G. Canti

The epr spectra of the inhibitor derivatives of cobalt carbonic anhydrase.

The epr spectra at 4.2 K of inhibitor derivatives of cobalt carbonic anhydrase have been recorded. The spectra can be grouped into two classes according to whether the low-field signal is broad or sharp and with g ranges of 6.1-6.8, 2.3-2.9, 1.6-1.8, and 5.8-6.2, 2.2-2.8, 1.5-1.8, respectively, The two kinds of spectra have been empirically related to the features of the room-temperature solution electronic spectra. A third kind of epr spectrum with a single broad signal is obtained when the inhibitor is in large excess. The possibility of using the epr spectra for deducing the geometry of cobalt enzymes is discussed.

Evidence of exchangeable protons in the donor groups of the acidic form of cobalt bovine carbonic anhydrase B

T1 relaxation measurements on water protons of solutions containing cobalt(II) bovine carbonic anhydrase have been found to be affected by the paramagnetic center. T1 shortening has been found to be substantially pH independent. These data are diagnostic of the presence of exchangeable protons in the donor groups of the enzyme, and consistent with a water molecule in the donor set at low pH values. Previous researchers failed to reveal exchangeable protons at low pH values because of the presence of Tris-sulfate buffer which interacts with the metal ion.

Water in the coordination sphere of metallocarbonic anhydrases: A solvent proton longitudinal relaxation study at several frequencies

Abstract The proton longitudinal relaxation rates of water solutions of carbonic anhydrase derivatives in which the native zinc(II) ion has been substituted with the paramagnetic metal ions cobalt(II), copper(II) and manganese(II) have been measured at magnetic resonance frequencies betweeen 4 and 60 MHz. The investigation was extended to a number of systems containining inhibitors of the metal enzyme. The rate enhancements due to coupling between resonating protons and unpaired electrons vary significantly with magnetic field. For the cobalt and copper derivatives, correlation times has been estimated and structural information on the water in the enzymatic cavity have been obtained. The electronic relaxation times for the cobalt systems are related to the stereochemistry, four and five coordinated, of the metal ion. For the manganese derivative the electronic correlation times depend on the magnetic field. Such dependence has been analyzed in terms of structural data and the correlation time of the modulation of the zero field splitting.

Investigation of the system copper(II) carbonic anhydrase and HCO3−1/CO2

Copper(II) substituted human and bovine carbonic anhydrases B in the presence of bicarbonate have been investigated in solution through water-solvent proton nuclear magnetic resonance (nmr) at variable magnetic fields. HCO3-, contrary to all the other monoanionic inhibitors, partially reduces the water proton relaxation rates. This has been accounted for on the basis of the availability within the active cavity of two coordination positions partially overlapping. 13C-nmr measurements on both CO2 and HCO3- confirm that HCO3- binds the metal, whereas CO2 interacts with the paramagnetic center at nonbonding distance. The upper limit for the CO2 in equilibrium HCO3- interconversion has been estimated to be 10 sec-1.

Spectroscopic characterization of copper(II) thermolysin

The compound obtained from zinc-free thermolysin and copper(II) sulphate, which is biologically inactive, has been characterized together with some inhibitor derivatives of the native enzyme through electronic, e.s.r., and n.m.r. spectroscopies. The co-ordination at the metal is suggested to be pseudo-tetrahedral. A comparison with the similar copper(II)-substituted metalloenzyme carboxypeptidase is presented.

Characterization of oxovanadium(IV) substituted bovine carbonic anhydrase B

Abstract The compound obtained by combining the oxovanadium(IV) ion and apocarbonic anhydrase in water has been further characterized by means of electronic and nmr spectroscopy. The electronic spectra are typical of tetragonal complexes whereas water proton T 1 values indicate that a water molecule is bound to the paramagnetic center. The coordinating capability of the active cavity with respect to the various metal ions is comparatively discussed.

17O NMR investigation of copper(II) substituted carbonic anhydrases

Abstract The water 17 O longitudinal and transverse relaxation rates have been measured in solutions of copper substituted bovine and human carbonic anhydrases B and of some of their inhibitor derivatives, as well as in solutions of the corresponding native zinc enzymes. The sizeable transverse relaxation rate enhancement observed when the paramagnetic copper(II) ion is substituted for zinc(II) is ascribed to contact electron-nuclear coupling, typical of water coordinated to a copper(II) chromophore in equatorial position. Exchangeable H 2 17 O is present in the metal coordination sphere all over the pH range 5–10, as well as in the adducts with the monodentate anionic inhibitors, while it is removed in the adducts with the bidentate oxalate and with p -toluene-sulfonamide. For other sulfonamide derivatives the lack of paramagnetic effect on H 2 17 O is ascribed either to a more axial position of the coordinated water or to a slower exchange rate with the bulk solution.

Investigation of the system cobalt(II) bovine carbonic anhydrase b-trichloroacetaldehyde

The interactions between hydrated trichloroacetaldehyde and cobalt(II)bovine carbonic anhydrase B have been investigated as a function of pH by means of electronic spectroscopy of FT nmr spectroscopy. The hydrated aldehyde is bound to the metal ion and its apparent affinity constant is pH dependent with a bell-shaped profile. The kinetic parameters of the dissociation process have also been determined.

Cyanometallates and cobalt(II) bovine carbonic anhydrase B. Five coordination with dicyanoaurate(I)

Abstract The binding affinities of various cyanometallates for cobalt(II) bovine carbonic anhydrase B have been investigated by means of optical and magnetic resonance spectroscopies. Dicyanoaurate(I) and dicyanoargentate(I) ions show a large affinity for the enzyme, giving rise to a five coordinate adduct in the former case and to equilibria between four and five coordinate species in the latter case. Five coordination is reached through three histidine nitrogens, a cyanide nitrogen and a water molecule. Hg(CN) 2 , Fe(CN) 4− 6 , M(CN) 2− 4 (M = Ni, Pd) and Au(CN) − 4 do not show appreciable affinity for the enzyme either on account of the charge (Hg(CN) 2 ) or of the ionic dimensions.

Spectroscopic investigation of copper(II) bovine carbonic anhydrase and its inhibitor derivatives

Cooper(II) bovine carbonic anhydrase and its derivatives with ligands which act as inhibitors of the native zinc(II) and of the cobalt(II) enzyme have been investigated by n.m.r. and e.s.r. spectroscopy. Measurements of T1 of the water protons of copper enzyme solutions have shown that in the donor set there is a group containing mobile hydrogens which is not substituted by anionic donors. The derivatives with anionic ligands have been assigned a five-co-ordinate structure, whereas the sulphonamide derivatives have been assigned a pseudo-tetrahedral stereo-chemistry. For the copper enzyme a five-co-ordinate structure is also suggested, in which the distance between the metal atom and one of the donors is longer than normal.