¹ H‐NMR Study of a Cobalt‐Substituted Blue Copper Protein: Pseudomonas Aeruginosa Co(II)‐Azurin

Abstract

Substitution of copper by cobalt in blue copper proteins gives a paramagnetic metalloderivative suitable for paramagnetic NMR studies. A thorough analysis of the 'H-NMR spectrum of Pseudornonas aeruginosa Co(I1)-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(I1)-azurin, i.e. His46, Hisll7, Cysll2, 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(I1) 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,S plane towards the Gly45 0 axial position and weakly interacting with the Met121 sulfur.Keywords. Azurin; paramagnetic NMR; metal substitution; type 1 copper proteins ; cobalt proteins.Substitution of the naturally occumng metals in metalloproteins is used as a common strategy in inorganic biochemistry [l, 21. In a first approach it can be considered as a kind of sitedirected mutation, the most simple we can think of in the case of a metalloprotein. In this sense, apart from the possible function of the metal, it can give an idea of the metal site selectivity and structural rigidity, the mechanism of metal uptake, some general chemical properties of the metalloprotein, etc. In some special cases metal substitution is used to probe the metal site [ 1 -31. This is possible when the chromophore that brings along the exogenous metal has peculiar spectroscopic properties. For the blue copper proteins, like azurin, the natural copper(I1) center has itself singular, informative and in some way exciting spectroscopic properties (41. These are principally EPR and electronic spectral characteristics whose extensive study throughout the last few decades has achieved a detailed knowledge of type 1 copper sites [4-61. But even in this case metal substitution has proved to be useful in solving fundamental questions such as the assignment of the main visible bands of the blue copper proteins as S(Cys)-metal charge transfer bands Abbreviations. COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy ; WEFT, water-eliminated Fo...

Manganese and Cobalt in the Nonheme-Metal-Binding Site of a Biosynthetic Model of Heme-Copper Oxidase Superfamily Confer Oxidase Activity through Redox-Inactive Mechanism

“…Built upon the above success in elucidating the role of nonheme Cu and Fe ions in HCO activity, we explored further if the nonheme metal ion can be replaced with other first-row transition metal ions such as Mn and Co with different d-electron configurations and examined whether their presence imparted O 2 reduction activity. Cobalt and manganese have been used previously as robust probes to uncover necessary electronic structural properties and structure–activity relationships in other proteins, − including superoxide dismutase, − catechol dioxygenase, myoglobin, , P 1B -type ATPases, and metallothioneins . Understanding the roles of nonheme metal ions beyond Cu and Fe in native HCO and NOR will provide deeper insights into nature’s choice of metal ion and allow finer control of the activity beyond native enzymes.…”