José M. Moratal

Low-spin six-co-ordinate cobalt(II) complexes. A solution study of tris(violurato)cobaltate(II) ions

The formation of complexes of CoII with violuric acid (1H,3H-pyrimidine-2,4,5,6-tetrone 5-oxime), H3vi, and its monomethyl (H2mvi) and dimethyl (Hdmvi) derivatives in dimethyl sulphoxide–water (80 : 20) has been investigated. Potentiometric, calorimetric, magnetic, and e.s.r. measurements clearly evidence that [Co(H2vi)3]–, [Co(Hmvi)3]–, and [Co(dmvi)3]– are low-spin octahedral complexes. The high spin–low spin change occurs upon binding of the third ligand.

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

Abstract 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 ligand is enhanced in the amicyanin derivatives. Resonances belonging to the weak axial methionine ligand exhibit much larger shifts in the amicyanin derivatives, indicative of shorter M(II)-S(Met) distances. The presence of shorter axial M(II)-S(Met) and equatorial M(II)-S(Cys) distances in both Co(II) and Ni(II) amicyanin is ascribed to the absence of a second axially interacting amino acid at the active site of this cupredoxin.

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 by1H 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 the proportion of both type 1 species at the expense of the type 1.5 species. This process, characterized by a pK a = 4.3, is assigned to the protonation of His121. At high pH the NMR spectrum of the Co(II)-M121H azurin experiences an additional transition, which is not observed in the case of the Cu(II) protein. The dynamic properties of the M121H metal site appear to be related to changes in the coordination geometry and the strength of the axial interaction between the Nδ1(His121) and the metal.

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 carbonyl and the amino-NH2 groups of the side chain.

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. The strong tendency of the cat 2− anion to be coordinated to Fe(III) in chelate form only can be sterically hindered. On the basis of these results the suggested structure of the active site of catechol-1,2-dioxygenase is discussed.

Solution chemistry of NN′-ethylenebis(salicylideneiminato)iron(III). Part 1. Deprotonation equilibria and reversible decomposition in acid medium of NN′- ethylenebis(salicylideneimine). Stability constant of NN′- ethylenebis(salicylideneiminato)iron(III)

NN′-Ethylenebis(salicylideneimine), H2 salen, behaves as a weak diprotic acid in dimethyl sulphoxide (dmso)–H2O (80 : 20 w/w) solution. The values of the overall association constants are βj1=(1.63 ± 0.05)× 1012 dm3 mol–1 and βj2=(2.04 ± 0.02)× 1023 dm6 mol–2. H2 salen undergoes slow hydrolytic decomposition in acid medium [equations (i) and (ii)](Hsal = salicylaldehyde, en = ethylenediamine); βh1=(2.60 ± 0.05)× 104 and βh2=(1.60 ± 0.02)× 107. H2 salen + H+ [graphic omitted] Hsal + HOC6H4CHN(CH2)2NH3+(i), H2 salen + 2 H+ [graphic omitted] 2 Hsal + H2en2+(ii) The favourable thermodynamics for the hydrolysis are provided by the protonation of en. In neutral or basic media the hydrolysis is not spontaneous in spite of the large water content of the solvent. Solutions of [{Fe(salen)Cl}2] in dmso–H2O contain the cation [Fe(salen)]+. The chloride is completely displaced from the co-ordination sphere of FeIII by a solvent molecule, and [Fe(salen)]+ decomposes, in acid medium, very slowly [equation (iii)]. [Fe(salen)]++ 4 H+⇄ Fe3++ 2 Hsal + H2en2+(iii) The study of this equilibrium allows the stability constant of the complex, Fe3++ salen2–⇄[Fe(salen)]+, β=(7.1 ± 0.1)× 1025 dm3 mol–1 to be obtained. This is the first reported stability constant of a metal complex of H2 salen. All equilibrium constants were determined at 25 °C and 0.1 mol dm–3 KClO4 in dmso–H2O (80 : 20 w/w).

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 D2O/H2O 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 protons. In contrast, the last loop (including a short α‐helix) and the last β‐strand (where the other three ligands of the metal ion are located) form the most rigid domain of the protein. The results taken as a whole suggest that the first ligand detaches from the metal ion when the protein unfolds, while the other three ligands remain bound to it. The implications of these findings for the biological folding process of Rc are also discussed.

Metal coordination of azurin in the unfolded state

1H 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 the metal ion in azurin.

1H NMR and UV-vis spectroscopic characterization of sulfonamide complexes of nickek(II)-carbonic anhydrase. Resonance assignments based on NOE effects

The binding of acetazolamide, p-fluorobenzensulfonamide, p-toluenesulfonamide, and sulfanilamide to nickel(II)-substituted carbonic anhydrase II has been studied by 1H NMR and electronic absorption spectroscopies. These inhibitors bind to the metal ion forming 1:1 complexes and their affinity constants were determined. The 1H NMR spectra of the formed complexes show a number of isotropically shifted signals corresponding to the histidine ligands. The complexes with benzene-sulfonamides gave rise to very similar 1H NMR spectra. The NMR data suggest that these aromatic sulfonamides bind to the metal ion altering its coordination sphere. In addition, from the temperature dependence of 1H NMR spectra of the p-fluorobenzenesulfonamide adduct, a conformational change is suggested. The T1 values of the meta-like protons of the coordinated histidines have been measured and resonance assignments based on NOE experiments were performed.

Spectroscopic studies of nickel(II) carbonic anhydrase and its adducts with inorganic anions

Nickel(II) carbonic anhydrase, NiBCA II, and its adducts with nitrate, acetate, cyanate and azide, have been investigated through 1H NMR and electronic absorption spectroscopies. From the pH dependence of the molar absorbance the acidity constants of NiBCA were determined. The anions bind to the metal ion forming 1 : 1 adducts, and the corresponding affinity constants have been determined. The 1H NMR spectra of NiBCA and its adducts have been recorded, and the signals corresponding to the meta-like protons of the co-ordinated histidines followed by 1H NMR titration. The T1 values of these signals were measured and resonance assignments made based on nuclear Overhauser enhancement experiments. The co-ordination geometry of the metal ion in NiBCA and its adducts is discussed on the basis of the temperature dependence of the isotropic shifts, molar absorbance, and longitudinal relaxation times.