Ligands with Incomplete Double Cubane Core

The reaction of 2,6-diformyl-4-methylphenol (DFMF) with 1-amino-2-propanol (AP) and tris(hydroxymethyl)aminomethane (THMAM) was investigated in the presence of Cobalt(II) salts, (X = ClO₄⁻, CH₃CO₂⁻, Cl⁻, NO₃⁻), sodium azide (NaN₃), and triethylamine (TEA). In one pot, the variation in Cobalt(II) salt results in the self-assembly of dinuclear, tetranuclear, and H-bonding-directed polynuclear coordination complexes of Cobalt(III), Cobalt(II), and mixed-valence Co^IICo^III: [Co₂^III(H₂L⁻¹)₂(AP⁻¹)(N₃)](ClO₄)₂ (1), [Co₄(H₂L⁻¹)₂(µ₃-1,1,1-N₃)₂(µ-1,1-N₃)₂Cl₂(CH₃OH)₂]·4CH₃OH (2), [Co₂^IICo₂^III(HL⁻²)₂(µ-CH₃CO₂)₂(µ₃-OH)₂](NO₃)₂·2CH₃CH₂OH (3), and [Co₂^IICo₂^III (H₂L1²⁻)₂(THMAM⁻¹)₂](NO₃)₄ (4). In 1, two cobalt(III) ions are connected via three single atom bridges; two from deprotonated ethanolic oxygen atoms in the side arms of the ligands and one from the1-amino-2-propanol moiety forming a dinuclear unit with a very short (2.5430(11) Å) Co-Co intermetallic separation with a coordination number of 7, a rare feature for cobalt(III). In 2, two cobalt(II) ions in a dinuclear unit are bridged through phenoxide O and μ₃-1,1,1-N₃ azido bridges, and the two dinuclear units are interconnected by two μ-1,1-N₃ and two μ₃-1,1,1-N₃ azido bridges generating tetranuclear cationic [Co₄(H₂L⁻¹)₂(µ₃-1,1,1-N₃)₂(µ-1,1-N₃)₂Cl₂(CH₃OH)₂]²⁺ units with an incomplete double cubane core, which grow into polynuclear 1D-single chains along the a-axis through H-bonding. In 3, HL²⁻ holds mixed-valent Co(II)/Co(III) ions in a dinuclear unit bridged via phenoxide O, μ-1,3-CH₃CO₂⁻, and μ₃-OH⁻ bridges, and the dinuclear units are interconnected through two deprotonated ethanolic O in the side arms of the ligands and two μ₃-OH⁻ bridges generating cationic tetranuclear [Co₂^IICo₂^III(HL⁻²)₂(µ-CH₃CO₂)₂(µ_3-OH)₂]²⁺ units with an incomplete double cubane core. In 4, H₂L1⁻² holds mixed-valent Co(II)/Co(III) ions in dinuclear units which dimerize through two ethanolic O (μ-RO⁻) in the side arms of the ligands and two ethanolic O (μ₃-RO⁻) of THMAM bridges producing centrosymmetric cationic tetranuclear [Co₂^IICo₂^III (H₂L1⁻²)₂(THMAM⁻¹)₂]⁴⁺ units which grow into 2D-sheets along the bc-axis through a network of H-bonding. Bulk magnetization measurements on 2 demonstrate that the magnetic interactions are completely dominated by an overall ferromagnetic coupling occurring between Co(II) ions.     

Mechanosynthesis and Thermoelectric Properties of Fe,Zn, and Cd-Doped P-Type Tetrahedrite: Cu12-xMxSb4S13

This contribution deals with the mechanochemical synthesis, characterization, and thermoelectric properties of tetrahedrite-based materials, Cu_12-xM_xSb₄S₁₃ (M = Fe²⁺, Zn²⁺, Cd²⁺; x = 0, 1.5, 2). High-energy mechanical milling allows obtaining pristine and substituted tetrahedrites, after short milling under ambient conditions, of stoichiometric mixtures of the corresponding commercially available binary sulfides, i.e., Cu₂S, CuS, Sb₂S₃, and MS (M = Fe²⁺, Zn²⁺, Cd²⁺). All the target materials but those containing Cd were obtained as single-phase products; some admixture of a hydrated cadmium sulfate was also identified by XRD as a by-product when synthesizing Cu₁₀Cd₂Sb₄S₁₃. The as-obtained products were thermally stable when firing in argon up to a temperature of 350–400 °C. Overall, the substitution of Cu(II) by Fe(II), Zn(II), or Cd(II) reduces tetrahedrites’ thermal and electrical conductivities but increases the Seebeck coefficient. Unfortunately, the values of the thermoelectric figure of merit obtained in this study are in general lower than those found in the literature for similar samples obtained by other powder processing methods; slight compositional changes, undetected secondary phases, and/or deficient sintering might account for some of these discrepancies.