Open Access1 H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin
Author(s) -
Salgado Jesus,
Jiménez Hermas R.,
Donaire Antonio,
Moratal Jose M.
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0358e.x
Subject(s) - azurin , cobalt , paramagnetism , crystallography , copper , chemistry , copper protein , coordination sphere , ligand (biochemistry) , histidine , metal , tetracoordinate , stereochemistry , inorganic chemistry , organic chemistry , physics , crystal structure , biochemistry , receptor , enzyme , computer graphics (images) , quantum mechanics , computer science , planar
Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 1 H‐NMR spectrum of Pseudomonas aeruginosa Co(II)‐azurin is presented here. All the observable contact‐shifted signals as well as many other paramagnetic signals from protons placed up to about 1.0 nm around the metal center, including some residues belonging to functionally important parts of the protein like the hydrophobic patch and the His35 region, have been assigned. The results obtained permit the detection and study of structural variations like those originated by the His35 ionization, and allow us to draw a feasible picture of the metal coordination site. Contact‐shifted signals correspond to the same five residues which are found in the coordination sphere of the native Cu(II)‐azurin, i.e. His46, His117, Cys112, Met121 and Gly45. Among them, the histidine residues present a pattern of resonances typical for histidines coordinated to cobalt in other cobalt protein derivatives, and the cysteine signals clearly indicate a strong interaction with the paramagnetic Co(II) ion. In contrast, the Met121 signals indicate a weak but still existent contact interaction with the metal center. On the other hand, the very weak copper ligand, Gly45, appears here as clearly coordinated to cobalt. Results are consistent with a distorted tetrahedral metal site with the cobalt deviated from the N 2 S plane towards the Gly45 O axial position and weakly interacting with the Met121 sulfur.