Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

Synthesis and reactivity with amines of new diiron alkynyl methoxy carbene complexes

The new diiron alkynyl methoxy carbene complexes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(OMe)CCR′}(Cp)₂]⁺ (R = 2,6-Me₂C₆H₃ (Xyl), R′ = Tol, 3a; R = Xyl, R′ = Ph, 3b; R = Xyl, R′=Buⁿ, 3c; R = Xyl, R′=SiMe₃, 3d; R = Me, R′ = Tol, 3e; R = Me, R′ = Ph, 3f) are obtained in two steps by addition of R′CCLi (R′ = Tol, Ph, Buⁿ, SiMe₃) to the carbonyl aminocarbyne complexes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO)₂(Cp)₂]⁺ (R = Xyl, 1a; Me, 1b), followed by methylation of the resulting alkynyl acyl compounds [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(O)CCR′}(Cp)₂] (R = Xyl, R′ = Tol, 2a; R = Xyl, R′ = Ph, 2b; R = Xyl, R′ = Buⁿ, 2c; R = Xyl, R′ = SiMe₃, 2d; R = Me, R′ = Tol, 2e; R = Me, R′ = Ph, 2f). Complexes 3 react with secondary amines (i.e., Me₂NH, C₅H₁₀NH) to give the 4-amino-1-metalla-1,3-dienes [Fe₂{μ-CN(Me)(R)}(μ-CO)(CO){C(OMe)CHC(R′)(NMe₂)}(Cp)₂]⁺ (R = Xyl, R′ = Tol, 4a; R = Xyl, R′ = Ph, 4b; R = Me, R′ = Ph, 4c) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(Tol)(NC₅H₁₀)}(Cp)₂]⁺, 5. The addition occurs stereo-selectively affording only the E-configured products. Analogously, addition of primary amines R′NH₂ (R′ = Ph, Et, Prⁱ) affords the 4-(NH-amino)-1-metalla-1,3-diene complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(R)(NHR′)}(Cp)₂]⁺ (R = Ph, 6a; Et, 6b; Prⁱ, 6c). In the case of 6a, only the E isomer is formed, whereas a mixture of the E and Z isomers is present in the case of 6b,c, with prevalence of the latter. Moreover, the two isomeric forms exist under dynamic equilibrium conditions, as shown by VT NMR studies. Complexes 6 are deprotonated by strong bases (e.g., NaH) resulting in the formation of the neutral vinyl imine complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CHC(NR)(Tol)}(Cp)₂] (R = Ph, 7a; Et, 7b; Prⁱ, 7c); the reaction can be reverted by addition of strong acids. X-ray crystal structures have been determined for 3a[CF₃SO₃] · Et₂O, 4c[CF₃SO₃], 6a[BF₄] · CH₂Cl₂, 6c[CF₃SO₃] · 0.5Et₂O and 7a · CH₂Cl₂.     

Addition of protic nucleophiles to alkynyl methoxy carbene ligands in diiron complexes

Different protic nucleophiles (i.e.Ph₂CNH, PhSH, MeCO₂H, PhOH) can be added to the CC bond of [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C(OMe)CCTol}(Cp)₂][SO₃CF₃] (1), affording new diiron alkenyl methoxy carbene complexes.The additions of Ph₂CNH and MeCO₂H are regio and stereoselective, resulting in the formation of the 5-aza-1-metalla-1,3,5-hexatriene [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)(NCPh₂)}(Cp)₂][SO₃CF₃] (2), and the 2-(acyloxy)alkenyl methoxy carbene complex [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)OC(O)Me)}(Cp)₂][CF₃SO₃] (5); the E isomer of the former and the Z of the latter are formed exclusively.Conversely, the addition of PhSH is regio but not stereoselective; thus, both the E and Z isomers of [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(OMe)C_βHC_γ(Tol)(SPh)}(Cp)₂][SO₃CF₃] (3) are formed in comparable amounts.Compounds 3 and 5 are demethylated upon chromatography through Al₂O₃, resulting in the formation of the acyl complexes [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(O)C_βHC_γ(Tol)(SPh)}(Cp)₂] (4) and [Fe₂{μ-CN(Me)(Xyl)}(μ-CO)(CO){C_α(O)C_βHC_γ(Tol)OC(O)Me}(Cp)₂] (6), respectively, both with a Z configured C_βC_γ bond.Finally, the reaction of 1 with PhOH proceeds only in the presence of an excess of Et₃N affording the 2-(alkoxy)alkenyl acyl complex [Fe₂{μ-CN(Me)(Xyl)}(μ- CO)(CO){C_α(O)C_βHC_γ(Tol)(OPh)}(Cp)₂] (7). The crystal structures of 4 · CH₂Cl₂ and 7 · 0.5CH₂Cl₂ have been determined by X-ray diffraction experiments.     

Electrochemical study of triscyclopentadienyluranium complexes

The electrochemical behaviour of Cp₃UBH₄ and Cp₃UNEt₂ in THF has been studied using cyclic voltammetry and chronoamperometry. Cp₃UBH₄ undergoes a simple one-electron quasi-reversible reduction, while chemical reactions between the product of the charge-transfer reaction and the starting electroactive species occur both after oxidation and reduction of Cp₃UNEt₂.     

Synthesis, crystal structures and magnetic properties of the copper(II) complexes containing isophthalate anion as coligand

Two new dinuclear isophthalato-bridged copper(II) complexes [Cu₂(ntb)₂(μ-ipt)](ClO₄)₂·4CH₃OH·0.33H₂O (1), [Cu₂(bbma)₂(μ-ipt)(NO₃)(CH₃OH)]NO₃·CH₃OH (2) and one mononuclear complex [Cu(bbma)(ipt)(CH₃OH)_0.67(H₂O)_0.33]·2CH₃OH (3) containing tetradentate and tridentate poly-benzimidazole ligands were synthesized, where ntb is tris(2-benzimidazolylmethyl)amine, bbma is bis(benzimidazol-2-yl-methyl)amine and ipt is isophthalate dianion. All of the complexes were characterized by elemental analysis, IR spectra and X-ray crystallography. The structures of complexes 1 and 2 consist of μ-ipt bridging two Cu(II) centers in a bis(monodentate) bonding fashion. The coordination geometry around the Cu(II) ions of both compounds has a distorted square pyramidal geometry. The Cu···Cu distances are 9.142 and 10.435 Å for 1 and 2, respectively. Complex 3 has a distorted square pyramidal geometry achieved by the three N-atoms of the bbma ligand, one isophthalate-oxygen atom and one oxygen atom from a coordinated methanol molecule. The magnetic susceptibility measurements at variable temperature over the 2-300 K range for complexes 1 and 2 are reported, with J values to be −0.013 and −0.32 cm⁻¹, respectively. The results show that the two complexes exhibit very weak antiferromagnetic interactions between the dinuclear copper(II) centers.     

Electrochemical,UV-Visible and EPR studies on nitrofurantoin: Nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Electrochemical, UV--visible and EPR studies on nitrofurantoin: nitro radical anion generation and its interaction with glutathione

This paper deals with the reactivity of the nitro radical anion electrochemically generated from nitrofurantoin with glutathione. Cyclic voltammetry (CV) and controlled potential electrolysis were used to generate the nitro radical anion in situ and in bulk solution, respectively and cyclic voltammetry, UV-Visible and EPR spectroscopy were used to characterize the electrochemically formed radical and to study its interaction with GSH.

By cyclic voltammetry on a hanging mercury drop electrode, the formation of the nitro radical anion was possible in mixed media (0.015M aqueous citrate/DMF, 40/60, pH 9) and in aprotic media. A second order decay of the radicals was determined, with a k₂ value of 201 and 111M^-1 s^-1, respectively. Controlled potential electrolysis generated the radical and its detection by cyclic voltammetry, UV-Visible and EPR spectroscopy was possible. When glutathione (GSH) was added to the solution, an unambiguous decay in the signals corresponding to a nitro radical anion were observed and using a spin trapping technique, a thiyl radical was detected.

Electrochemical and spectroscopic data indicated that it is possible to generate the nitro radical anion from nitrofurantoin in solution and that GSH scavenged this reactive species, in contrast with other authors, which previously reported no interaction between them.     

Study of the physico-chemical properties of prostaglandin F2 alpha and its complexes

To study the intracellular action mechanisms of prostaglandin F2 alpha its interaction with lipid components of biological membranes was investigated. It has been found that prostaglandin forms a complex with phosphatidylcholine and cholesterol. Immobilization ability of phospholipids is changed in the course of complex formation. Ability of prostaglandin F2 alpha for complex formation with insulin was also observed. Combination of monolayer technique and electron microscopy made possible to discover molecular reconstructions when prostaglandin is incorporated into monolayer of biologically active compounds.     

Electrochemical behavior of some bioorganometallic complexes

An electrochemical investigation of several complexes of the type Ru(II)Cp^*(Ar) and Rh(III)Cp^*(Ar) (Cp^* = η⁵-pentamethylcyclopentadienyl, Ar = aromatic ring) has been performed. The electrochemical results, although very complicated from a mechanistic point of view, nonetheless suggest a possible application of RuCp^* and RhCp^* fragments as labels of steroids for polarographic assay.     

Effect of the synergistic anion on electron paramagnetic resonance spectra of iron-transferrin anion complexes is consistent with bidentate binding of the anion.

Continuous wave (cw) X-band EPR spectra at approximately 90 K were obtained for iron-transferrin-anion complexes with 18 anions. Each anion had a carboxylate group and at least one other polar moiety. As the second polar group was varied from hydroxyl to carbonyl to amine to carboxylate, the EPR spectra changed from a dominant signal at g' approximately 4.3 with a second smaller peak at g' approximately 9 to a broad signal with intensity between g' approximately 5 and 7. Computer simulation indicated that the changes in the EPR spectra were due to changes in the zero field splitting parameter ratio, E/D, from approximately 1/3 for carbonate anion to approximately 0.04 for malonate anion. Observation of iron-13C coupling in the electron spin echo envelope modulation (ESEEM) for iron transferrin [1-13C]pyruvate indicated that the carboxylate group was bound to the iron. It is proposed that all of the anions behave as bidentate ligands, with coordination to the iron through both the carboxylate and proximal groups, and the carboxyl group serves as a bridge between the iron and a positively charged group on the protein.     

Oxidation of diiron and triiron sulfurdithiolato complexes: mimics for the active site of [FeFe]-hydrogenase

The oxidation of the hexacarbonyl(1,3-dithiolato-S,S')diiron complexes 4a-4c with varying amounts of dimethyldioxirane (DMD) was systematically studied. The chemoselectivity of the oxidation products depended upon the substituent R (R=H, Me, 1/2 (CH2)(5)). For R=H, four oxidation products, 6a-6d, have been obtained. In the case of R=Me, three products, 7a-7c, were formed, and for R=1/2 (CH2)(5), only complex 8 was observed. These observations are due to steric and electronic effects caused by the substituent R. Additionally, oxidation of the triiron complex 5 with DMD was performed to yield the products 9a and 9b. X-Ray diffraction analyses were performed for 6a-6d, 7a, and 7c, as well as for 9a and 9b. The electronic properties were determined by density-functional theory (DFT) calculations.     

A re-investigation of the reactions between superoxide anion and metal picolinate complexes

Rate constants for the reactions of superoxide with the α-picolinate ion and its complexes with copper(II), iron(III) and zinc(II), and for the reaction of α-picolinate with the hydrated electron, were measured using pulse radiolysis. The rate constant for the reaction of superoxide with copper(II)picolinate at pH 9 [(4.1 ± 0.4) × 10⁷l mol⁻¹ s⁻¹] was an order of magnitude higher than that determined previously (W. H. Bannister, J. V. Bannister, A. J. F. Searle and P. J. Thornally, Inorg. Chim. Acta, 78, 139 (1983)) using a less direct competitive inhibition method. The corresponding rate constant for iron(III)picolinate [(7.5 ± 1.5) X 10³ l mol⁻¹ s⁻] was an order of magnitude lower than a previous pulse radiolysis determination (same reference as above). We are not able to reconcile these two values for iron(III)picolinate, although a possible source of spuriously high results is contamination with the kinetically active copper(II) complex. The likely roles of iron(III)picolinate and other low molecular weight iron complexes as potential catalysts of an in vivo superoxide-driven Fenton reaction are discussed in the light of present measurements.     

Composition and optical properties of reaction centre core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum

Photosynthetically active reaction centre core (RCC) complexes were isolated from two species of green sulfur bacteria, Prosthecochloris (Ptc.) aestuarii strain 2K and Chlorobium (Chl.) tepidum, using the same isolation procedure. Both complexes contained the main reaction centre protein PscA and the iron–sulfur protein PscB, but were devoid of Fenna–Matthews–Olson (FMO) protein. The Chl. tepidum RCC preparation contained in addition PscC (cytochrome c). In order to allow accurate determination of the pigment content of the RCC complexes, the extinction coefficients of bacteriochlorophyll (BChl) a in several solvents were redetermined with high precision. They varied between 54.8 mM⁻¹ cm⁻¹ for methanol and 97.0 mM⁻¹ cm⁻¹ for diethylether in the Q_Y maximum. Both preparations appeared to contain 16 BChls a of which two are probably the 13²-epimers, 4 chlorophylls (Chls) a 670 and 2 carotenoids per RCC. The latter were of at least two different types. Quinones were virtually absent. The absorption spectra were similar for the two species, but not identical. Eight bands were present at 6 K in the BChl a Q_Y region, with positions varying from 777 to 837 nm. The linear dichroism spectra showed that the orientation of the BChl a Q_Y transitions is roughly parallel to the membrane plane; most nearly parallel were transitions at 800 and 806 nm. For both species, the circular dichroism spectra were dominated by a strong band at 807–809 nm, indicating strong interactions between at least some of the BChls. The absorption, CD and LD spectra of the four Chls a 670 were virtually identical for both RCC complexes, indicating that their binding sites are highly conserved and that they are an essential part of the RCC complexes, possibly as components of the electron transfer chain. Low temperature absorption spectroscopy indicated that typical FMO–RCC complexes of Ptc. aestuarii and Chl. tepidum contain two FMO trimers per reaction centre. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spectroscopic properties of reaction center pigments in photosystem II core complexes: revision of the multimer model

Absorbance difference spectra associated with the light-induced formation of functional states in photosystem II core complexes from Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 (e.g., ) are described quantitatively in the framework of exciton theory. In addition, effects are analyzed of site-directed mutations of D1-His¹⁹⁸, the axial ligand of the special-pair chlorophyll P_D1, and D1-Thr¹⁷⁹, an amino-acid residue nearest to the accessory chlorophyll Chl_D1, on the spectral properties of the reaction center pigments. Using pigment transition energies (site energies) determined previously from independent experiments on D1-D2-cytb559 complexes, good agreement between calculated and experimental spectra is obtained. The only difference in site energies of the reaction center pigments in D1-D2-cytb559 and photosystem II core complexes concerns Chl_D1. Compared to isolated reaction centers, the site energy of Chl_D1 is red-shifted by 4 nm and less inhomogeneously distributed in core complexes. The site energies cause primary electron transfer at cryogenic temperatures to be initiated by an excited state that is strongly localized on Chl_D1 rather than from a delocalized state as assumed in the previously described multimer model. This result is consistent with earlier experimental data on special-pair mutants and with our previous calculations on D1-D2-cytb559 complexes. The calculations show that at 5 K the lowest excited state of the reaction center is lower by ∼10 nm than the low-energy exciton state of the two special-pair chlorophylls P_D1 and P_D2 which form an excitonic dimer. The experimental temperature dependence of the wild-type difference spectra can only be understood in this model if temperature-dependent site energies are assumed for Chl_D1 and P_D1, reducing the above energy gap from 10 to 6 nm upon increasing the temperature from 5 to 300 K. At physiological temperature, there are considerable contributions from all pigments to the equilibrated excited state P*. The contribution of Chl_D1 is twice that of P_D1 at ambient temperature, making it likely that the primary charge separation will be initiated by Chl_D1 under these conditions. The calculations of absorbance difference spectra provide independent evidence that after primary electron transfer the hole stabilizes at P_D1, and that the physiologically dangerous charge recombination triplets, which may form under light stress, equilibrate between Chl_D1 and P_D1.     

Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex

The exchange coupling of reduced uteroferrin has been measured (19.8(5) cm-1 S1.S2) using recently developed techniques for studying metalloprotein magnetization. A spin Hamiltonian describing the coupled binuclear Fe(II).Fe(III) center has been used to fit the low and high field magnetization data, the EPR g values, and the highly anisotropic effective hyperfine tensor of the ferric site. The exchange coupling of the phosphate complex of reduced uteroferrin has also been measured (6.0(5) cm-1 S1.S2) using the same techniques. The smaller exchange coupling of the phosphate complex is comparable with the zero field splittings of the iron sites. This results in increased sensitivity of the system g values (found by calculation from the spin Hamiltonian) to variations of the zero field splitting parameters arising from heterogeneities in the protein microenvironment. Consequently, there is a very significant (9-fold) increase in the "effective g strain" of the system compared to the situation in the absence of phosphate. This, together with the larger g anisotropy (g = (1.06, 1.51, 2.27)), gives rise to an EPR signal for the phosphate complex of reduced uteroferrin which is extremely broad and difficult to detect but which has now been identified for the first time.     

Evolution of the soluble diiron monooxygenases

Based on structural, biochemical, and genetic data, the soluble diiron monooxygenases can be divided into four groups: the soluble methane monooxygenases, the Amo alkene monooxygenase of Rhodococcus corallinus B-276, the phenol hydroxylases, and the four-component alkene/aromatic monooxygenases. The limited phylogenetic distribution of these enzymes among bacteria, together with available genetic evidence, indicates that they have been spread largely through horizontal gene transfer. Phylogenetic analyses reveal that the alpha- and beta-oxygenase subunits are paralogous proteins and were derived from an ancient gene duplication of a carboxylate-bridged diiron protein, with subsequent divergence yielding a catalytic alpha-oxygenase subunit and a structural beta-oxygenase subunit. The oxidoreductase and ferredoxin components of these enzymes are likely to have been acquired by horizontal transfer from ancestors common to unrelated diiron and Rieske center oxygenases and other enzymes. The cumulative results of phylogenetic reconstructions suggest that the alkene/aromatic monooxygenases diverged first from the last common ancestor for these enzymes, followed by the phenol hydroxylases, Amo alkene monooxygenase, and methane monooxygenases.