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.

Resonance Raman studies of native and mesohemereconstituted horseradish peroxidase and their catalytic intermediates, known as Compounds I and II, have been conducted using both near UV (ϳ350 nm) and visible (406.7 nm) excitation. Careful power studies indicate that the authentic Compound I spectra are obtainable using near UV excitation, but that use of visible excitation results in contamination of the Compound I spectrum with the spectrum of a Compound II-like photoproduct. Using H 2 18 O 2 , the (Fe‫؍‬O) stretching modes for both systems are unambiguously identified, for the first time, at ϳ790 cm ؊1 . The authentic Compound I spectra are indicative of an 2 A 1u -like ground state for both the native and the mesoheme-reconstituted proteins. Finally, the possible biological implications of such information are briefly discussed.

Resonance Raman spectroscopy has been used to investigate the properties of cyanide-bound beef liver catalase (BLC) and Aspergillus niger catalase (ANC) in the pH range 4.9-11.5. Evidence has been obtained for the binding of cyanide to both BLC and ANC in two binding geometries. The first conformer, exhibiting the nu[Fe-CN] stretching mode at a higher frequency than the delta[Fe-C-N] bending mode, exists as an essentially linear Fe-C-N linkage. For both BLC-CN and ANC-CN, the nu[Fe-CN] and delta[Fe-C-N] frequencies of this conformer were practically identical and observed at approximately 434 and approximately 413 cm-1, respectively. The second conformer exhibits a nu[Fe-CN] mode at lower frequency than the delta[Fe-C-N] mode, and is thus characteristic of a bent Fe-C-N linkage. The nu[Fe-CN] and delta[Fe-C-N] modes were identified at 349 and 445 cm-1, respectively, for BLC-CN, and at 350 and 456 cm-1, respectively, for ANC-CN. The two conformers persist in the pH range 4.9-11.5. Furthermore, upon raising the pH to 11.5, the nu[Fe-CN] mode of the linear conformer of BLC-CN downshifts to 429 cm-1 while that of the bent conformer remains unchanged. The observed pH-dependent shift is attributed to the deprotonation of a distal-side amino acid residue, probably a distal histidine. The Fe-C-N axial vibrations of the two conformers identified for ANC-CN did not show any significant pH-dependent shifts, indicating a more stable hydrogen bonding interaction relative to BLC-CN.

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