FACTORING OF THE HYPERFINE SHIFTS IN THE CYANIDE ADDUCT OF LIGNIN PEROXIDASE FROM P-CHRYSOSPORIUM

We have used NMR spectroscopy to study the cyanide derivative of lignin peroxidase with the goal of characterizing the electronic structure of this derivative of peroxidases. By using various homonuclear and heteronuclear 2D NMR techniques and by making use of the available X-ray structure of the cyanide-free protein, it has been possible to extend the assignment of the heme substituents and of the protons of some active-site residues. An estimate of the anisotropy and direction of the magnetic susceptibility anisotropy tenser has been obtained from the pseudocontact shifts of protons of residues not directly bound to the heme iron ion. Finally, a factoring of the hyperfine shifts of the heme and proximal histidine protons, as well as of the C-13 heme methyls and N-15 of the cyanide moiety, is obtained. The contact shift pattern of the heme protons is related to the orientation of the histidine plane. Very large upfield contact shifts are experienced by the aromatic protons of the proximal histidine. The axial magnetic anisotropy is smaller than in metmyoglobin-CN and slightly larger than in horseradish peroxidase-CN. This may reflect the order of the donor strength of the proximal histidine. The z axis of the magnetic susceptibility tenser is found essentially perpendicular to the heme plane.