Analysis of the paramagnetic copper(II) site of amicyanin by 1H NMR spectroscopy.
Application of the tailored pulse sequences like super-WEFT allows the direct observation of the hyperfine-shifted signals of the paramagnetic Cu(II) forms of blue copper proteins in solution. The signals can be assigned by applying 2D NMR techniques, like EXSY, to solutions containing a mixture of reduced and oxidized species. The Fermi contact shift is separated from the pseudocontact shift on the basis of the known g-tensor anisotropy of the Cu(II) state, allowing the determination of a number of hyperfine-splitting constants between protons on the Cu ligands and the unpaired electron. These results are used to quantify the spin density distribution over the Cu ligands. In amicyanin about 50%-60% of the unpaired electron density is found on the ligands. It appears possible to quantify the Cu-S(Met) interaction on the basis of the NMR results. Application of the technique to the wild type forms of amicyanin and azurin and to two active site mutants of amicyanin (His96Asp and a plastocyanin-amicyanin loop exchange mutant) shows that the Cu-S(Met) interaction parallels the rhombicity and axial distortion of the Cu site.
Kalverda
A P
AP
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Salgado
J
J
Dennison
C
C
Canters
G W
GW
D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D013855:Thiobacillus / Q000378:metabolism*
D001426:Bacterial Proteins; D008667:Metalloproteins; D011994:Recombinant Proteins; D012996:Solutions; C017079:mauC protein, Methylobacterium extorquens; D001400:Azurin; D003300:Copper; D006859:Hydrogen