A ¹H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site

Abstract

The 'H NMR spectrum of Co(H) stellacyanin is reported, in which four signals not previously observed have been detected. NOE experiments were performed to assign the hyperfine shifted signals corresponding to a Cys and two His residues. Both His residues are solventaccessible and are shown to bind the metal ion through their NJ1 atoms. The j?-CH, Cys proton shifts indicate the presence of a strong axial ligand.

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic 1H NMR study of Co(II)-pseudoazurin

“…All the hyperfine shifted resonances follow a Curie temperature dependence, as observed in other Co(II)-substituted blue copper proteins [15,24,25,26,31,32,33,34,35,36,37]. Their chemical shifts and relaxation rates are typical of signals corresponding to proton nuclei from metal ligands ( Table 1).…”

“…Paramagnetic NMR investigations on cupredoxins include studies of the native Cu(II) species [19,20,21,22,23] and of their Co(II) and Ni(II) derivatives [15,24,25,26,27,28,29,30,31,32,33,34,35,36,37]. These studies have afforded evidence on the interplay between the strength of the metal-axial ligand interaction and the metal-Cys covalence [15,20,21].…”

The Structure of a Phytocyanin, the Basic Blue Protein from Cucumber, Refined at 1.8 Å Resolution

“…In a model for stellacyanin, generated later on the assumption that a high level of sequence identity with CBP indicates a high level of structural homology, the Cu site was just as exposed as in CBP (Fields et al, 1991). The correctness of this prediction was subsequently confirmed experimentally by 1 H NMR measurements on Co II -substituted stellacyanin (Vila, 1994).…”

Investigating metal-binding in proteins by nuclear magnetic resonance

“…Metal substitution in the blue copper proteins has also proved valuable. As amply demonstrated, the paramagnetic 1 H NMR spectra of Co 2+ -and Ni 2+ -substituted blue copper proteins possess hyperfine-shifted resonances which are much sharper than their counterparts in the Cu 2+ proteins [127,128]. These sharper signals can be assigned, and detailed analyses of the active-site structure of several blue copper proteins have been carried out [127 -132].…”