Complexes of tetra(parasulphonatephenyl)porphyrinato ferrate(III), FeTPPs, with 1,10 phenanthroline and 2,2′ bipyridine ligands

Abstract Iron(III) porphyrin, FeTPPS, gives high-spin 1:1 monomeric complexes with the ligands 1,10 phenanthroline, 2,9 dimethylphenanthroline and 2,2 bipyridine in aqueous solution. Absorption spectrum of the complexes is very different to the one of the monomer metalloporphyrin, but nearly identical to the visible spectrum of the μ-oxo dimer Fe2(TPPS)2O. A spectrophotometric study of the formation equilibria yields the following values of the stability constants: Me2phen 4.3 × 104, phen 1.1 × 104, bipy 2.0 × 102 (25 °C, 0.1 M NaNO3). Analysis of the experimental data strongly suggest that the ligands are hydrogen bonded to one coordinated water molecule (TPPS)FeOH2 → (LL). Although the …

Violurato complexes of transition metals.

NMR and homology modeling studies of copper(II)-halocyanin from Natronobacterium pharaonis bacteria

Abstract Halocyanin from the haloalkaliphilic archaean Natronobacterium pharaonis is a peripheral membrane type 1 blue copper protein with a single polypeptide chain of 163 amino acid residues. Halocyanin participates as putative electron carrier protein associated to an electron acceptor role for a terminal oxidase and has the lowest redox potential value reported to date for a BCP. NMR studies and homology modeling calculations were performed to evaluate the electronic properties of Cu(II)-halocyanin from Natronobacterium pharaonis . The copper coordination site properties of Cu(II)-halocyanin are discussed. The 1 H NMR spectra, isotropic chemical shifts and relaxation times for halocyani…

Paramagnetic NMR investigations of Co(II) and Ni(II) amicyanin.

The paramagnetic 1H NMR spectra of the Co(II) and Ni(II) substituted forms of the type 1 blue copper protein (cupredoxin) amicyanin have been assigned. This is the first such analysis of a cupredoxin, which has a distorted tetrahedral active site with the ligands provided by two histidines, a cysteine and a methionine. The isotropic shifts of the resonances in these spectra are compared with those of Co(II) and Ni(II) azurin. A number of interesting similarities and differences are found. The coordination of the metal by the two equatorial histidine ligands is very similar in both proteins. The interaction between the introduced metal and the thiolate sulfur of the equatorial cysteine ligan…

Catecholato complexes of o-phenylenebis(salicylideneiminato)iron(III) and meso-tetra(parasulphonatephenyl)porphyrinatoferrate(III). A comment on the structure of the active site in catechol-1,2-dioxygenase

Abstract Formation of catecholato complexes of Fe(saloph) + and Fe(TPPS) 3− in solution is studied. Fe(saloph)(cat) − contains a cat 2− bidentate ligand. Its formation in solution competes efficiently with the hydrolysis and dimerization of Fe(saloph) + to give Fe 2 (saloph) 2 O. This behaviour shows that the planar saloph 2− ligand, as the analogous salen 2− , is easily distorted, and is not as rigid as generally considered. Iron(III) porphyrin Fe(TPPS) 3− with catechol gives the complex [Fe(TPPS)(Hcat)] 4− . Deprotonation of the unidentate Hcat − ligand cannot be studied because the smaller stability of the complex, and the dimerization of the metalloporphyrin dominates in basic medium. T…

Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to esti…

Protein Unfolding:1H-NMR Studies of Paramagnetic Ferricytochrome c-550 from Horse Heart

Electronic transfer protein cytochrome c-550 from horse heart is studied in the unfolded state by means of paramagnetic 1H NMR. The protein contains 104 aminoacid residues and a heme group with low spin FeIII ion in the oxidized form of protein. The global secondary structure is of the α-helix type as occurs in the case of very other cytochromes c investigated such as cyt c-550 from Thiobacillus versutus or cyt c-551 from Pseudomonas aeruginosa. We have studied the coordination characteristic and electronic properties of heme iron horse heart ferricytochrome c-550 at increasing denaturing conditions (up to 3.1 M GuHCl and 288-323 K). The 1H T1 values of the signals were measured and some re…

Metal coordination of azurin in the unfolded state.

Abstract1H NMR data applied to the paramagnetic cobalt(II) derivative of azurin from Pseudomonas aeruginosa have made it possible to show that the metal ion is bound to the protein in the unfolded state. The relaxation data as well as the low magnetic anisotropy of the metal ion indicate that the cobalt ion is tetrahedral in the unfolded form. The cobalt ligands have been identified as the residues Gly45, His46, Cys112 and His117. Met121 is not coordinated in the unfolded state. In this state, the metal ion is not constrained to adopt a bipyramidal geometry, as imposed by the protein when it is folded. This is clear confirmation of the rack-induced bonding mechanism previously proposed for …

Phenolate complexes of iron(III) in dimethylsulphoxide solution

The formation of complexes between Fe3+ and 2,4-dinitrophenol, 4-nitrophenol and 4-methylphenol is studied in dimethylsulphoxide solution. The reaction proceeds almost to completion and the occurrence, in solution, of complexes with higher stoichiometry than 1∶1 is reported for the first time. The following stability constants are determined (25 °C, 0.1 M KClO4): FeIII-2,4-dinitrophenolate β1=1.8×103, β2=4.4×105; FeIII-4-nitrophenolate β1=1.10×107, β2=2.5×1012 β3=3.9×1016; FeIII-4-methylphenolate β=1.7×1012.

1H-NMR study of a cobalt-substituted blue copper protein: Pseudomonas aeruginosa Co(II)-azurin.

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1H-NMR spectrum of Pseudomonas aeruginosa Co(II)-azurin is presented here. All the observable contact-shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible pictur…

VIOLURATO COMPLEXES OF Cr(III). SYNTHESIS AND CHARACTERIZATION. PROTONATION-DEPROTONATION EQUILIBRIA OF THE COORDINATED LIGANDS. EVIDENCE OF THE COORDINATION OF VIOLURIC ACID AS A NEUTRAL LIGAND

Abstract The formation of violurato complexes of Cr(III) has been studied in aqueous solution. Two compounds, [Cr(H2V)3].5H2O and Na[Cr(H2V)2(OH)2].4H2O have been synthesized and characterized. [Cr(H2V)3] behaves in aqueous solution as a triprotic acid but it can also add a proton yielding [Cr(H2V)2(H3V)]+. The acidity constants of this species have been determined: pKa1 =3.32, pKa2 =4.25, pKa3 =4.83 and pKa4 =6.99 (25°C, 0.1 M NaC1O4). [Cr(H2V)2(OH)2]− undergoes a deprotonation of the two H2V− ligands and a protonation of the hydroxo ligands. The acidity constants of [Cr(H2V)2(H2O)2]+ have been also determined: pKa1 =3.5, pKa2 =4.6, pKa3 =7.1 and pKa4 =9.2 (25°C, 0.1 M NaC1O4). The coordin…

Violurato complexes of nickel(II). Formation equilibria. Deprotonation equilibria of the coordinated ligands and related stereochemical changes

The formation equilibria of nickel(II) violurato-complexes in water and dimethylsulphoxide-water (80∶20) as well as deprotonation of the coordinated ligands and related stereochemical changes are reported. The stability constants of the Ni2+-H2V− complexes logβ1=5.06, Iogβ2=9.38, logβ3=12.98 as well as the acidity constants of the [Ni(H2V)3]− complex, logβj1= 8.37, logβj2=15.76, logβj3=22.37 are determined at 25 °C and 0.1M NaClO4. A new violurato-complex of Ni2+, Na4[Ni(HV)3]5H2O, is isolated and characterized.

1H NMR studies of paramagnetic ferricytochrome c-551 from Pseudomonas aeruginosa at high pH: The role of histidine 16 in the spin transition

Abstract Cytochrome c-551 from the mesophile Pseudomonas aeruginosa is an electronic transfer protein that contains 82 amino-acid residues and a c-type heme as the prosthetic group with low spin Fe(II) in the reduced form and low spin Fe(III) in the oxidized form of cytochrome c-551. We have studied the electronic properties of ferricytochrome c-551 from P. aeruginosa at high pH (9–11.4) by means of paramagnetic 1H NMR spectra and the T1 and T2 values of isotropically shifted proton resonances. We have also analyzed the temperature dependence of the hyperfine-shifts. Resonance assignment of some signals was based on 2D saturation transfer experiments, EXSY. These results indicate the existe…

Two-dimensional 1 H NMR spectra of ferricytochrome c 551 from Pseudomonas aeruginosa

AbstractThe full assignment of 1H NMR signals of heme proton resonances of ferricytochrome c551 from Pseudomonas aeruginosa has been performed by means of 2D NMR experiments. This technique allows the complete and unequivocal assignment of all heme resonances, including methylene resonances of the propionic groups, directly implicated in the pH dependence of the redox properties of cytochrome c551.

An NMR view of the unfolding process of rusticyanin: Structural elements that maintain the architecture of a β-barrel metalloprotein

The unfolding process of the blue copper protein rusticyanin (Rc) as well as its dynamic and D(2)O/H(2)O exchange properties in an incipient unfolded state have been studied by heteronuclear NMR spectroscopy. Titrations of apo, Cu(I), and Cu(II)Rc with guanidinium chloride (GdmCl) show that the copper ion stabilizes the folded species and remains bound in the completely unfolded state. The oxidized state of the copper ion is more efficient than the reduced form in this respect. The long loop of Rc (where the first ligand of the copper ion is located) is one of the most mobile domains of the protein. This region has no defined secondary structure elements and is prone to exchange its amide p…

Azide and chloride binding to carboxypeptidase A in the presence of L-phenylalanine

The interaction of chloride with native and cobalt (Co)-substituted carboxypeptidase-A (CPD) has been investigated by 35Cl nuclear magnetic resonance (NMR) spectroscopy in the presence and absence of L-Phe. The affinity constants of azide and chloride toward the Co(II)CPD·L-Phe complex have been measured by electronic spectroscopy. The correlation times determining T1 and T2 for the 35Cl nuclei are related to movements inside the cavity. In the presence of L-Phe, the anions bind to the metal with a relatively high affinity at pH values below 6. Anion binding to the Co enzyme can be analyzed in terms of the three protonation state model for the enzyme (EH2 α EH α E). In the presence of L-Phe…

Paramagnetic Cobalt and Nickel Derivatives of Alcaligenes denitrificans Azurin and Its M121Q Mutant. A 1H NMR Study

Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His…

Interaction of sulphate and chloride with cobalt(II)-carbonic anhydrase

The interaction between Cobalt(II)-Bovine Carbonic Anhydrase II and the inhibitors sulphate and chloride have been investigated through 1H NMR and electronic absorption spectroscopies. Both inhibitors bind to the metal ion forming a 1:1 adduct and the corresponding affinity constants have been determined. These inhibitors interact weakly with CoBCA II and this interaction only occurs at low pH values. The T1 values of the meta-like protons of the coordinated histidines have been measured. The coordination number of the metal ion in the adducts is discussed on the basis of temperature dependence of the isotropic shifts, T1, and molar absorbance values.

The Crystal Structure of Nickel(II)-Azurin

The nickel(II)-azurin metalloderivative has been crystallized and its structure solved at 0.205-nm resolution by X-ray diffraction. The overall structure is not modified by the metal exchange and the only differences with regard to the native copper(II)-azurin occur in the metal site region. These variations affect principally the axial ligands. Nickel co-ordinates more strongly to the carbonyl oxygen of Gly45 while its distance to the Met121 S4 enlarges up to 0.330 nm. The resulting metal center structure is intermediate between those of the Cu(II) and Zn(II) azurins, and can be described as distorted tetrahedral. However, the existence of contact interaction between Met121 and the nickel …

1H-NMR spectroscopic studies of paramagnetic superstructured iron(III) porphyrinsThis article is dedicated to the memory of Professor Michel Momenteau of Institut Curie.Abbreviations. 1D and 2D: one-dimensional and two-dimensional; COSY: correlated spectroscopy; NOESY: nuclear Overhauser effect spectroscopy; TPP: 5,10,15,20-tetraphenylporphyrinate dianion.

The electronic properties of several superstructured iron(III) porphyrins, in which the two faces of the porphyrin are protected by two chains linked at the opposite phenyl rings, have been investigated. The paramagnetic 1H NMR spectra of three iron(III) “basket-handle” porphyrins such as FeIII[e-BHP(C12)2], FeIII[a-BHP(C12)2] and FeIII[e-BHP(C12)(ImC11)] in organic solvent have been assigned. The 1H T1 values of the signals were measured and resonance assignments made based on NOESY and COSY experiments. The electronic structure of the iron(III) is discussed on the basis of the temperature dependence of the isotropic shifts and relaxation times. The hyperfine-shifted resonances in these sp…

The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives

The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by 1H NMR of the Cu(II) and Co(II) paramagnetic metalloderivatives. In both cases up to three species are observed in slow exchange, the proportions of which are different for the two metalloderivatives. Above pH 5 the major species displays a tetrahedral coordination in which the His121 can be observed as a coordinated residue. Its metal site corresponds to a new type of site that is defined as a type 1.5 site. The second and third species resemble the wild type (type 1) azurin and, above pH 4.5, they are present only at a low concentration. At low pH a protonation process increases…

Coordinating properties of the Cephalexine antibiotic. A potentiometric study of the complexes formation between Cephalexine and Co(II), Ni(II) and Cu(II) metal ions

Abstract The formation of complexes between Co(II), Ni(II) and Cu(II) with Cephalexine has been investigated using potentiometric techniques. The stability constants of the complexes formed were calculated using the non-linear least-squares computer program SUPERQUAD. The obtained values were: Co(II) logβ1=2.40, logβMLOH=8.89; Ni(II) logβ1=2.80, logβ2=5.10, logβML2OH=12.09; Cu(II) logβ1=4.094 (25 °C, 0.1 M KNO3). The compound [Ni(CEX)(OH2)4]BPh4 has been synthesized and characterized by electronic, IR and NMR spectroscopies as well as by magnetic measurements. From these studies it is proposed that the Cephalexinate anion acts as a bidentate ligand and is bound to the metal ion through the …