G. Lanini

The electron-nucleus dipolar coupling in slow rotating systems. 3. The effect of isotropic exchange coupling of the electron spin with a second paramagnetic center

Abstract The solvent proton NMRD of a solution containing bis(ethylenediamine)copper(II) and hexacyanoferrate(III) has been measured at magnetic fields between 0.01 and 60 MHz. The two metal ions are known to be weakly magnetically coupled. The NMRD dispersion is similar to that expected on the basis of the simple Solomons approach although the values are sensibly smaller than those observed in solutions containing bis(ethylenediamine)copper(II) and diamagnetic hexacyanocobaltate(III). A theoretical treatment of the nucleus-unpaired electrons coupling, the latter under isotropic magnetic coupling conditions, is capable of accounting for the observed pattern.

1H NMR relaxation rate and coordination number in high spin cobalt(II) complexes

Abstract 1H T−11 values were measured for bis-salicylaldiminate cobalt(II) complexes with overall coordination numbers of four, five and six. It was found that the nuclear relaxing capabilities decrease in the order four- > five- > six-coordinate complexes. A semiquantitative analysis of the various contributions to the proton relaxation rates was performed. The data are discussed in terms of their relevance for determining the coordination number of cobalt(II) substituted zinc proteins.

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase

Abstract1H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition 13C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the β-CH2 protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The 1H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3-and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD+ only the resonances of the β-CH2 protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation. The only changes observed are the β-CH2 proton resonances of the metal-coordinating cysteines which are sensitive to local conformational changes within the ternary complex Co(II)-HLADH · Imidazole · NADH in the open conformation or global changes in the ternary complex Co(II)-HLADH · Pyrazole · NAD+ in the closed conformation. Acetate which can be regarded as a substrate model was shown to induce a similar change in the optical spectra of the Co(II) enzyme as all other anions observed so far. From the optical changes a dissociation constant of acetate at the catalytic metal site of 200±50 mM was calculated and from the changes of the 13C-NMR linewidth of 13C acetate direct bonding of the anion to the catalytic Co(II) ion can be demonstrated to occur under the conditions of rapid exchange. The implications of these data for the assessment of tetracoordination around the catalytic metal ion as well as the chemical nature of intermediates occurring along the catalytic pathway are discussed.

Investigation of cobalt(II) substituted carboxypeptidase a interacting with azide and cyanate ions

Abstract Cobalt(II)-substituted carboxypeptidase A has been found to reversibly bind N3 − and NCO − , but not NCS − , in the pH range 5–10, thus including the pH range of activity of the enzyme. The pH dependence of the anion binding constant is affected by two ionizations, which are assigned as those regulating k cat and K M . The electronic and 1 H NMR spectra are consistent with a substantially pseudotetrahedral geometry of the anion derivatives.

Are there other acidic groups capable of affecting the electronic spectra of cobalt(II) substituted carbonic anhydrase

Abstract The electronic spectra of NCS − and I − adducts of cobalt(II) human carbonic anhydrase I are pH dependent at pH values below 7. The pK a of such equilibrium is dependent on the anion concentration and varies between 4.6 and 6.6. The 1 H NMR spectra show that the three histidine residues are bound to the metal ion over the entire pH range investigated. It is supposed that a Glu residue triggers the change in stereochemistry around the metal ion. It is possible that such a Glu residue is Glu 106 present in the active cavity.

A water 1H and 17O N.M.R. study on PHG-modified SOD

The low-activity phenylglyoxal (PHG) modified bovine copper zinc superoxide dismutase (SOD) has been studied through 1H and 17O NMR of solvent water. Water 1H NMR T1−1 values have been measured at magnetic fields between 4 and 60 MHz. The data for PHG modified SOD provide evidence that water is still semicoordinated in the axial position, though possibly at a greater distance than in native SOD. Water 17O NMR data support this finding.

Differences between high activity bovine carbonic anhydrase B and low activity human carbonic anhydrase B monitored through metal substitution

The properties of cobalt(II) and copper(II) substituted bovine B and human B isoenzymes of carbonic anhydrase have been compared; the different coordination number of the metal ion in the low pH forms of the two isoenzymes, the different behavior of small anions like NCS− and I− and other molecules like imidazole and related ligands, and the different dissociation rate of HCO3− with respect to the two isoenzymes reflect some differences in the active site flexibility of the human and bovine isoenzymes.

The investigation of cobalt(II) substituted carbonic anhyrase and carboxypeptidase A

It is generally accepted that information obtained on cobalt(II) complexes can be transferred to the analogous zinc compounds. Therefore substitution of zinc(II) with cobalt(II) in zinc containing enzymes allows one to investigate these systems through spectroscopic techniques. The data reported here are concerned with carbonic anhydrase (CA) and carboxypeptidase A (CPA). 1H NMR data in D2O on CoCa reveal a relatively sharp signal assigned to the β proton of the histidine bound to the metal. Its T−1 value is constant with pH for the bovine CA isoenzyme B (BCAB) whereas it s lower at low [H in the case of human CA isoenzyme (HCAB). These data parallel the pH dependence of the water 1H NMR data at every magnetic field [1, 2]. It is proposed that in the latter case (HCAB) a change in coordination chemistry occurs at low pH, and in particular that five coordinate species are obtained, whereas CoBCAB at every pH and CoHCAB at high pH are pseudotetrahedral. The general equilibrium is Model compounds show that nuclear longitudinal relaxation decreases from tetra- to five- to six-coordination in cobalt(II) complexes. The above equilibrium is consistent with the difference in the electronic spectra and in the pKa between the two isoenzymes. Water 1H NMR data, coordinated histidine 1H NMR data, electronic and EPR data are in our opinion consistent with five-coordination in CoCPA [3]. We believe that five coordination is reached through two water molecules. N3− is found to bind cobalt(II) providing a derivative the electronic spectrum of which can be interpreted as being due to tetracoordination. The affinity of N3− for the enzyme decreases with pH, the pKa being around 9. This behavior of N3− is analogous to that shown with CoHCAB both with respect to change in coordination number and pH dependence of the affinity constants.

The binding of cobalt(II) to apo alkaline phosphatase

Abstract Alkaline phosphatase from Escherichia Coli is a dimeric zinc enzyme which catalyzes the hydrolysis of monophosphoric esters; it contains four zinc(II) and two magnesium(II) ions per molecule. Each subunit contains a ‘catalytic’ zinc ion, a ‘structural’ zinc ion and a ‘regulatory’ magnesium ion. The low resolution crystal structure shows that the catalytic and structural zinc ions are about 5 A apart, while the magnesium ion is about 30 A away from the above-mentioned ions [1]. The enzyme can be totally depleted of metal ions and reconstituted by the addition of two, four or six like or unlike metal ions. Since a high resolution X-ray structure is not yet available, the geometry of the metal sites and the donor group should be argued only from different spectroscopic data. Otvos et al . proposed, on the basis of 113 Cd and 13 C NMR data, that the catalytic metal ion could be coordinated by four histidine nitrogens [2]. Previous water proton NMR data indicated that a water molecule is present in the first coordination sphere of Mn 2+ , Cu 2+ and Co 2+ alkaline phosphatase, substituted st the catalytic sites [3]. Combining these data, the catalytic metal ion should be five-coordinated; this hypothesis is in agreement with our recent proposal of five coordination of cobalt(II) ion in the catalytic site, based on the relatively low molar absorbance of the electronic spectra [4]. Structural and regulatory sites were assigned as pseudo-octahedral on the basis of the low molar absorbance of the cobalt(II) derivative. We have titrated apoalkaline phosphatase solutions at different pH values with increasing amounts of cobalt(II) ions in order to shed light on the distribution of metal ions among the various metal sites, which is still a matter of discussion. We worked with unbuffered and unsalted solutions either in the presence or absence of magnesium(II) ions. When excess Mg 2+ is present, only two cobalt(II) ions are required to develop fully the electronic spectrum typical of the catalytic site, while in its absence four cobalt(II) ions are required. We thus propose that when magnesium is present, cobalt(II) has a higher affinity for the catalytic site than for the structural sites, while in the absence of magnesium the affinity of cobalt(II) ions for the two sites is comparable; this holds both for the low and high pH limits. We also recorded the spectrum of cobalt(II) in the structural site at low pH in the presence of a 2:1 ratio of copper(II) ions to apoenzyme molecule, where copper is selectively bound only to the structural sites [5]; the difference spectrum gives a molar absorbance of around 10 per cobalt, which we assign to the cobalt(II) ions bound in the pseudo-octahedral environment of the structural site. It was previously reported that for the system M 2 AP only one mol of inorganic phosphate is required to obtain to limit spectrum for the phosphate adduct [6], while for the M 4 AP system, two equivalents of phosphate are required. We have titrated both Co 2 Mg 4 AP and CO 4 Mg 2 AP with inorganic phosphate and we found that in both cases only one mol of phosphate is required to develop fully the spectrum of the phosphate adduct. This result is in contrast with the model worked out for the enzymatic activity of AP [7].