title: NMR spectroscopic identification of a hexacyanochromate(III) binding site on Pseudomonas azurin.
abstract: Studies of redox reactions between the blue copper protein azurin and inorganic reagents have suggested the formation of discrete complexes between the reaction partners prior to the actual electron-transfer step. To get an insight into the structural nature of the complexes formed, we have studied the interaction (i) between oxidized Pseudomonas aeruginosa azurin and the anion Fe(CN)6(3-) and (ii) between reduced azurin and Cr(CN)6(3-). At low ionic strengths, stoichiometric binding of one Fe(CN)6(3-) ion to the oxidized protein is observed. In the high-resolution proton magnetic resonance spectra of the reduced protein, specific broadening of the assigned residues is observed upon titration with the redox-inert Cr(CN)6(3-) ion. Analysis of this paramagnetic spectral broadening in terms of the three-dimensional structure of the protein has led to the proposal that the binding site of the anions lies approximately midway between lysine residues 85 and 92. Evidence in support of this conclusion is provided by parallel studies on Alcaligenes faecalis azurin, which lacks these lysine residues. A similar site on the surface of Pseudomonas azurin has recently been identified by affinity labeling with chromous ions as an electron-transfer locus [ Farver , O., & Pecht , I. (1981) Isr . J. Chem. 21, 13-17]. The results presented here suggest that this region on the protein surface also may be employed by anionic electron-transfer agents.
mesh_terms: D000421:Alcaligenes / Q000378:metabolism*; D001400:Azurin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002840:Chromates; D003486:Cyanides; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002840:Chromates; D003486:Cyanides; D001400:Azurin; C021038:hexacyanochromate