Structure of metal site in azurin, met 121 mutants of azurin, and stellacyanin investigated by 111m Cd Perturbed Angular Correlation (PAC)

The geometries of the metal sites in cadmium-substituted azurins have been investigated by Cd-111m perturbed angular correlation (PAC), The study includes wild type azurin as well as Met(121) mutants of azurin, where methionine has been substituted by Ala, Asn, Asp, Gin, Glu, and Leu.The nuclear quadrupole interaction of wild type azurin analyzed in the angular overlap model is well described as coordination of His(46), His(117), and Cys(112) and cannot be described by coordination of Met(121) and/or Gly(45).For most of the mutants, there exist two coordination geometries of the cadmium ion, With the exception of the Gau and Asp mutants, one of the conformations is similar to the wild type conformation. The other coordination geometries are either best described by a coordinating water molecule close to the original methionine position or by coordination by the substituting amino acid, These experiments show that even though the methionine does not coordinate it plays an important role for the geometry of the metal site.The nuclear quadrupole interaction of stellacyanin was also measured, The value resembles the most prominent nuclear quadrupole interaction of the Met(121) --> Gin mutant of Alcaligenes denitrificans azurin, indicating that the structures of the two metal sites are similar.Macromolecular Biochemistr

1H and 15N nuclear magnetic resonance assignments, secondary structure in solution, and solvent exchange properties of azurin from Alcaligenes denitrificans

Complete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14 000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32-degrees-C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in N H chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C(alpha) protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3J(HNalpha) values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N(epsilon2)H of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.Macromolecular Biochemistr

Isolation and sequencing of the Alcaligenes denitrificans azurin-encoding gene: comparison with the genes encoding blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis

Macromolecular Biochemistr

The mutation Met121His creates a type-1.5 copper site in Alcaligenes denitrificans azurin.

The Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pK(a) of 3.8 due to the ionization of His121. The [M121H]azurin mutant exhibits two major distinct metal-site-coordination geometries which coexist in solution according to a pH-dependent equilibrium. Both species have been spectroscopically characterized by ultraviolet-visible, EPR and resonance Raman spectroscopies. At neutral DH. His121 is deprotonated and acts as the fourth ligand of the Cu; the spectroscopic characteristics of the Cu site at this pH are halfway between those of a type-1 and a type-2 Cu site, and the site is referred to as a type-1.5 or intermediate Cu site. The spectral data are compatible with a tetrahedral geometry of this site. At low pH, the spectroscopic data indicate that [M121H]azurin has a trigonal type-1 rhombic Cu site.Macromolecular Biochemistr