Structural states of the binuclear copper cluster of Cancer magister methemocyanin.

The binuclear cupric copper cluster of $\text{Cancer}\text{magister}$ methemocyanin prepared from hemocyanin and hydrogen peroxide is diamagnetic (1). Upon treatment with azide, it is transformed into magnetic dipolar coupled (paramagnetic) Cu(II) pairs and then into magnetically isolated Cu(II) complexes. This progressive uncoupling of the binuclear cupric pairs in methemocyanin is interpreted in terms of a relaxation of superexchange through one or more bridging ligands.     

Resonance Raman spectroscopie investigations on phenolate bridged binuclear Cu(II) complexes: A basis for the identification of the endogenous bridging

Phenolate bridged binuclear Cu(II) complexes were used to monitor the contribution of the endogenous bridging ligand to the resonance Raman spectrum of the oxygen transport protein such as hemocyanin. From the excitation profile of the intensity enhanced phenolate v_c-o the phenolate-to-Cu(II) charge-transfer transition of the four studied complexes has been located between 380 nm and 430 run, similar to the low-temperature absorption band in the hemocyanin spectra. By approaching the exciting laser frequency to this electronic transition, the whole spectral range 1250–1650 cm⁻¹ is strongly intensity enhanced and considered as the typical feature of this kind of phenolate bridged Cu(II) complexes. Comparison to mononuclear analogues pointed out no significant differences of the resonance enhanced Raman lines. The results are discussed in relation to the resonance Raman spectra of hemocyanin.     

Fluorescence and circular dichroism studies with hemocyanin from taiwan snails

Several derivatives of hemocyanin from Taiwan snails (Achatina fulica) have been prepared. The reconstituted protein (R-HcO₂) has lower Cu content, lower circular dichroism intensity, and higher fluorescence intensity than native oxyhemocyanin (HcO₂). The Co(II) derivative (CoHc) does not take up molecular oxygen and only 50% of the total sites for Cu in native hemocyanin is taken up by Co. The half-apo derivative (half-apo-Hc) contains a single Cu per active site. Divalent cations quench the tryptophan fluorescence in the hemocyanin species and also quench the fluorescence from Tb³⁺ bound to the protein. The collisional quenching constants decrease in the order Co²⁺ > Mn²⁺ > Ca²⁺. The static component is negligible. For carboxy hemocyanin (HcCO), fluorescence originates from a Cu(I) CO complex and was used to study reaction of Hc CO with CN⁻.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Reactions and interconversion of met and dimer hemocyanin.

The regenerable methemocyanin of $\text{Busycon canaliculatum}$ is shown to be EPR-nondetectable. The small EPR signal present in met preparations is a nonregenerable binuclear cupric unit accounting for ～ 6% of the active sites. A variety of anions are found to bind to met with the reactions being complicated by both reduction and competitive binding of buffer. The metastable dimer hemocyanin is shown to rearrange either directly to EPR-nondectable met or through an EPR-detectable regenerable binuclear cupric form obtained on reaction of dimer hemocyanin with azide. These results combined with previous results on half met derivatives are used to support the presence of an endogenous protein bridge between the two coppers in hemocyanin.     

Oxygen binding to hemocyanin: a resonance Raman spectroscopic study

Oxygenation of hemocyanin gives rise to resonance Raman peaks at 742 and 282 cm^?1. The 742 cm^?1 peak which is in resonance with the 575 nm charge transfer band shifts to 704 cm^?1 when ¹⁸O₂ is substituted for ¹⁶O₂. Our results establish that the bound oxygen is in the form of peroxide (O₂^2?). The 282 cm^?1 peak which is in resonance with the 340 nm optical transition is insensitive to isotopic substitution, suggesting that the 282 cm^?1 peak corresponds to a vibration involving the magnetically-coupled Cu(II)··Cu(II) centers.     

Magnetostructural correlations and catecholase-like activities of μ-alkoxo-μ-carboxylato double bridged dinuclear and tetranuclear copper(II) complexes

Two μ-alkoxo-μ-carboxylato bridged dinuclear copper(II) complexes, [Cu₂(L¹)(μ-HCO₂)] (1) ((H₃L¹ = 1,3-bis(5-bromosalicylideneamino)-2-propanol)), [Cu₂(L²)(μ-HCO₂)] · dmf (2) (H₃L² = 1,3-bis(3,5-chlorosalicylideneamino-2-propanol)), and two μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear copper(II) complexes, [{Cu₂(L³)}₂(μ-O₂C–C(CH₃)₂–CO₂)] · 5H₂O · 3CH₃OH (3) ((H₃L³ = 1,3-bis(salicylid-deneamino)-2-propanol)) and [{Cu₂(L³)}₂(μ- O₂CCH₂–C₆H₄–CH₂CO₂)] · 2H₂O (4) have been prepared and characterized. The single crystal X-ray analysis shows that the structures of complexes 1 and 2 are dimeric with two adjacent copper(II) atoms bridged by μ-alkoxo-μ-carboxylato ligands with the Cu⋯Cu distances and Cu–O(alkoxo)–Cu angles are 3.511 Å and 132.85° for 1, 3.517 Å and 131.7° for 2, respectively. Complexes 3 and 4 consist of μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear Cu(II) complexes with mean Cu–Cu distances and Cu–O–Cu angles of 3.158 Å and 108.05° for 3 and 3.081 Å and 104.76° for 4, respectively. Magnetic measurements reveal that 1 and 2 are strong antiferromagnetically coupled with 2J = −156 and −152 cm⁻¹, respectively, while 3 and 4 exhibit ferromagnetic coupling with 2J = 86 and 155.2 cm⁻¹, respectively. The 2J values of 1–4 are linearly correlated to the Cu–O–Cu angles. Dependence of the pH at 25 °C on the reaction rate of oxidation of 3,4-di-tert-butylcatechol (3,5-dtbc) to the corresponding quinone catalyzed by 1–4 was studied. Complexes 1–4 exhibit high catecholase-like activity at pH 9.0 and 25 °C for oxidation of 3,5-di-tert-butylcatechol.     

Preparation and properties of hydrated uranium(III) complex chlorides. Part II. Uranium trichloride monomethylcyanide pentahydrate

The preparation and characterization of a new uranium(III) compound, UCl₃·CH₃CN·5H₂O, is reported. The complex chloride belongs to the monoclinic system with a=1226.(2), b=1298.(3), c=662.(1) pm, γ=101.7(2)° and Z=4. Magnetic susceptibility measurements were carried out by the Faraday method in the 6.5–300 K range. An antiferromagnetic transition is observed at T_N=12 K. In the 65–300 K region the compound exhibits Curie-Weiss paramagnetism with the paramagnetic constants C=1.430 emu K mol⁻¹ and θ=−65.7 K. The electronic spectra of thin layers of the compound have been recorded in the 4000–24000 cm⁻¹ region and discussed.     

A binuclear copper(II) complex of the schiff base derived from 1,1′-(2,6-pyridyl)-bis-1,3-butanedione and 3-amino-1-propanol

The reaction of Cu(ClO₄)₂·6H₂O with the Schiff base derived from 1,1′-(2,6-pyridyl)-bis-1,3-butanedione and 3-amino-1-propanol, (H₄L²), yields the complex Cu(H₄L²)(ClO₄)₂·H₂O. The crystal structure of this complex is triclinic, R = 0.0521, 5602 reflections. The species is dimeric leading to a binuclear copper(II) complex in which the well- separated (8.93 Å intramolecular and 5.46 Å inter- molecular) copper(II) atoms are in distorted square pyramidal geometries.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.     

Spectroscopic studies on the active site of Sepioteuthis lessoniana hemocyanin.

The absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra in the visible region have been measured for $\text{Sepioteuthis lessoniana}$ hemocyanin at 77, 198, and 293K. From the temperature dependence of the CD spectra of oxyhemocyanin, the bands observed at 450, 565, and 700 nm were resolved into those centered at 430, 490, 565, 600, and 700 nm. Since these five peaks are most probably due to the d-d transitions, the two copper ions at the oxygenated active center are inferred to be Cu(II) ions each in a non-equivalent coordination geometry of very low symmetry. The MCD spectral data confirm the view and reasonably explain the diamagnetism of oxyhemocyanin.     

Metal complexes with 2-(α-hydroxy-benzyl) thiamin pyrophosphate (HBTPP). Models for metal binding of thiamin enzymes

The reactions of MCl₂ (M = Zn²⁺, Cd²⁺, Hg²⁺) with 2-(α-hydroxy-benzyl)thiamin pyrophosphate (HBTPP) at various pH values (different protonation states) were studied in methanolic solutions. Solid complexes of formulae K[Zn(HBTPP) ⁻Cl₂ · H₂O_n, K₂[Cd(HBTPP)²⁻Cl₂ · 3H₂O_n, K₂[Hg(HBTPP)₂⁻Cl₂ · 3H₂O and Zn(HBTPP)₂⁰Cl₂ were isolated and characterized by elemental analysis and various NMR techniques, namely ¹³C NMR, ³¹P NMR, ¹¹³Cd NMR, ¹⁹⁹Hg NMR and ¹H NMR ROESY spectra in D₂O. The data provide evidence that Zn(II) in K[Zn(HBTPP) ⁻Cl2 · H₂O_n, and Cd(II) in K₂[Cd(HBTPP)²⁻Cl₂ · 3H₂O_n, are coordinated both to the pyrimidine N(1′) and to the pyrophosphate group. In contrast, Hg(II) in K₂[Hg(HBTPP)₂⁻Cl₂ · 3H₂O and Zn(II) in Zn(HBTPP)₂⁰Cl₂ are bound only to the N(1′) atom or to the pyrophosphate group, respectively.     

Spectroscopic studies of ligand perturbation effects on the half oxidized action site of Busycon canaliculatum hemocyanin.

We report the preparation and characterization of an important new series of hemocyanin derivatives containing a variety of anions bound to the cupric ion in a singly oxidized active site. Spectroscopic studies on these half met-L derivatives, Cu(I)…Cu(II)-L, where L = CN^?, NO₂^?, CH₃CO₂^?, and N₃^? reveal that the unpaired electron is localized on one copper in an approximately tetragonal site with a d_{x² ? y²} ground state. Ligand variation is used as a perturbation on the spectral features to show that the small molecule (and by analogy, oxygen in oxyhemocyanin) is bound in the equatorial plane of the copper.     

Exchange rates of pyridine on ferro- and ferriprotoporphyrin (IX) dimethylester in chloroform.

Nuclear magnetic resonance line-widths data have been used to determine the rate of solvent exchange from the first coordination sphere of ferro-and ferriprotoporphyrin(IX) dimethylester (Fe-PPD) in pyridine/chloroform. The average values of kinetic parameters for pyridine (PY) exchange indicate an SN2 mechanism tor Fe(III)-PPD(ΔH^&;#; = 36 kJ · mol⁻¹ ; ΔS^&;#; = −53 J·mol⁻¹K⁻¹; T_M(298 K) = 0.07 msec) and an SNI mechanism for Fe(II)-PPD (ΔH^&;#; = 67 kJ·mol⁻¹; ΔS^&;#; = 42 J · mol⁻¹K⁻¹; T_M(298 K) = 0.06 msec). Parallel to the accelerated ligand exchange rate at rising temperatures a redistribution of the electrons causing a transition of the metal porphyrin from the low-spin state to the high-spin state is observed. Enthalpy and entropy of the thermodynamic equilibrium between low- and high-spin Fe-PPD have been determined from experimental values of the average magnetic moment. A mean lifetime of low-spin Fe(III)-PPD was estimated from line. widths changes (T_L→H(298 K)≈ 20 msec) and the corresponding activation parameters have been obtained (ΔH^&;#;_L→H(298 K) = 26 kJ · mol⁻¹; ΔS^&;#;_L→H(298K) = −125 J · mol⁻¹K⁻¹).     

Adsorption of Mn(II) ions to human low density lipoproteins: Magnetic resonance studies

Mn(II) ions were used to study ion-binding properties of human low density lipoproteins (LDL). From the intensity of the EPR lines corresponding to the unbound Mn(II) ions the percentage of the ions bound to LDL is determined. By the titration of LDL with Mn(II) the binding parameters, dissociation constant, K_d, and the number of binding sites, n, could be derived. It has been found that there are at least two types of binding site on the LDL surface: ‘strong’ sites characterized by n = 6, K_d = 1.5 · 10⁻⁵M · l⁻¹, and ‘weak’ sites characterized by n = 145 and K_d = 6.6 · 10⁻³M · l⁻¹ for the sample in 0.01 M Tris-HCI buffer at 10°C. At very low Mn(II) concentrations binding to the ‘strong’ sites exhibits a cooperative behaviour. In the 0.1 M buffer the ‘strong’ sites are almost completely occupied or blocked by the monovalent buffer cations. The number of the ‘weak’ sites remains unaltered and K_d is decreased slightly (K_d = 4.9 · 10⁻³M · l⁻¹). The location, chemical nature and the structural and functional relevance of the binding sites are discussed.     

Continuous ethanol production by yeast immobilized on to channeled alumina beads

Vertical and near-horizontal (15° angle) packed-bed columns were compared for continuous ethanol fermentation using an alcohol- and glucose-tolerant Saccharomyces cerevisiae strain immobilized on to channeled alumina beads (5·0 × 10⁹ cells g⁻¹ beads). Spaces between beads (1·0–6·5 mm) and angle (15°) of near-horizontal reactor columns (with six ports in each) efficiently removed CO₂ and increased ethanol productivity. Malt-glucose-yeast-extract broth containing 16·7% glucose at 35°C fed at a dilution rate of 3· h⁻¹ to thw two horizontal columns (in series) yielded maximum ethanol productivity of 40·0 g liter⁻¹ h⁻¹. Feedstock flow rate and other factors (temperature, pH, nutrients, and glucose levels) affected productivities. The immobilized-cell system showed operational stability for >3 months without plugging, and could be stored for at least one year with no loss of bioreactor performance. Scanning electron micrographs of the beads revealed large numbers of yeast-cells attached on to internal and external surfaces of beads.     

Studies on the reactions of PtCl2en,cis-Pt(NH3)2Cl2 and their aqua species with adenosine,deoxyadenosine and adenine using ion-pair HPLC

The reactions of PtCl₂en or cis-Pt(NH₃)₂Cl₂ and their aqua species with adenine and adenosine were studied by means of ion-pair HPLC. From the chromatograms, it was found that the first binding site of Pt(II) was the N(7) site of adenine under both acidic and neutral conditions. The rates of Pt(II) binding at the (N7) site of adenosine and deoxyadenosine were measured. The rate constants, k₁, were obtained for the reactions of PtCl₂en or cis-Pt(NH₃)₂Cl₂ with adenosine and deoxyadenosine at pH 3 and 7 over the temperature range 9–25 °C. The k₁ values were 6.8–7.7 × 10⁻⁴ dm³ mol⁻¹ s⁻¹ at 25 °C. For the aqua species, the rate of [cis-Pt(NH₃)₂ClH₂O]⁺ with adenosine N(7) was measured. The rate constants, k₂ which were found to be smaller than those of hydrolysis, k_h, were calculated at pH 3 over the temperature range 25–40 °C. The k₂ value obtained at 25 °C was 1.1 × 10⁻² dm³ mol⁻¹ s⁻¹, 15 time larger than k¹. The activation parameters were also calculated.