AN INTERPRETIVE BASIS OF THE HYPERFINE SHIFTS CYANIDE-INHIBITED HORSERADISH-PEROXIDASE BASED ON THE MAGNETIC AXES AND LIGAND TILT - INFLUENCE OF SUBSTRATE-BINDING AND EXTENSIONS TO OTHER PEROXIDASES

The H-1 NMR hyperfine shift pattern for nonheme resonances in low-spin (S = 1/2) cyanide-inhibited horseradish peroxidase, HRP-CN, and cytochrome c peroxidase, CcP-CN, are interpreted in terms of the anisotropy and orientation of the paramagnetic susceptibility tenser in the molecular framework. The orientation of the axes is obtained from a least-squares search for the Euler angles which will rotate the metal-centered crystal coordinates to a coordinate system (the magnetic axes) which quantitatively account for the observed dipolar shifts. Previous 2D NMR has shown that;the key active site residues in HRP-CN (Chen, Z.; de Ropp, J. S.; Hernandez, G.; La Mar, G. N. J. Am. Chem. Sec. 1994, 116, 8772) occupy essentially the same position in the active site as in the: crystal structure of CcP-CN, such that the crystal coordinates for select protons of CcP-CN could be used for both HRP-CN and CcP-CN NMR analysis. The validity of the magnetic axes description for HRP-CN is supported by the excellent correlation between observed and predicted dipolar shifts, by the ability to predict the shifts for residues not conserved between CcP and HRP, by the quantitative prediction of the influence of substrate binding on the hyperfine shifted pattern in HRP-CN, and by accounting for the significant variation in hyperfine shift pattern among cyanide-inhibited heme peroxidases genetic variants which possess very similar molecular structure. The major magnetic axis is found tilted (similar to 20 degrees) from the heme normal in both CcP-CN and HRP-CN, and is similar to that of the Fe-CN tilt in the CcP-CN crystal structure. The effect of binding the substrate benzhydroxaminic acid to HRP-CN is to decrease the tilt of the Fe-CN unit by 9 degrees away from the BHA binding site. The distinct patterns of hyperfine shifted for the cyanide-inhibited lignin and manganese peroxidases indicate that these proteins exhibit significantly less tilt (similar to 5 degrees) of the Fe-CN unit from the heme normal than CcP-CN or HRP-CN.