0000000000154632
AUTHOR
J. M. Moratal
COBALT SUBSTITUTED PROTEINS
Cobalt(II) has been extensively used as a spectroscopic probe in many proteins, mainly replacing zinc, but also substituting iron, manganese and copper ions. The relatively short electronic relaxation times of high spin cobalt(II) makes this ion suitable as a paramagnetic probe for Nuclear Magnetic Resonance spectroscopy. A survey of the NMR studies performed in cobalt substituted proteins is shown. In the zinc enzymes Carboxypeptidase A, Carbonic Anhydrase and Superoxide Dismutase the implications of these studies on their catalytic mechanisms are commented. Finally, a further insight in the research of the blue copper protein Azurin by applying NMR to its cobalt derivative is also reporte…
The Mechanism of Action of Carbonic Anhydrase
A survey of the structure-function relationship in the enzyme carbonic anhydrase is presented. The coordination number around the metal ion in derivatives inhibited with some anions is stressed as well as the role of the cavity in determining the coordination number. Inferences on the enzyme-substrate interactions are presented.
1H 2D-NMR characterization of Ni(II)-substituted azurin fromPseudomonas aeruginosa
1 H two-dimemional NMR experiments on nickel(II)-substituted azurin have been succesfully applied. Despite the short relaxation time of the hyperfine-shifted resonances, the combined use of NOESY and COSY spectra allowed the alignment of 15 resonances belonging to the metal-coordinated residues Gly-45, His-46, His-117 and Met-121. Even in the case of the two broad and furthest downfield resonances, the NOESY spectra were successful in assigning these signals to the β-CH 2 protons of Cys-112. The protons of the non-coordinated residues Met-13, Phe-15 and Trp-48 were also assigned via NOESY, COSY and TOCSY experiments
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 s…
Electronic characterisation of the oxidized state of the blue copper protein Rusticyanin by 1 H NMR: Is the axial methionine the dominant influence for the high redox potential
The oxidized state of rusticyanin, the blue copper protein with the highest redox potential in its class, has been investigated through (1)H nuclear magnetic resonance applied to its cobalt(II) derivative. The assignment of the protons belonging to the coordinated residues has been performed. Many other amino acids situated in the vicinity of the metal ion, including six hydrophobic residues (isoleucine140 and five phenylalanines) have also been identified. The orientation of the main axes of the magnetic susceptibility tensor for the cobalt(II)-rusticyanin as well as its axial, Deltachi(ax), and rhombic, Deltachi(rh), magnetic susceptibility anisotropy components have been determined. A co…
Backbone dynamics of rusticyanin: the high hydrophobicity and rigidity of this blue copper protein is responsible for its thermodynamic properties.
Local dynamics and solute-solvent exchange properties of rusticyanin (Rc) from Thiobacillus ferrooxidans have been studied by applying heteronuclear ((1)H, (15)N) NMR spectroscopy. (15)N relaxation parameters have been determined for the reduced protein, and a model-free analysis has been applied. The high average value of the generalized order parameter, S(2) (0.93), indicates that Rc is very rigid. The analysis of cross correlation rates recorded in both the reduced and the oxidized forms conclusively proves that Rc possesses the same dynamic features in both oxidation states. The accessibility of backbone amide protons to the solvent at different time scales has also been studied by appl…