Resonance Raman Study of Cyanide-Ligated Horseradish Peroxidase.Detection of Two Binding Geometries and Direct Evidence for the "Push-Pull" Effect

Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of the basic isoenzymes of horseradish peroxidase (HRP) in the pH range 5.5-12.5. Evidence for the binding of cyanide in two forms, characterized by the reversal of ordering of the Fe-CN stretching and Fe-C-N bending vibrations, is observed. Moreover, it is shown that both conformers exhibit an acid-alkaline transition in the pH range employed. In the first conformer, the Fe-C-N linkage is essentially linear, exhibiting axial Fe-CN stretching and Fe-C-N bending frequencies at 453 and 405 cm-1, respectively (at pH 5.5) (Lopez-Garriga et al., 1990). In the second conformer, the Fe-C-N fragment is bent, and the axial stretching and bending modes have been identified at 360 and 422 cm-1 at pH 5.5. At pH 12.5, the v[Fe-CN] stretching mode of the linear conformer shifts down by 9 cm-1 to 444 cm-1 while the bending frequency remains unchanged. For the bent conformer at this pH, the stretching mode shifts to 355 cm-1 (-5 cm-1), and the bending vibration shifts slightly to lower frequency by 2 cm-1 to 420 cm-1. The observed pH-dependent shift of the v[Fe-CN] stretching mode of the linear conformer is attributed to the direct effect of deprotonation of a distal-side amino acid residue while the shift of v[Fe-CN] of the bent conformer is most reasonably ascribable to indirect alteration of the iron-proximal histidine linkage induced by the distal-side deprotonation, a spectral response which reflects a protein-coupled "push-pull" mechanism for heterolytic O-O bond cleavage.