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

Abstract

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.

Validation of a two‐parameter quantitative structure–activity relationship as a legitimate tool for rational re‐design of horseradish peroxidase

“…Thus, the use of the average characteristic binding distance (m HIS ) as a second QSAR parameter unifies the two classes of chemical substrates investigated as indicated in Figure 2 (Colosi et al, 2006). This is consistent with the current understanding of the ''push-pull'' mechanism by which HRP Compound I is thought to oxidize a phenolic substrate, whereby formation of a hydrogen bond and subsequent proton transfer between the substrate proton and the dN of the HIS42 imidazole facilitates electron transfer to the heme (Al-Mustafa and Kincaid, 1994;Dunford, 1991).…”

Catalytic Activities and Structural Properties of Horseradish Peroxidase Distal His42 → Glu or Gln Mutant

“…Figure 5 illustrates resonance Raman spectra of (A) C 14 N, (B) C 15 N-ligated states, and (C) their difference spectra with 413.1 nm excitation. The Raman band at 451 cm -1 of native cyanide-ligated HRP has been assigned to the axial Fe-CN stretching Raman frequency (Al-Mustafa & Kincaid, 1994). Although the corresponding Raman bands for the cyanideligated H42E and H42Q mutants were almost diminished, as shown in Figure 5A, the difference spectra ( Figure 5C) clearly showed an isotope shift of the Fe-CN mode.…”

Crystallographic Snapshots of Cyanide- and Water-Bound C-Clusters from Bifunctional Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase,

“…Here, we similarly observe Ile591 impart a potential steric clash that cannot be overcome during refinement (Figure 2f). There is precedence for a bent cyanide metal ligand in several heme-containing metalloproteins, including catalases (49), peroxidases (50,51), sulfite reductase hemoprotein (52), hemoglobin and myoglobin (53), cytochrome cd 1 nitrite reductase (54), and cytochrome P450 enzymes (55,56). A study of the non-heme mononuclear iron enzyme superoxide reductase also identifies bent CN geometry, where the Fe−C−N bond angle was observed as 123° and 133° for two different substrates (57).…”